US 20050214276 A9
The invention relates to Δ4,5 glycuronidase, related compositions, and methods of use thereof.
1. A substantially pure Δ4,5 unsaturated glycuronidase.
2. The glycuronidase of
3. The glycuronidase of
4. The glycuronidase of
5. An isolated polypeptide comprising:
Δ4,5 unsaturated glycuronidase having an amino acid sequence of SEQ ID NO: 1 or a functional variant thereof.
6. The isolated polypeptide of
7. A composition comprising, an isolated Δ4,5 unsaturated glycuronidase having a higher specific activity than native glycuronidase.
8. The composition of
9. The composition of
10. The composition of
11. The composition of
12. An isolated nucleic acid molecule selected from the group consisting of
(a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ ID NO:2, and which code for Δ4,5 unsaturated glycuronidase having an amino acid sequence set forth as SEQ ID NO:1,
(b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code, and
(c) complements of (a) or (b).
13. The isolated nucleic acid molecule of
14. The isolated nucleic acid molecule of
15. An expression vector comprising the isolated nucleic acid molecule of
16. A host cell comprising the expression vector of
17. An expression vector comprising the isolated nucleic acid molecule of
18. A host cell comprising the expression vector of
19. A pharmaceutical preparation comprising:
a composition or vector of any of claims 1-15 and a pharmaceutically acceptable carrier.
20. A method of cleaving a glycosaminoglycan, comprising:
contacting a glycosaminoglycan with the glycuronidase of any one of claims 1-10 in an effective amount to cleave the glycosaminoglycan.
21. A glycosaminoglycan prepared according to the method of
22. A method of removing heparin from a heparin containing fluid, comprising:
contacting a heparin containing fluid with the glycuronidase of any one of claims 1-10 in an effective amount to removing heparin from the heparin containing fluid.
23. The method of
24. The method of
25. A method of analyzing of a glycosaminoglycan, comprising:
contacting a glycosaminoglycan with the glycuronidase of any one of claims 1-10 in an effective amount to analyze the glycosaminoglycan.
26. The method of
27. The method of
28. The method of
29. The method of
30. The method of
31. A method of cleaving a glycosaminoglycan comprised of at least one disaccharide unit, comprising:
contacting a glycosaminoglycan comprised of at least one disaccharide unit with the glycuronidase of any one of claims 1-10 in an effective amount to cleave the glycosaminoglycan.
32. The method of
33. The method of
34. The method of
35. The method of
36. The method of
37. The method of
38. The method of
39. The method of
40. A method of inhibiting angiogenesis, comprising
administering to a subject in need thereof an effective amount of the pharmaceutical preparation of
41. A method of treating cancer, comprising
administering to a subject in need thereof an effective amount of the pharmaceutical preparation of
42. A method of inhibiting cellular proliferation, comprising
administering to a subject in need thereof an effective amount of the pharmaceutical preparation of
43. A method of treating a coagulation disorder, comprising
administering the composition of
44. The method of
45. A pharmaceutical preparation comprising:
a composition of
46. A method of inhibiting angiogenesis, comprising
administering the composition of
47. A method of treating cancer, comprising
administering the composition of
48. A method of inhibiting cellular proliferation, comprising
administering the composition of
49. A method of treating a coagulation disorder, comprising
administering the composition of
This application claims priority under 35 U.S.C. §119 from U.S. provisional application serial No. 60/377,488 filed May 3, 2002, the entire contents of which is incorporated by reference.
Aspects of the invention may have been made using funding from National Institutes of Health Grant number NIHGM57073 and CA090940. Accordingly, the Government may have rights in the invention.
The invention relates to Δ4, 5 glycuronidase and uses thereof. In particular, the invention relates to substantially pure Δ4, 5 glycuronidase which is useful for a variety of purposes, including analysis of glycosaminoglycans (GAGs), sequencing, identifying, quantifying and purifying glycosaminoglycans present in a sample, removing glycosaminoglycans, such as heparin, from a solution and inhibiting angiogenesis, controlling coagulation, etc. The invention also relates to methods of treating cancer and inhibiting cellular proliferation and/or metastasis using Δ4, 5 glycuronidase and/or GAG fragments produced by enzymatic cleavage with Δ4, 5 glycuronidase.
Glycosaminoglycans (GAGs) are linear, acidic polysaccharides that exist ubiquitously in nature as residents of the extracellular matrix and at the cell surface of many different organisms of divergent phylogeny [Habuchi, O. (2000) Biochim Biophys Acta 1474, 115-27; Sasisekharan, R., Bulmer, M., Moremen, K. W., Cooney, C. L., and Langer, R. (1993) Proc Natl Acad Sci USA 90, 3660-4]. In addition to a structural role, GAGs act as critical modulators of a number of biochemical signaling events [Tumova, S., Woods, A., and Couchinan, J. R. (2000) Int J Biochem Cell Biol 32, 269-88] requisite for cell growth and differentiation, cell adhesion and migration, and tissue norphogenesis.
Heparan sulfate like glycosaminoglycans (GAGS or HSGAGs) are present both at the cell surface and in the extracellular matrix. Heparin-like glycosaminoglycans are important components of the extracellular matrix that are believed to regulate a wide variety of cellular activities including invasion, migration, proliferation and adhesion (Khodapkar, et al. 1998; Woods, et al., 1998). HSGAGs accomplish some of these functions by binding to and regulating the biological activities of diverse molecules, including growth factors, morphogens, enzymes, extracellular proteins. HSGAGs are a group of complex polysaccharides that are variable in length, consisting of a disaccharide repeat unit composed of glucosamine and an uronic acid (either iduronic or glucuronic acid). The high degree of complexity for HSGAGs arises not only from their polydispersity and the possibility of two different uronic acid components, but also from differential modification at four positions of the disaccharide unit. Three positions, viz., C2 of the uronic acid and the C3, C6 positions of the glucosamine can be O-sulfated. In addition, C2 of the glucosamine can be N-acetylated or N-sulfated. Together, these modifications could theoretically lead to 32 possible disaccharide units, making HSGAGs potentially more information dense than either DNA (4 bases) or proteins (20 amino acids). It is this enormity of possible structural variants that allows HSGAGs to be involved in a large number of diverse biological processes, including angiogenesis (Sasisekharan, R., Moses, M. A., Nugent, M. A., Cooney, C. L. & Langer, R. (1994) Proc Natl Acad Sci USA, 1524-8.), embryogenesis (Binari, R. et al (1997) Development, 2623-32; Tsuda, M., et al. (1999) Nature, 276-80.; and Lin, X., et al (1999) Development, 3715-23.) and the formation of β-fibrils in Alzheimer's disease (McLaurin, J., et al (1999) Eur J Biochem, 1101-10. and Lindahl, B., et al (1999) J Biol Chem, 30631-5).
One specific example of an HSGAG is heparin. Heparin, a highly sulphated HSGAG produced by mast cells, is a widely used clinical anticoagulant, and is one of the first biopolymeric drugs and one of the few carbohydrate drugs. Heparin primarily elicits its effect through two mechanisms, both of which involve binding of antithrombin III (AT-III) to a specific pentasaccharide sequence, HNAc/S,6SGHNS,3S,6SI2SHNs,6S contained within the polymer. HSGAGs have also emerged as key players in a range of biological processes that range from angiogenesis [Folkman, J., Taylor, S., and Spillberg, C. (1983) Ciba Found Symp 100, 132-49] and cancer biology [Blackhall, F. H., Merry, C. L., Davies, E. J., and Jayson, G. C. (2001) Br J Cancer 85, 1094-8] to microbial pathogenesis [Shukla, et al (1999) Cell 99, 13-22]. HSGAGs have also recently been shown to play a fundamental role in multiple aspects of development [Perrimon, N. and Bernfield, M. (2000) Nature 404, 725-8]. The ability of HSGAGs to orchestrate multiple biological events is again likely a consequence of its structural complexity and information density [Sasisekharan, R. and Venkataraman, G. (2000) Curr Opin Chem Biol 4, 626-31].
Although the structure and chemistry of HSGAGs are fairly well understood, information on how specific HSGAG sequences modulate different biological processes has proven harder to obtain. Determination of these HSGAG sequence has been technically challenging. HSGAGs are naturally present in very limited quantities, which, unlike other biopolymers such as proteins and nucleic acids, cannot be readily amplified. Second, due to their highly charged character and structural heterogeneity, HSGAGs are not easily isolated from biological sources in a highly purified state. Additionally, the lack of sequence-specific tools to cleave HSGAGs in a manner analogous to DNA sequencing or restriction mapping has made sequencing a challenge.
Recently, in an effort to develop an understanding of HSGAG structure, focus has been placed on the cloning and characterization of the enzymes involved in HSGAG biosynthesis. Another, strategy for elucidating the structure of HSGAGs has been to employ specific HSGAG degradation procedures, including chemical or enzymatic cleavage, in conjunction with analytical methodologies, including gel electrophoresis or HPLC, to sequence HSGAGs. Recently, we have introduced a sequencing procedure that couples a bioinformatics framework with mass spectrometric and capillary electrophoretic procedures to sequence rapidly biologically important HSGAGs, including saccharide sequences involved in modulating anticoagulation. The sequencing methodology uses chemical and enzymatic tools to modify or degrade an unknown glycosaminoglycan polymer in a sequence-specific manner. (Venkataraman, G., et al., Science, 286, 537-542 (1999), and U.S. patent application Ser. Nos. 09/557,997 and 09/558,137, both filed on Apr. 24, 2000, having common inventorship).
Δ4, 5 glycuronidase has been cloned from the F. heparinum genome and its subsequent recombinant expression in E. coli as a soluble, highly active enzyme has been accomplished. Thus, in one aspect the present invention provides for a substantially pure Δ4,5 glycuronidase. In one embodiment of the invention the substantially pure Δ4,5 glycuronidase is a recombinantly produced glycuronidase. Recombinant expression may be accomplished in one embodiment with an expression vector. An expression vector may be a nucleic acid for SEQ ID NO:2, optionally operably linked to a promoter. In another embodiment the expression vector may be a nucleic acid for SEQ ID NO:4 or a variant thereof also optionally linked to a promoter. In one embodiment the substantially pure Δ4,5 glycuronidase is produced using a host cell comprising the expression vector. In another embodiment the substantially pure Δ4,5 glycuronidase is a synthetic glycuronidase.
In another aspect the glycuronidase of the invention is a polypeptide having an amino acid sequence of SEQ ID NO:1, or a functional variant thereof. In yet another aspect the polypeptide has an amino acid sequence of SEQ ID NO:3, or a functional variant thereof.
In yet another aspect of the invention the polypeptide of the Δ4,5 glycuronidase is an isolated polypeptide. The isolated polypeptide in some embodiments is set forth in SEQ ID NO:1 or is a functional variant thereof. In other embodiments the isolated polypeptide is set forth in SEQ ID NO:3 or a functional variant thereof.
In one aspect, the invention is a composition comprising, an isolated Δ4,5 unsaturated glycuronidase having a higher specific activity than native glycuronidase. In some embodiments, the specific activity is at least about 60 picomoles of substrate hydrolyzed per minute per picomole of enzyme. In one embodiment the Δ4,5 glycuronidase has a specific activity that is about 2 fold higher than the native enzyme. In another embodiment the Δ4,5 glycuronidase has a specific activity that is about 3 fold higher. The specific activity of the Δ4,5 glycuronidase in other embodiments may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any integer therebetween fold higher than the activity of the native enzyme.
In yet another aspect of the invention an isolated nucleic acid molecule is provided. The nucleic acid is (a) nucleic acid molecules which hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ ID NO:2 or SEQ ID NO:4, and which code for Δ4,5 unsaturated glycuronidase having an amino acid sequence set forth as SEQ ID NO:1 or SEQ ID NO:3, respectively, (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code, or (c) complements of (a) or (b). In one embodiment the isolated nucleic acid molecule codes for SEQ ID NO:1. In another embodiment the isolated nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:2. In still other embodiments the isolated nucleic acid molecule codes for SEQ ID NO:3 and in yet other embodiments the isolated nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:4.
Pharmaceutical compositions of any of the compositions or vectors described herein are also encompassed in the invention.
In other aspects the invention relates to a method of cleaving a glycosaminoglycan with a Δ4,5 unsaturated glycuronidase. The method may be performed by contacting a glycosaminoglycan with the glycuronidase in an effective amount to cleave the glycosaminoglycan. In one embodiment the invention is a glycosaminoglycan prepared according to this method.
In other aspects the invention also provides a method of cleaving a glycosaminoglycan comprised of at least one disaccharide unit. The method may be performed by contacting the glycosaminoglycan with a glycuronidase of the invention in an effective amount to cleave the glycosaminoglycan. In some embodiments the glycosaminoglycan is a long chain saccharide. In other embodiments the glycosaminoglycan does not contain a 2-O sulfated uronidate or it does not contain N-substituted glycosamine. In yet another embodiment the glycosaminoglycan is 6-0 sulfated. The disaccharide units in some embodiments are ΔUHNAc; ΔUHNAc,6S; ΔUHNS,6S; or ΔUHNS. In another embodiment the invention also provides for the products of the cleavage of a glycosaminoglycan with the Δ4,5 glycuronidase. In some embodiments the glycuronidase is used to generate a LMWH.
The present invention also provides methods for the analysis of glycosaminoglycan. In one aspect the invention is a method of analyzing a glycosaminoglycan by contacting a glycosaminoglycan with the glycuronidase of the invention in an effective amount to analyze the glycosaminoglycan. In one embodiment the method is a method for identifying the presence of a particular glycosaminoglycan in a sample. In another embodiment the method is a method for determining the identity of a glycosaminoglycan in a sample. In yet another embodiment the method is a method for determining the purity of a glycosaminoglycan in a sample. In still a further embodiment the method is a method for determining the composition of a glycosaminoglycan in a sample. In another embodiment the method is a method for determining the sequence of saccharide units in a glycosaminoglycan. In other embodiments, these methods may also comprise an additional analytical technique such as mass spectrometry, gel electrophoresis, capillary electrophoresis and HPLC. In some embodiments the glycosaminoglycan is LMWH.
In other aspects the invention is a method of removing heparin from a heparin containing fluid by contacting a heparin containing fluid with a glycuronidase of the invention in an effective amount to remove heparin from the heparin containing fluid. In one embodiment the glycuronidase is immobilized on a solid support. In another embodiment a heparinase is also provided and the heparinase is also immobilized on the solid support.
In another aspect the invention is a method of inhibiting angiogenesis by administering to a subject in need thereof an effective amount of any of the pharmaceutical preparations described herein for inhibiting angiogenesis.
In another aspect a method of treating cancer by administering to a subject in need thereof an effective amount of any of the pharmaceutical preparations described herein for treating cancer is also provided.
Yet another aspect of the invention is a method of inhibiting cellular proliferation by administering to a subject in need thereof an effective amount of any of the pharmaceutical preparations described herein for inhibiting cellular proliferation.
In another aspect a method of treating a coagulation disease by administering to a subject in need thereof a LMWH prepared using the glycuronidase of the invention.
In some embodiments of the methods of the invention the glycuronidase is used concurrently with or following treatment with heparinase.
In other aspects of the invention, the pharmaceutical compositions and therapeutic methods are provided using the Δ4,5 unsaturated glycuronidase and the cleaved GAG fragments alone or in combination.
Other aspects of the invention provide compositions that include other enzymes such as heparinase with the Δ4,5 unsaturated glycuronidase.
In other aspects a pharmaceutical preparation of a composition or vector of the invention in a pharmaceutically acceptable carrier is provided.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.
The invention in some aspects relates to Δ4, 5 glycuronidase, substantially pure forms thereof and uses thereof. In particular the invention arose, in part, from the cloning of Δ4, 5 glycuronidase that now enables one of skill in the art to produce the enzyme in large quantities and in substantially pure form. The invention also provides another tool that may be used to determine the structure of glycosaminoglycans and to help elucidate their role in cellular processes. It has now also been discovered that substantially pure preparations of Δ4, 5 glycuronidase having higher specific activity than the enzyme produced from culture may be produced. The invention also provides for cleavage of glycosaminoglycans (GAGs) as well as for the analysis of a sample of GAGs and for their sequencing. This present invention also provides treatment and prevention methods for cancer through the control of cellular proliferation, angiogenesis and/or coagulation disorders with the enzyme and/or its cleavage products (GAG fragments).
One aspect of the invention enables one of ordinary skill in the art, in light of the present disclosure, to produce substantially pure preparations of the Δ4, 5 glycuronidase by standard technology, including recombinant technology, direct synthesis, mutagenesis, etc. For instance, using recombinant technology one may produce substantially pure preparations of the Δ4, 5 glycuronidase having the amino acid sequences of SEQ ID NO:1 or encoded by the nucleic acid sequence of SEQ ID NO:2. In other aspects of the invention substantially pure preparations of the Δ4, 5 glycuronidase having the amino acid sequences of SEQ ID NO:3 or encoded by the nucleic acid sequence of SEQ ID NO:4 can be prepared. One of skill in the art may also substitute appropriate codons to produce the desired amino acid substitutions in SEQ ID NOS:1 or 3 by standard site-directed mutagenesis techniques. One may also use any sequence which differs from the nucleic acid equivalents of SEQ ID NO:1 or 3 only due to the degeneracy of the genetic code as the starting point for site directed mutagenesis. The mutated nucleic acid sequence may then be ligated into an appropriate expression vector and expressed in a host such as E. coli. The resultant Δ4, 5 glycuronidase may then be purified by techniques, including those disclosed below.
As used herein, the term “substantially pure” means that the proteins are essentially free of other substances to an extent practical and appropriate for their intended use. In particular, the proteins are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, protein sequencing, or producing pharmaceutical preparations.
As used herein, a “substantially pure Δ4,5 unsaturated glycuronidase” is a preparation of Δ4,5 unsaturated glycuronidase which has been isolated or synthesized and which is greater than about 90% free of contaminants. A contaminant is a substance with which the Δ4,5 unsaturated glycuronidase is ordinarily associated in nature that interfere with the activity of the enzyme. Preferably, the material is greater than about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even greater than about 99% free of contaminants. The degree of purity may be assessed by means known in the art. One method for assessing the purity of the material may be accomplished through the use of specific activity assays. The native Δ4,5 glycuronidase which has been described in the prior art as being isolated from F. heparinum has low specific activity because of impurities inherent in harvesting the enzyme from bacterial cultures of F. heparinum.
The invention also provides isolated polypeptides (including whole proteins and partial proteins), of Δ4, 5 glycuronidase having the amino acid sequence of SEQ ID NO:1 and functional variants thereof. Isolated polypeptides are also provided by the invention that have the amino acid sequence of SEQ ID NO:3. Polypeptides can be isolated from biological samples, and can also be expressed recombinantly in a variety of prokaryotic and eukaryotic expression systems by constructing an expression vector appropriate to the expression system, introducing the expression vector into the expression system, and isolating the recombinantly expressed protein. Polypeptides can also be synthesized chemically using well-established methods of peptide synthesis.
As used herein with respect to polypeptides, “isolated” means separated from its native environment and present in sufficient quantity to permit its identification or use. Isolated, when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis. Isolated proteins or polypeptides may be, but need not be, substantially pure. Because an isolated polypeptide may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the polypeptide may comprise only a small percentage by weight of the preparation. The polypeptide is nonetheless isolated in that it has been separated from the substances with which it may be associated in living systems, i.e., isolated from other proteins.
Thus the term “Δ4, 5 glycuronidase polypeptides” embraces variants as well as the natural Δ4, 5 glycuronidase polypeptides. As used herein, a “variant” of a Δ4, 5 glycuronidase polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of a naturally occurring Δ4, 5 glycuronidase polypeptide. Variants include modified Δ4, 5 glycuronidase polypeptides that do not have altered function relative to the polypeptide of the unmodified (naturally occurring) sequence. Variants also include Δ4, 5 glycuronidase polypeptides with altered function. Modifications which create a Δ4, 5 glycuronidase polypeptide variant are typically made to the nucleic acid which encodes the Δ4, 5 glycuronidase polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and addition of amino acids or non-amino acid moieties to: 1) enhance a property of a Δ4, 5 glycuronidase polypeptide, such as protein stability in an expression system or the stability of protein-protein binding; 2) provide a novel activity or property to a Δ4, 5 glycuronidase polypeptide, such as addition of a detectable moiety; or 3) to provide equivalent or better interaction with other molecules (e.g., heparin). Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the Δ4, 5 glycuronidase amino acid sequence. One of skill in the art will be familiar with methods for predicting the effect on protein conformation of a change in protein sequence, and can thus “design” a variant Δ4, 5 glycuronidase polypeptide according to known methods. One example of such a method is described by Dahiyat and Mayo in Science 278:82-87, 1997, whereby proteins can be designed de novo. The method can be applied to a known protein to vary a only a portion of the polypeptide sequence. By applying the computational methods of Dahiyat and Mayo, specific variants of a polypeptide can be proposed and tested to determine whether the variant retains a desired conformation.
Variants can include Δ4, 5 glycuronidase polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, certain amino acids can be changed to enhance expression of a Δ4, 5 glycuronidase polypeptide by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present).
Mutations of a nucleic acid which encodes a Δ4, 5 glycuronidase polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the variant polypeptide.
Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Variant polypeptides are then expressed and tested for one or more activities to determine which mutation provides a variant polypeptide with the desired properties. Further mutations can be made to variants (or to non-variant Δ4, 5 glycuronidase polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli, are well known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a Δ4, 5 glycuronidase gene or cDNA clone to enhance expression of the polypeptide.
One type of amino acid substitution is referred to as a “conservative substitution.” As used herein, a “conservative amino acid substitution” or “conservative substitution” refers to an amino acid substitution in which the substituted amino acid residue is of similar charge as the replaced residue and is of similar or smaller size than the replaced residue. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) the small non-polar amino acids, A, M, I, L, and V; (b) the small polar amino acids, G, S, T and C; (c) the amido amino acids, Q and N; (d) the aromatic amino acids, F, Y and W; (e) the basic amino acids, K, R and H; and (f) the acidic amino acids, E and D. Substitutions which are charge neutral and which replace a residue with a smaller residue may also be considered “conservative substitutions” even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine). The term “conservative amino acid substitution” also refers to the use of amino acid analogs or variants.
Methods for making amino acid substitutions, additions or deletions are well known in the art. The terms “conservative substitution”, “non-conservative substitutions”, “non-polar amino acids”, “polar amino acids”, and “acidic amino acids” are all used consistently with the prior art terminology. Each of these terms is well-known in the art and has been extensively described in numerous publications, including standard biochemistry text books, such as “Biochemistry” by Geoffrey Zubay, Addison-Wesley Publishing Co., 1986 edition, which describes conservative and non-conservative substitutions, and properties of amino acids which lead to their definition as polar, non-polar or acidic.
One skilled in the art will be able to predict the effect of a substitution by using routine screening assays, preferably the biological assays described herein. Modifications of peptide properties including thermal stability, enzymatic activity, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan. For additional detailed description of protein chemistry and structure, see Schulz, G. E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1979, and Creighton, T. E., Proteins: Structure and Molecular Principles, W. H. Freeman & Co., San Francisco, 1984.
Additionally, some of the amino acid substitutions are non-conservative substitutions. In certain embodiments where the substitution is remote from the active or binding sites, the non-conservative substitutions are easily tolerated provided that they preserve a tertiary structure characteristic of, or similar to, native Δ4, 5 glycuronidase, thereby preserving the active and binding sites. Non-conservative substitutions, such as between, rather than within, the above groups (or two other amino acid groups not shown above), which will differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
In another set of embodiments an isolated nucleic acid equivalent of SEQ ID NO:2 encode the substantially pure Δ4, 5 glycuronidase of the invention and functional variants thereof. In still further embodiments isolated nucleic acid equivalents of SEQ ID NO:4 are also given. According to the invention, isolated nucleic acid molecules that code for a Δ4, 5 glycuronidase polypeptide are provided and include: (a) nucleic acid molecules which hybridize under stringent conditions to a molecule selected from a group consisting of the nucleic acid equivalent of SEQ ID NO:2 or 4 and which code for a Δ4, 5 glycuronidase polypeptide or parts thereof, (b) deletions, additions and substitutions of (a) which code for a respective Δ4, 5 glycuronidase polypeptide or parts thereof, (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and (d) complements of (a), (b) or (c).
The invention also includes degenerate nucleic acids which include alternative codons to those present in the naturally occurring materials. For example, serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purposes of encoding a serine residue. Thus, it will be apparent to one of ordinary skill in the art that any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into an elongating Δ4, 5 glycuronidase polypeptide. Similarly, nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons). Other amino acid residues may be encoded similarly by multiple nucleotide sequences. Thus, the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.
As used herein with respect to nucleic acids, the term “isolated” means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5′ and 3′ restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its naturally occurring state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.
One embodiment of the invention provides Δ4, 5 glycuronidase that is recombinantly produced. Such molecules may be recombinantly produced using a vector including a coding sequence operably joined to one or more regulatory sequences. As used herein, a coding sequence and regulatory sequences are said to be “operably joined” when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein the coding sequences are operably joined to regulatory sequences. Two DNA sequences are said to be operably joined if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5′ non-transcribing and 5′ non-translating sequences involved with initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5′ non-transcribing regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Promoters may be constitutive or inducible. Regulatory sequences may also include enhancer sequences or upstream activator sequences, as desired.
As used herein, a “vector” may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium, or just a single time per host as the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques. Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
As used herein, the tern “stringent conditions” refers to parameters known to those skilled in the art. One example of stringent conditions is hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin (BSA), 25 mM NaH2PO4 (pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS is sodium dodecylsulphate; and EDTA is ethylene diamine tetra acetic acid. There are other conditions, reagents, and so forth which can be used, which result in the same degree of stringency. A skilled artisan will be familiar with such conditions, and thus they are not given here.
The skilled artisan also is familiar with the methodology for screening cells for expression of such molecules, which then are routinely isolated, followed by isolation of the pertinent nucleic acid. Thus, homologs and alleles of the substantially pure Δ4, 5 glycuronidase of the invention, as well as nucleic acids encoding the same, may be obtained routinely, and the invention is not intended to be limited to the specific sequences disclosed. It will be understood that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of the Δ4, 5 glycuronidase nucleic acids of the invention. The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and sequencing.
In general homologs and alleles typically will share at least about 40% nucleotide identity and/or at least about 50% amino acid identity with the equivalents of SEQ ID Nos: 2 and 1, respectively. Homologs and alleles of the invention are also intended to encompass the nucleic acid and amino acid equivalents of SEQ ID Nos: 4 and 3, respectively. In some instances sequences will share at least about 50% nucleotide identity and/or at least about 65% amino acid identity and in still other instances sequences will share at least about 60% nucleotide identity and/or at least about 75% amino acid identity. The homology can be calculated using various, publicly available software tools developed by NCBI (Bethesda, Md.) that can be obtained through the internet (ftp:/ncbi.nlm.nih.gov/pub/). Exemplary tools include the BLAST system available at http://wwww.ncbi.nim.nih.gov. Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis can be obtained using the MacVetor sequence analysis software (Oxford Molecular Group). Watson-Crick complements of the foregoing nucleic acids also are embraced by the invention.
In screening for Δ4, 5 glycuronidase related genes, such as homologs and alleles of Δ4, 5 glycuronidase, a Southern blot may be performed using the foregoing conditions, together with a radioactive probe. After washing the membrane to which the DNA is finally transferred, the membrane can be placed against X-ray film or a phosphoimager plate to detect the radioactive signal.
For prokaryotic systems, plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host may be used. Examples of suitable plasmid vectors include pBR322, pUC18, pUC19 and the like; suitable phage or bacteriophage vectors include λgt10, λgt11 and the like; and suitable virus vectors include pMAM-neo, pKRC and the like. Preferably, the selected vector of the present invention has the capacity to autonomously replicate in the selected host cell. Useful prokaryotic hosts include bacteria such as E. coli, Flavobacterium heparinum, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like.
To express the substantially pure Δ4, 5 glycuronidase of the invention in a prokaryotic cell, it is desirable to operably join the nucleic acid sequence of a substantially pure Δ4, 5 glycuronidase of the invention to a functional prokaryotic promoter. Such promoter may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of the β-lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ (PL and PR), the trp, recA, lacZ, lacI, and gal promoters of E. coli, the α-amylase (Ulmanen et al., J. Bacteriol. 162:176-182 (1985)) and the 4-28-specific promoters of B. subtilis (Gilman et al., Gene sequence 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward et al., Mol. Gen. Genet. 203:468-478 (1986)).
Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo (Biochimie 68:505-516 (1986)); and Gottesman (Ann. Rev. Genet. 18:415-442 (1984)).
Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream of the encoding sequence. Such ribosome binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404 (1981)).
Because prokaryotic cells may not produce the Δ4, 5 glycuronidase of the invention with normal eukaryotic glycosylation, expression of the Δ4, 5 glycuronidase of the invention of the eukaryotic hosts is useful when glycosylation is desired. Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, and mammalian cells, either in vivo or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or the myeloma P3x63Sg8, and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332 that may provide better capacities for correct post-translational processing. Embryonic cells and mature cells of a transplantable organ also are useful according to some aspects of the invention.
In addition, plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences.
Another preferred host is an insect cell, for example in Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science 240:1453-1459 (1988)). Alternatively, baculovirus vectors can be engineered to express large amounts of the Δ4, 5 glycuronidase of the invention in insect cells (Jasny, Science 238:1653 (1987); Miller et al., In: Genetic Engineering (1986), Setlow, J. K., et al., eds., Plenum, Vol. 8, pp. 277-297).
Any of a series of yeast gene sequence expression systems which incorporate promoter and termination elements from the genes coding for glycolytic enzymes and which are produced in large quantities when the yeast are grown in media rich in glucose may also be utilized. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. Yeast provide substantial advantages in that they can also carry out post-translational peptide modifications. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number plasmids which can be utilized for production of the desired proteins in yeast. Yeast recognize leader sequences on cloned mammalian gene sequence products and secrete peptides bearing leader sequences (i.e., pre-peptides).
A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, and the like, may be employed. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated. Of interest are regulatory signals that are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or which are subject to chemical (such as metabolite) regulation.
As discussed above, expression of the Δ4, 5 glycuronidase of the invention in eukaryotic hosts is accomplished using eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein 1 gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, Cell 31:355-365 (1982)); the SV40 early promoter (Benoist et al., Nature (London) 290:304-310 (1981)); the yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).
As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the Δ4, 5 glycuronidase of the invention does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in the formation of a fusion protein (if the AUG codon is in the same reading frame as the Δ4, 5 glycuronidase of the invention coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the Δ4, 5 glycuronidase of the invention coding sequence).
In one embodiment, a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may, for example, provide for prototrophy to an auxotrophic host or may confer biocide resistance to, e.g., antibiotics, heavy metals, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of the Δ4, 5 glycuronidase mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, Molec. Cell. Biol. 3:280 (1983).
In a preferred embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species. Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, Co1E1, pSC101, pACYC 184, and πVX). Such plasmids are, for example, disclosed by Sambrook, et al. (Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989)). Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids are disclosed by Gryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include pIJ101 (Kendall et al., J. Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages such as φC31 (Chater et al., In: Sixth International Symposium on Aclinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis. 8:693704 (1986)), and Izaki (Jpn. J. Bacteriol. 33:729-742 (1978)).
Preferred eukaryotic plasmids include, for example, BPV, EBV, SV40, 2-micron circle, and the like, or their derivatives. Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274 (1982); Broach, In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, Cell 28:203-204 (1982); Bollon et al., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, In: Cell Biology. A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980)). Other preferred eukaryotic vectors are viral vectors. For example, and not by way of limitation, the pox virus, herpes virus, adenovirus and various retroviruses may be employed. The viral vectors may include either DNA or RNA viruses to cause expression of the insert DNA or insert RNA.
Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, and the like. Additionally, DNA or RNA encoding the Δ4, 5 glycuronidase of the invention may be directly injected into cells or may be impelled through cell membranes after being adhered to microparticles. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the Δ4, 5 glycuronidase of the invention. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).
The present invention also provides for the use of Δ4, 5 glycuronidase as an enzymatic tool due to its substrate specificity and specific activity. In a direct and more rigorous comparison between the recombinant and native enzymes, it was found that at least some of the recombinant enzyme (Δ4,5Δ20) possessed at least about two-fold higher and in some cases a roughly about three-fold higher specific activity relative to the native Flavobacterial enzyme when measured under identical reaction conditions. Additionally, the activity of a cloned enzyme is not compromised by its recombinant expression in E. coli.
The recombinant Δ4,5 glycuronidase exhibited a sharp ionic strength dependence. These results are interesting given both the ionic character of the disulfated heparin disaccharide used in the experiments described below as well as the many ionic residues present within the enzyme that may function in substrate binding and/or catalysis; many of these charged residues are conserved in structurally and functionally related enzymes. From a substrate perspective, all of the unsaturated disaccharides examined possess a negative charge (at pH 6.4) due to the C6 carboxylate of the uronic acid. It is possible that this acid acts as a critical structural determinant, especially given its proximity to the Δ4,5 bond. Charge neutralization of 6-O sulfate (e.g., in ΔUHNS,6S) could possibly be another contributing factor. From the enzyme perspective, the recombinant glycuronidase (Δ4,5Δ20) does possess 47 basic residues (theoretical p1 of 8.5), including R151 whose position is invariantly conserved among the different glycuronidases examined. R151 may possibly interact with the uronic acid carboxylate. At the same time, Δ4,5 also possesses 44 acidic residues. At least ten of these positions are highly conserved. Charge masking of some of these ionic residues (either acidic or basic) by increasing salt concentration might interfere with enzymatic activity. A similar observation of this ionic strength dependency has been made for the heparinases [Ernst S, et al Expression in Escherichia coli, purification and characterization of heparinase 1 from Flavobacterium heparinum. Biochem J. Apr. 15, 1996; 315 (pt 2): 589-97.]
A bell-shaped pH profile with a 6.4 optimum was also observed in the present invention. The 6.4 pH optimum generally agrees with results originally reported for the F. heparinum Δ4,5 as well as for more recent results published for an unsaturated glucuronyl hydrolase purified from Bacillus sp. GL1 [Hashimoto, W., et al. (1999) Arch Biochem Biophys 368, 367-74]. While there are 11 histidines present within the primary sequence, three histidines (H115, H201, and H218) appear to be highly conserved. Interestingly, catalytically critical histidines also exist in all three heparin lyases [Pojasek, K., et al. (2000) Biochemistry 39, 4012-9] as well as chondroitin AC lyase [Huang, W., et al. (2001) Biochemistry 40, 2359-72] from Flavobacterium heparinum. While these two classes of enzymes cleave glycosaminoglycans by somewhat different mechanisms (i.e., β-elmination vs. hydrolysis), both would presumably involve acid-base catalysis, viz the imidazole.
The question of substrate specificity has now been considered from three structural perspectives: (1) the nature of the glycosidic linkage; (2) the relative sulfation pattern of the unsaturated disaccharide; and (3) the role of saccharide chain length (e.g., di- vs. tetrasaccharide). Our results indicate that for the recombinant Δ4,5 glycuronidase, there is an unambiguous preference for the 1→4 linkage over the 1→3 linkage making heparin rather than chondroitin/dernatan and/or hyaluronan the best substrate. It should be noted, however, that while this linkage position is important, it is not absolute. Both chondroitin and hyaluronan Δ4,5 disaccharides were hydrolyzed, albeit at much slower rates and using higher enzyme concentrations than were required to hydrolyze heparin disaccharides.
We also present a kinetic pattern of the Δ4,5 glycuronidase with regard to the specific sulfation within a heparin disaccharide. First and foremost, we find that unsaturated saccharides containing a 2-O-sulfated uronidate (ΔU2S) at the non-reducing end are in general not cleaved by the Δ4,5 glycuronidase. Furthermore, the inability of a 2-O-sulfated disaccharide to competitively inhibit the hydrolysis of non 2-O-containing disaccharide substrates (such as ΔUHNAc) further suggests that the presence of a 2-O sulfate precludes binding of this saccharide to the enzyme.
In considering the effect of specific sulfate groups present on the glucosamine, the enzyme may be loosely summarized as having a graded preference for 6-O-sulfation but a clear selection against unsubstituted or sulfated amines. This hierarchy is not an absolute distinction given the fact that all the non 2-O-containing heparin disaccharides examined were cleaved by the enzyme. Instead, it is based on relative kinetic parameters. This apparent substrate discrimination at the N and 6 positions of the glucosamine appears to be somewhat contextual, especially in the case of 6-O-sulfation. That is, while 6-0 sulfation may bestow a favorable selectivity to a saccharide substrate, this positive effect may be offset by the presence of a deacetylated amine (e.g., ΔUHNAc6S vs. ΔUHNH26S or ΔUHNS,6S).
The structural preference the Δ4,5 demonstrates against 2-O-sulfated uronidates along with a so-called “N-position” discrimination for the glucosamine may be exploited for use of the glycuronidase as an analytical tool for the compositional analyses of glycosaminoglycans. We were able to predict the extent and relative rates by which specific disaccharide “peaks” would disappear (i.e., due to the glycuronidase-dependent loss of absorbance at 232 nm.), based entirely on our kinetically defined substrate specificity determinations described in the Examples below. All 2-O-sulfate containing disaccharides tested were refractory to hydrolysis by the Δ4,5 glycuronidase. On the other hand, the remaining disaccharides were hydrolyzed in a time-dependent fashion that corresponded to their relative substrate specificities (i.e., ΔUHNAc6S>ΔUHNS,6S>ΔUHNS).
From this experiment, another important and surprising observation was made, namely that the Δ4,5 glycuronidase also hydrolyzes Δ4,5 unsaturated tetrasaccharides. It is also very interesting to note that this particular tetrasaccharide is as good of a substrate as the disaccharide ΔUHNS. This observation may argue against a substrate discrimination used by the enzyme that is negatively based on increasing molecular weight as was first reported [Hovingh, P. and Linker, A. (1977) Biochem J 165, 287-93].
Therefore, the invention also provides for the cleavage of glycosaminoglycans using the substantially pure Δ4,5 glycuronidase described herein. The Δ4,5 glycuronidase of the invention may be used to specifically cleave an HSGAG by contacting the HSGAG substrate with the Δ4,5 glycuronidase of the invention. The invention is useful in a variety of in vitro, in vivo and ex vivo methods in which it is useful to cleave HSGAGs.
As used herein the terms “HSGAG”, “GAG”, and “glycosaininoglycans” are used interchangeably to refer to a family of molecules having heparin-like/heparan sulfate-like structures and properties. These molecules include but are not limited to low molecular weight heparin (LMWH), heparin, biotechnologically prepared heparin, chemically modified heparin, synthetic heparin, and heparan sulfate. The term “biotechnological heparin” encompasses heparin that is prepared from natural sources of polysaccharides which have been chemically modified and is described for example in Razi et al., Bioche. J. Jul. 15, 1995;309 (Pt 2): 465-72. Chemically modified heparin is described in Yates et al., Carbohydrate Res (1996) November 20;294:15-27, and is known to those of skill in the art. Synthetic heparin is well known to those of skill in the art and is described in Petitou, M. et al., Bioorg Med Chem Lett. (1999) April 19;9(8):1161-6.
Analysis of a sample of glycosaminoglycans is also possible with Δ4,5 glycuronidase alone or in conjunction with other enzymes. Other HSGAG degrading enzymes include but are not limited to heparinase-I, heparinase-II, heparinase-III, heparinase-IV, D-glucuronidase and L-iduronidase, modified versions of heparinases, variants and functionally active fragments thereof.
The methods that may be used to test the specific activity of Δ4,5 glycuronidase of the present invention are known in the art, e.g., those described in the Examples. These methods may also be used to assess the function of variants and functionally active fragments of Δ4,5 glycuronidase. The kcat value may be determined using any enzymatic activity assay to assess the activity of a Δ4,5 glycuronidase enzyme. Several such assays are well-known in the art. For instance, an assay for measuring kcat is described in (Ernst, S. E., Venkataraman, G., Winkler, S., Godavarti, R., Langer, R., Cooney, C. and Sasisekharan. R. (1996) Biochem. J. 315, 589-597. The “native Δ4,5 glycuronidase kcat value” is the measure of enzymatic activity of the native Δ4,5 glycuronidase obtained from cell lysates of F. heparinum also described in the Examples below. Therefore, based on the disclosure provided herein, those of ordinary skill in the art will be able to identify other Δ4,5 glycuronidase molecules having altered enzymatic activity with respect to the naturally occurring Δ4,5 glycuronidase molecule such as functional variants.
The term “specific activity” as used herein refers to the enzymatic activity of a preparation of Δ4,5 glycuronidase. In general, it is preferred that the substantially pure and/or isolated Δ4,5 glycuronidase preparations of the invention have a specific activity of at least about 60 picomoles of substrate hydrolized per minute per picomole of enzyme. This generally corresponds to a kcat of at least about 10 per second for the enzyme using a substrate such as heparin disaccharide ΔUHNAc.
Due to the activity of Δ4,5 glycuronidase on glycosaminoglycans, the product profile produced by a Δ4,5 glycuronidase may be determined by any method known in the art for examining the type or quantity of degradation product produced by Δ4,5 glycuronidase alone or in combination with other enzymes. One of skill in the art will also recognize that the Δ4,5 glycuronidase may also be used to assess the purity of glycosaminoglycans in a sample. One preferred method for determining the type and quantity of product is described in Rhomberg, A. J. et al., PNAS, v. 95, p. 4176-4181, (April 1998), which is hereby incorporated in its entirety by reference. The method disclosed in the Rhomberg reference utilizes a combination of mass spectrometry and capillary electrophoretic techniques to identify the enzymatic products produced by heparinase. The Rhomberg study utilizes heparinase to degrade HSGAGs to produce HSGAG oligosaccharides. MALDI (Matrix-Assisted Laser Desorption Ionization) mass spectrometry can be used for the identification and semiquantitative measurement of substrates, enzymes, and end products in the enzymatic reaction. The capillary electrophoresis technique separates the products to resolve even small differences amongst the products and is applied in combination with mass spectrometry to quantitate the products produced. Capillary electrophoresis may even resolve the difference between a disaccharide and its semicarbazone derivative. Detailed methods for sequencing polysaccharides and other polymers are disclosed in co-pending U.S. patent application Ser. Nos. 09/557,997 and 09/558,137, both filed on Apr. 24, 2000 and having common inventorship. The entire contents of both applications are hereby incorporated by reference.
For example, the method is performed by enzymatic digestion, followed by mass spectrometry and capillary electrophoresis. The enzymatic assays can be performed in a variety of manners, as long as the assays are performed identically on the Δ4,5 glycuronidase, so that the results may be compared. In the example described in the Rhomberg reference, enzymatic reactions are performed by adding 1 mL of enzyme solution to 5 mL of substrate solution. The digestion is then carried out at room temperature (22° C.), and the reaction is stopped at various time points by removing 0.5 mL of the reaction mixture and adding it to 4.5 mL of a MALDI matrix solution, such as caffeic acid (approximately 12 mg/mL) and 70% acetonitrile/water. The reaction mixture is then subjected to MALDI mass spectrometry. The MALDI surface is prepared by the method of Xiang and Beavis (Xiang and Beavis (1994) Rapid. Commun. Mass. Spectrom. 8, 199-204). A two-fold lower access of basic peptide (Arg/Gly)15 is premixed with matrix before being added to the oligosaccharide solution. A 1 mL aliquot of sample/matrix mixture containing 1-3 picomoles of oligosaccharide is deposited on the surface. After crystallization occurs (typically within 60 seconds), excess liquid is rinsed off with water. MALDI mass spectrometry spectra is then acquired in the linear mode by using a PerSeptive Biosystems (Framingham, Mass.) Voyager Elite reflectron time-of-flight instrument fitted with a 337 nanometer nitrogen laser. Delayed extraction is used to increase resolution (22 kV, grid at 93%, guidewire at 0.15%, pulse delay 150 ns, low mass gate at 1,000, 128 shots averaged). Mass spectra are calibrated externally by using the signals for proteinated (Arg/Gly)15 and its complex with the oligosaccharide.
Capillary electrophoresis may then be performed on a Hewlett-Packard3D CE unit by using uncoated fused silica capillaries (internal diameter 75 micrometers, outer diameter 363 micrometers, Idet 72.1 cm, and Itot 85 cm). Analytes are monitored by using UV detection at 230 nm and an extended light path cell (Hewlett-Packard). The electrolyte is a solution of 10 mL dextran sulfate and 50 millimolar Tris/phosphoric acid (pH2.5). Dextran sulfate is used to suppress nonspecific interactions of the heparin oligosaccharides with a silica wall. Separations are carried out at 30 kV with the anode at the detector side (reversed polarity). A mixture of a 1/5-naphtalenedisulfonic acid and 2-naphtalenesulfonic acid (10 micromolar each) is used as an internal standard.
Other methods for assessing the product profile may also be utilized. For instance, other methods include methods which rely on parameters such as viscosity (Jandik, K. A., Gu, K. and Linhardt, R. J., (1994), Glycobiology, Q, 4:284-296) or total UV absorbance (Ernst, S. et al., (1996), Biochem. J., 315:589-597) or mass spectrometry or capillary electrophoresis alone.
The Δ4,5 glycuronidase molecules of the invention are also useful as tools for sequencing HSGAGs. Detailed methods for sequencing polysaccharides and other polymers are disclosed in co-pending U.S. patent application Ser. Nos. 09/557,997 and 09/558,137, both filed on Apr. 24, 2000 and having common inventorship. These methods utilize tools such as heparinases in the sequencing process. The Δ4,5 glycuronidase of the invention is useful as such a tool.
One of ordinary skill in the art, in light of the present disclosure, is enabled to produce substantially pure preparations of HSGAG and/or GAG fragment compositions utilizing the Δ4, 5 glycuronidase molecules alone or in conjunction with other enzymes. These GAG fragments have many therapeutic utilities. The glycuronidase molecules and/or GAG fragments can be used for the treatment of any type of condition in which GAG fragment therapy has been identified as a useful therapy, e.g., preventing coagulation, inhibiting angiogenesis, inhibiting proliferation. The GAG fragment preparations are prepared from HSGAG sources. A “HSGAG source” as used herein refers to heparin-like/heparan sulfate-like glycosaminoglycan composition which can be manipulated to produce GAG fragments using standard technology, including enzymatic degradation etc. As described above, HSGAGs include but are not limited to isolated heparin, chemically modified heparin, biotechnology prepared heparin, synthetic heparin, heparan sulfate, and LMWH. Thus HSGAGs can be isolated from natural sources, prepared by direct synthesis, mutagenesis, etc.
Thus, the methods of the invention enable one of skill in the art to prepare or identify an appropriate composition of GAG fragments, depending on the subject and the disorder being treated. These compositions of GAG fragments may be used alone or in combination with the Δ4,5 glycuronidase and/or other enzymes. Likewise Δ4, 5 glycuronidase and/or other enzymes may also be used to produce GAG fragments in vivo.
The compositions of the invention can be used for the treatment of any type of condition in which GAG fragment therapy has been identified as a useful therapy. Thus, the invention is useful in a variety of in vitro, in vivo and ex vivo methods in which therapies are useful. For instance, it is known that GAG fragments are useful for preventing coagulation, inhibiting cancer cell growth and metastasis, preventing angiogenesis, preventing neovascularization, preventing psoriasis. The GAG fragment compositions may also be used in in vitro assays, such as a quality control sample.
Each of these disorders is well-known in the art and is described, for instance, in Harrison's Principles of Internal Medicine (McGraw Hill, Inc., New York), which is incorporated by reference.
In one embodiment the preparations of the invention are used for inhibiting angiogenesis. An effective amount for inhibiting angiogenesis of the GAG fragment preparation is administered to a subject in need of treatment thereof. Angiogenesis as used herein is the inappropriate formation of new blood vessels. “Angiogenesis” often occurs in tumors when endothelial cells secrete a group of growth factors that are mitogenic for endothelium causing the elongation and proliferation of endothelial cells which results in a generation of new blood vessels. Several of the angiogenic mitogens are heparin binding peptides which are related to endothelial cell growth factors. The inhibition of angiogenesis can cause tumor regression in animal models, suggesting a use as a therapeutic anticancer agent. An effective amount for inhibiting angiogenesis is an amount of GAG fragment preparation which is sufficient to diminish the number of blood vessels growing into a tumor. This amount can be assessed in an animal model of tumors and angiogenesis, many of which are known in the art.
The Δ4, 5 glycuronidase is, in some embodiments, immobilized on a support. The glycuronidase may be immobilized to any type of support but if the support is to be used in vivo or ex vivo it is desired that the support is sterile and biocompatible. A biocompatible support is one which would not cause an immune or other type of damaging reaction when used in a subject. The Δ4, 5 glycuronidase may be immobilized by any method known in the art. Many methods are known for immobilizing proteins to supports. A “solid support” as used herein refers to any solid material to which a polypeptide can be immobilized.
Solid supports, for example, include but are not limited to membranes, e.g., natural and modified celluloses such as nitrocellulose or nylon, Sepharose, Agarose, glass, polystyrene, polypropylene, polyethylene, dextran, amylases, polyacrylamides, polyvinylidene difluoride, other agaroses, and magnetite, including magnetic beads. The carrier can be totally insoluble or partially soluble and may have any possible structural configuration. Thus, the support may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube or microplate well, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, bottom surface of a microplate well, etc.
The Δ4, 5 glycuronidase of the invention may also be used to remove active GAGs from a GAG containing fluid. A GAG containing fluid is contacted with the Δ4, 5 glycuronidase of the invention to degrade the GAG. The method is particularly useful for the ex vivo removal of GAGs from blood. In one embodiment of the invention the Δ4, 5 glycuronidase is immobilized on a solid support as is conventional in the art. The solid support containing the immobilized Δ4, 5 glycuronidase may be used in extracorporeal medical devices (e.g. hemodialyzer, pump-oxygenator) for systemic heparinization to prevent the blood in the device from clotting. The support membrane containing immobilized Δ4, 5 glycuronidase is positioned at the end of the device to neutralize the GAG before the blood is returned to the body.
Thus, the Δ4, 5 glycuronidase molecules are useful for treating or preventing disorders associated with coagulation. A “disease associated with coagulation” as used herein refers to a condition characterized by an interruption in the blood supply to a tissue due to a blockage of the blood vessel responsible for supplying blood to the tissue such as is seen for myocardial or cerebral infarction. A cerebral ischemic attack or cerebral ischemia is a form of ischemic condition in which the blood supply to the brain is blocked. This interruption in the blood supply to the brain may result from a variety of causes, including an intrinsic blockage or occlusion of the blood vessel itself, a remotely originated source of occlusion, decreased perfusion pressure or increased blood viscosity resulting in inadequate cerebral blood flow, or a ruptured blood vessel in the subarachnoid space or intracerebral tissue.
The Δ4, 5 glycuronidase or the GAG fragments generated therewith may be used alone or in combination with a therapeutic agent for treating a disease associated with coagulation. Examples of therapeutics useful in the treatment of diseases associated with coagulation include anticoagulation agents, antiplatelet agents, and thrombolytic agents.
Anticoagulation agents prevent the coagulation of blood components and thus prevent clot formation. Anticoagulants include, but are not limited to, heparin, warfarin, coumadin, dicumarol, phenprocoumon, acenocoumarol, ethyl biscoumacetate, and indandione derivatives.
Antiplatelet agents inhibit platelet aggregation and are often used to prevent thromboembolic stroke in patients who have experienced a transient ischemic attack or stroke. Antiplatelet agents include, but are not limited to, aspirin, thienopyridine derivatives such as ticlopodine and clopidogrel, dipyridamole and sulfinpyrazone, as well as RGD mimetics and also antithroinbin agents such as, but not limited to, hirudin.
Thrombolytic agents lyse clots which cause the thromboembolic stroke. Thrombolytic agents have been used in the treatment of acute venous thromboembolism and pulmonary emboli and are well known in the art (e.g. see Hennekens et al, J Am Coll Cardiol; v. 25 (7 supp), p. 18S-22S (1995); Holmes, et al, J Am Coll Cardiol; v.25 (7 suppl), p. 10S-17S(1995)). Thrombolytic agents include, but are not limited to, plasminogen, a2-antiplasmin, streptokinase, antistreplase, tissue plasminogen activator (tPA), and urokinase. “tPA” as used herein includes native tPA and recombinant tPA, as well as modified forms of tPA that retain the enzymatic or fibrinolytic activities of native tPA. The enzymatic activity of tPA can be measured by assessing the ability of the molecule to convert plasminogen to plasmin. The fibrinolytic activity of tPA may be determined by any in vitro clot lysis activity known in the art, such as the purified clot lysis assay described by Carlson, et. al., Anal. Biochem. 168, 428-435 (1988) and its modified form described by Bennett, W. F. et al., 1991, J. Biol. Chem. 266(8):5191-5201, the entire contents of which are hereby incorporated by reference.
The invention compositions of the invention are useful for the same purposes as heparinases and the degradation products of heparinases (HSGAG fragments). Thus, for instance, the compositions of the invention are useful for treating and preventing cancer cell proliferation and metastasis. Thus, according to another aspect of the invention, there is provided methods for treating subjects having or at risk of having cancer.
Critically, HSGAGs (along with collagen) are key components of the cell surface-extracellular matrix (ECM) interface. While collagen-like proteins provide the necessary extracellular scaffold for cells to attach and form tissues, the complex polysaccharides fill the space created by the scaffold and act as a molecular sponge by specifically binding and regulating the biological activities of numerous signaling molecules like growth factors, cytokines etc. It has recently been recognized that cells synthesize distinct HSGAG sequences and decorate themselves with these sequences, using the extraordinary information content present in the sequences to bind specifically to many signaling molecules and thereby regulate various biological processes.
The invention also contemplates the use of therapeutic GAG fragments for the treatment and prevention of tumor cell proliferation and metastasis. A “therapeutic GAG fragment” as used herein refers to a molecule or molecules which are pieces or fragments of a GAG that have been identified or generated through the use of the Δ4, 5 glycuronidase possibly along with other naturally occurring and/or modified heparinases. In some aspects the therapeutic GAG fragments have the same structure as commercially available LMWH, but are generated using the Δ4, 5 glycuronidase.
The invention also encompasses screening assays for identifying therapeutic GAG fragments for the treatment of a tumor and for preventing metastasis. The assays are accomplished by treating a tumor or isolated tumor cells with Δ4, 5 glycuronidase and/or other naturally occurring or modified heparinases and isolating the resultant GAG fragments. Surprisingly, these GAG fragments have therapeutic activity in the prevention of tumor cell proliferation and metastasis. Thus the invention encompasses individualized therapies, in which a tumor or portion of a tumor is isolated from a subject and used to prepare the therapeutic GAG fragments. These therapeutic fragments can be re-administered to the subject to protect the subject from further tumor cell proliferation or metastasis or from the initiation of metastasis if the tumor is not yet metastatic. Alternatively the fragments can be used in a different subject having the same type or tumor or a different type of tumor.
Therapeutic GAG fragments include GAG fragments which have therapeutic activity in that they prevent the proliferation and/or metastasis of a tumor cell. Such compounds may be generated using Δ4, 5 glycuronidase to produce therapeutic fragments or they may be synthesized de novo. Putative GAG fragments can be tested for therapeutic activity using any of the assays described herein or known in the art. Thus the therapeutic GAG fragment may be a synthetic GAG fragment generated based on the sequence of the GAG fragment identified when the tumor is contacted with Δ4, 5 glycuronidase, or having minor variations which do not interfere with the activity of the compound. Alternatively the therapeutic GAG fragment may be an isolated GAG fragment produced when the tumor is contacted with Δ4, 5 glycuronidase.
The invention is useful for treating and/or preventing tumor cell proliferation or metastasis in a subject. The terms “treat” and “treating” tumor cell proliferation as used herein refer to inhibiting completely or partially the proliferation or metastasis of a cancer or tumor cell, as well as inhibiting any increase in the proliferation or metastasis of a cancer or tumor cell.
A “subject having a cancer” is a subject that has detectable cancerous cells. The cancer may be a malignant or non-malignant cancer. Cancers or tumors include but are not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell and non-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas.
A “subject at risk of having a cancer” as used herein is a subject who has a high probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality, the presence of which has been demonstrated to have a correlative relation to a higher likelihood of developing a cancer and subjects exposed to cancer causing agents such as tobacco, asbestos, or other chemical toxins, or a subject who has previously been treated for cancer and is in apparent remission. When a subject at risk of developing a cancer is treated with a Δ4, 5 glycuronidase or degradation product thereof the subject may be able to kill the cancer cells as they develop.
Effective amounts of the Δ4, 5 glycuronidase, variant Δ4, 5 glycuronidase or therapeutic GAGs of the invention are administered to subjects in need of such treatment. Effective amounts are those amounts which will result in a desired improvement in the condition or symptoms of the condition, e.g., for cancer this is a reduction in cellular proliferation or metastasis, without causing other medically unacceptable side effects. Such amounts can be determined with no more than routine experimentation. It is believed that doses ranging from 1 nanogram/kilogram to 100 milligrams/kilogram, depending upon the mode of administration, will be effective. The absolute amount will depend upon a variety of factors (including whether the administration is in conjunction with other methods of treatment, the number of doses and individual patient parameters including age, physical condition, size and weight) and can be determined with routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. The mode of administration may be any medically acceptable mode including oral, subcutaneous, intravenous, etc.
In some aspects of the invention the effective amount of Δ4, 5 glycuronidase or therapeutic GAG is that amount effective to prevent invasion of a tumor cell across a barrier. The invasion and metastasis of cancer is a complex process which involves changes in cell adhesion properties which allow a transformed cell to invade and migrate through the extracellular matrix (ECM) and acquire anchorage-independent growth properties Liotta, L. A., et al., Cell 64:327-336, 1991. Some of these changes occur at focal adhesions, which are cell/ECM contact points containing membrane-associated, cytoskeletal, and intracellular signaling molecules. Metastatic disease occurs when the disseminated foci of tumor cells seed a tissue which supports their growth and propagation, and this secondary spread of tumor cells is responsible for the morbidity and mortality associated with the majority of cancers. Thus the term “metastasis” as used herein refers to the invasion and migration of tumor cells away from the primary tumor site.
The barrier for the tumor cells may be an artificial barrier in vitro or a natural barrier in vivo. In vitro barriers include but are not limited to extracellular matrix coated membranes, such as Matrigel. Thus the Δ4, 5 glycuronidase compositions or degradation products thereof can be tested for their ability to inhibit tumor cell invasion in a Matrigel invasion assay system as described in detail by Parish, C. R., et al., “A Basement-Membrane Permeability Assay which Correlates with the Metastatic Potential of Tumour Cells,” Int. J. Cancer, 1992, 52:378-383. Matrigel is a reconstituted basement membrane containing type IV collagen, laminin, heparan sulfate proteoglycans such as perlecan, which bind to and localize bFGF, vitronectin as well as transforming growth factor-β (TGF-β), urokinase-type plasmninogen activator (uPA), tissue plasminogen activator (tPA), and the serpin known as plasminogen activator inhibitor type I (PAl-1). Other in vitro and in vivo assays for metastasis have been described in the prior art, see, e.g., U.S. Pat. No. 5,935,850, issued on Aug. 10, 1999, which is incorporated by reference. An in vivo barrier refers to a cellular barrier present in the body of a subject.
In general, when administered for therapeutic purposes, the formulations of the invention are applied in pharmaceutically acceptable solutions. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
The compositions of the invention may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V); and phosphoric acid and a salt (0.8-2% W/V). Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).
The present invention provides pharmaceutical compositions, for medical use, which comprise Δ4, 5 glycuronidase, variant Δ4, 5 glycuronidase of the invention, or therapeutic GAG fragments together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The term “pharmaceutically-acceptable carrier” as used herein, and described more fully below, means one or more compatible solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other animal. In the present invention, the term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the Δ4, 5 glycuronidase of the present invention or other compositions, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular active agent selected, the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the drug without causing clinically unacceptable adverse effects. A preferred mode of administration is a parenteral route. The term “parenteral” includes subcutaneous injections, intravenous, intramuscular, intraperitoneal, intra sternal injection or infusion techniques. Other modes of administration include oral, mucosal, rectal, vaginal, sublingual, intranasal, intratracheal, inhalation, ocular, transdermal, etc.
For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.
The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active Δ4, 5 glycuronidase into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the polymer into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. The polymer may be stored lyophilized.
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the heparinases of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhydrides and polycaprolactone; nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. Specific examples include, but are not limited to: (a) erosional systems in which the polysaccharide is contained in a form within a matrix, found in U.S. Pat. No. 4,452,775 (Kent); U.S. Pat. No. 4,667,014 (Nestor et al.); and U.S. Pat. No. 4,748,034 and U.S. Pat. No. 5,239,660 (Leonard) and (b) diffusional systems in which an active component permeates at a controlled rate through a polymer, found in U.S. Pat. No. 3,832,253 (Higuchi et al.) and U.S. Pat. No. 3,854,480 (Zaffaroni). In addition, a pump-based hardware delivery system can be used, some of which are adapted for implantation.
A subject is any human or non-human vertebrate, e.g., dog, cat, horse, cow, pig.
When administered to a patient undergoing cancer treatment, the Δ4, 5 glycuronidase or therapeutic GAG compounds may be administered in cocktails containing other anti-cancer agents. The compounds may also be administered in cocktails containing agents that treat the side-effects of radiation therapy, such as anti-emetics, radiation protectants, etc.
Anti-cancer drugs that can be co-administered with the compounds of the invention include, but are not limited to Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carinustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexorinaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazoinycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Geincitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-1 a; Interferon Gamma-1 b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicainycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletinide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride.
The Δ4, 5 glycuronidase or therapeutic GAG compounds may also be linked to a targeting molecule. A targeting molecule is any molecule or compound which is specific for a particular cell or tissue and which can be used to direct the Δ4, 5 glycuronidase or therapeutic GAG to the cell or tissue. Preferably the targeting molecule is a molecule which specifically interacts with a cancer cell or a tumor. For instance, the targeting molecule may be a protein or other type of molecule that recognizes and specifically interacts with a tumor antigen.
Tumor-antigens include Melan-A/M-ART-1, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-AL, MAGE-A2, MAGE-A3, MAGE-Δ4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21 ras, RCAS 1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, cdc27, adenomatous polyposis coli protein (APC), fodrin, PIA, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, Imp-1, EBV-encoded nuclear antigen (EBNA)1, and c-erbB-2.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
Materials And Methods
Chemicals and reagents. Unless otherwise stated, biochemicals were purchased from Sigma Aldrich Chemical (St. Louis, Mo.). Disaccharides were purchased from Calbiochem (San Diego, Calif.). Reagents for λZAP II genomic library construction and screening were obtained from Stratagene (La Jolla, Calif.). Restriction endonucleases were purchased from New England Biolabs (Beverly, Mass.). DNA oligonucleotide primers were manufactured by Invitrogen/Life Technologies (Carlsbad, Calif.). Additional molecular cloning reagents were obtained from the manufacturers listed.
Bacterial strains and growth conditions. F. heparinum (Pedobacter heparinus) was obtained as a lyophilized stock from American Type Culture Collection (ATCC, Manassas, Va.), stock no. 13125. Rehydrated cultures were grown aerobically at 30° C. with moderate shaking to an optical density (A600) between 1.5 and 2 in defined media containing 6.4 mM NaH2PO4, 7.6 mM Na2HPO4, 12 mM KH2PO4, 14.3 mM K2HPO4, 1.7 mM NaCl, and 1.9 mM NH4Cl, pH 6.9 and supplemented with 0.1 mM trace metals CaCl2, FeSO4, CuSO4, NaMoO4, CoCl2, and MnSO4 (added from a 100× stock in 10 mM H2SO4), 0.8% glucose, 0.05% methionine, 0.05% histidine, 2 mM MgSO4, and 0.1% heparin all added under sterile conditions. E. coli strains included TOP10 (Invitrogen) or DH5α for PCR cloning and subcloning and BL21(DE3) (Novagen, Madison Wis.) for recombinant protein expression. Bacteriophage host strains XL1-Blue MRF′ and SOLR were obtained from Stratagene.
Purification of glycuronidase peptides and protein sequencing. The 4, 5 glycuronidase was purified from 10 liter fermentation cultures using a method such as those described in McLean, M. W., Bruce, J. S., Long, W. F., and Williamson, F. B., 1984, Eur J Biochem 145, 607-15.
Molecular cloning of the Δ4, 5 glycuronidase gene from F. heparinum genomic DNA.
Flavobacterial genomic DNA was isolated from 10 mL of Flavobacterial culture using the QIAGEN DNeasy DNA purification kit according to the Manufacturer's instructions for gram-negative bacteria using approximately 2×109 cells per column. Following purification, genomic DNA was ethanol precipitated and resuspended in TE, pH 7.5 at 0.5 mg/mL. The quality of genomic DNA was confirmed spectrophotometrically at 260/280 nm, by electrophoresis on a 0.5% agarose gel and by PCR using Flavobacterial specific primers.
The following degenerate primers were synthesized from peptides corresponding initially to peaks 19 and 24 (Example 1): 5′ GARACNCAYCARGGNYTNACNAAYGAR 3′ (SEQ ID NO. 5) (peak 19 forward), 5′ YTCRTTNGTNARNCCYTGRTGNGTYTC 3′ (SEQ ID NO. 6) (peak 19 reverse); 5′ AAYTAYGCNGAYTAYTAYTAY 3′ (SEQ ID NO. 7) (peak 24 forward); 5′ RTARTARTARTCNGCRTARTT 3′ (SEQ ID NO. 8) (peak 24 reverse). All four primers were screened in a PCR assay using all possible pairings (forward and reverse). The PCR reaction conditions included 200 ng of genomic DNA, 200 picomoles for each forward and reverse primer, 200 μM dNTPs, 1 unit of Vent DNA Polymerase (New England Biolabs) in a 100 μl reaction volume. 35 cycles were completed using a 52° C. annealing temperature and 1.5 minute extensions at 72° C. The 450 bp product amplified using primers 19 forward and 24 reverse was gel purified and subject to direct DNA sequencing which confirmed the inclusion of translated sequence corresponding to peptide peaks 19 and 24 in addition to peak 12. The same DNA was also 32P radiolabeled by random priming using 200 μCi α32P[dCTP] at 6000 Ci/mmole (NEN, Boston, Mass.), 50-100 ng of DNA and the Prime-it II random priming kit (Stratagene) (probel). Unincorporated 32P dNTPs were removed by gel filtration using G-50 Quick-spin columns (Roche Biochemicals, New Jersey). Labeling reactions typically yielded approximately 50 ng of radiolabeled DNA with specific activities exceeding 109 cpm/1 g.
DNA hybridization probe 2 was initially created by PCR as described above except using degenerate primer 26 5′CARACNTAYACNCCNGGNATGAAY 3′ (SEQ ID NO. 9) (peak 26 forward) and 20 picomoles of reverse, non-degenerate primer 54 (5′ TTCATGGTCGTAACCGCATG 3′) (SEQ ID NO. 10); the latter oligonucleotide corresponds to Δ4, 5 DNA sequence 3′ of peak 8. Direct sequencing of this PCR fragment confirmed the presence of peak 26 and peak 13 peptides. DNA probe 3 used in DNA southern hybridizations (below) was PCR amplified from genomic DNA using primer 68 (5′ TATACACCAGGCATGAACCC 3′) (SEQ ID NO. 11) and 74 (5′ CCCAGTATAAATACTCCAGGT 3′) (SEQ ID NO. 12).
Plaque hybridization screening of F. heparinum genomic library. A λ ZAP II genomic library (Stratagene) was constructed as described [Sasisekharan, R., Bulmer, M., Moremen, K. W., Cooney, C. L., and Langer, R. (1993) Proc Natl Acad Sci USA 90, 3660-4]. The amplified library (1×1010 pfu/mL) was plated out at approximately 1×106 pfu (˜50,000 pfu/plate) on 100×150 mm LB plates. Plaque lifts on to nylon membranes (Nytran Supercharge, Schleicher and Schuell) and subsequent hybridization screenings were completed using standard methods and solutions [Current Protocols in Molecular Biology, 1987, John Wiley and Sons, New York]. DNA was crosslinked to each filter by UV-irradiation (Stratagene Stratalinker) for 30 seconds at 1200 joules/cm3. Hybridizations were carried at 42° C. using 107-108 cpm of radiolabeled probe (at approximately 0.25 ng/mL). Low stringency washes were carried out at room temperature in 2×SSC, 0.1% SDS; high stringency washes carried out at 58-60° C. in 0.2×SSC and 0.1% SDS. Hybridized plaques were visualized by phosphor imaging (Molecular Dynamics) and/or 32P autoradiography. Tertiary screens of positive clones were completed and the recombinant phage was excised as a double-stranded phagemid (pBluescript) using the ExAssist interference-resistant helper phage and SOLR strain according to the manufacturer's protocol (Stratagene). Recombinants were characterized by DNA sequencing using both T7 and T3 primers.
Creation of a flavobacterium Bgl II-EcoR1 subgenomic library for isolation of the Δ4, 5 5′ terminus. 1 μg of genomic DNA was cut with 20 units of Eco R1, Bgl 11, and Hind III individually or as double digests. Restriction products were resolved by gel electrophoresis on 1% agarose gels run in 1× TAE buffer. Southern DNA hybridizations were completed according to standard protocols [Current Protocols in Molecular Biology, 1987, John Wiley and Sons, New York] using 32P radiolabeled probe 3. Based on this Southern analysis, 5 μg of Flavobacterial genomic DNA was digested with Bgl-II-Eco R1 and the DNA resolved on a preparative 1% agarose gel run under identical conditions as those described for the analytical gel. DNA ranging from approximately 1-2 kb was gel purified and ligated into pLITMUS as a Bgl Il-EcoR1 cassette. Positive clones were identified by PCR colony screening using primers 68 and 74 and confirmed by DNA sequencing.
PCR cloning of Δ4, 5 gene and recombinant expression in E. coli. The full-length glycuronidase gene was directly PCR amplified from genomic DNA using forward primer 85 5′ TGTTCTAGACATATGAAATCACTACTCAGTGC (SEQ ID NO. 13) 3′ and reverse primer 86 5′ GTCTCGAGGATCCTTAAGACTGATTAATTGTT 3′ (SEQ ID NO. 14) (with Nde 1 and Xho 1 restriction sites denoted in bold), 200 ng genomic DNA, and Vent DNA Polymerase for 35 cycles. dA overhangs were generated in a final 10 minute extension at 72° C. using AmpliTaq DNA polymerase (Applied Biosystems). PCR products were gel purified, ligated into the TOPO/TA PCR cloning vector (Invitrogen), and transformed into One-shot TOP10 chemically competent cells. Positive clones were identified by blue/white colony selection and confirmed by PCR colony screening. The 1.2 kb Δ4, 5 gene was subcloned into expression plasmid pET28a (Novagen) as an Nde 1-Xho 1 cassette. Final expression clones were confirmed by plasmid DNA sequencing.
For the expression of Δ4, 5 glycuronidase beginning with M21 (Δ4,5Δ20), the forward primer 95 5′ TGT TCT AGA CAT ATG ACA GTT ACG AAA GGC AA 3′ (SEQ ID NO. 15) (also containing an Nde 1 restriction site near its 5′ terminus) was used in place of primer 85 (above). 50 ng of the original expression plasmid pET28a/Δ4, 5 was used as the DNA template in PCR reactions involving a total of 20 cycles. Otherwise, cloning was as described for the full-length gene. Both pET28a/Δ4, 5 and pET28aΔ4,5Δ20 plasmids were transformed into BL21 (DE3) for expression as N-terminal 6×His tagged proteins. 1 liter cultures were grown at room temperature (˜20° C.) in LB media supplemented with 40 μg/mL kanamycin. Protein expression was induced with 500 μM IPTG added at an A600 of 1.0. Induced cultures were allowed to grow for 15 hours (also at room temperature).
Recombinant Δ4, 5 glycuronidase purification. Bacterial cells were harvested by centrifugation at 6000×g for 20 minutes and resuspended in 40 mL of binding buffer (50 mM Tris-HCL, pH 7.9, 0.5 M NaCl, and 10 mM imidazole). Lysis was initiated by the addition of 0.1 mg/mL lysozyme (20 minutes at room temperature) followed by intermittent sonication in an ice-water bath using a Misonex XL sonicator at 40-50% output. The crude lysate was fractionated by low-speed centrifugation (18,000×g; 4° C.; 15 minutes) and the supernatant was filtered through a 0.45 micron filter. The 6×-His tag Δ4, 5 glycuronidase was purified by Ni+2 chelation chromatography on a 5 mL Hi-Trap column (Pharmacia Biotech, New Jersey) pre-charged with 200 mM NiSO4 and subsequently equilibrated with binding buffer. The column was run at a flow rate of approximately 3-4 mL/minute that included an intermediate wash step with 50 mM imidazole. The Δ4, 5 enzyme was eluted from the column in 5 mL fractions using high imidazole elution buffer (50 mM Tris-HCL, pH 7.9, 0.5 M NaCl, and 250 mM imidazole). Peak fractions were dialyzed overnight against 4 liters of phosphate buffer (0.1M sodium phosphate, pH 7.0, 0.5 M NaCl) to remove the imidazole.
The 6×His tag was effectively cleaved by adding biotinylated thrombin at 2 units/milligram of recombinant protein, overnight at 4° C. with gentle inversion. Thrombin was captured by binding to streptavidin agarose at 4° C. for two hours using the Thrombin Capture Kit (Novagen). The cleaved peptide 5′ MGSSHHHHHHSSGLVPR 3′ (SEQ ID NO. 16) was removed by final dialysis against a 1000-fold volume of phosphate buffer.
Protein concentrations were determined by protein assay (Bio-Rad, Hercules, Calif.) and confirmed by UV spectroscopy using a theoretical molar extinction coefficient F=88,900 for Δ4,5Δ20. Protein purity was assessed by SDS-PAGE followed by Coomassie Brilliant Blue staining.
Computational methods. Signal sequence predictions were made by SignalP V1.1 using the von Heijne computational method [Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G., 1997, Protein Eng 10, 1-6] with maximum Y and S cutoffs set at 0.36 and 0.88, respectively. Glycuronidase multiple sequence alignments were made from select BLASTP database sequences (with scores exceeding 120 bits and less than 6% gaps) using the CLUSTAL W program (version 1.81) preset to an open gap penalty of 10.0, a gap extension penalty of 0.20, and both hydrophilic and residue-specific gap penalties turned on.
Assay for enzyme activity and determination of kinetic parameters. Standard reactions were carried out at 30° C. and included 100 mM sodi um phosphate buffer, pH 6. 4, 50 mM NaCl, 500 μM disaccharide substrate and 200 nM enzyme in a 100 μl reaction volume. Hydrolysis of heparin disaccharides was determined spectrophotometrically by measuring the loss of the Δ4, 5 chromophore measured at 232 nm. Substrate hydrolysis was calculated using the following molar extinction coefficients empirically determined for each disaccharide substrate: ΔUHNAc, ε232=4,524; ΔUHNAc6S, ε232=4,300; ΔUHNS, ε232=6,600; ΔUHNS,6S, ε232=6,075; ΔUHNH26S, Δ232=4,826; ΔU2SHNS, ε232=4,433. Initial rates (Vo) were extrapolated from linear activities representing<10% substrate turnover and fit to pseudo first-order kinetics. For kinetic experiments, disaccharide concentration for each respective substrate was varied from 48 to 400 μM concentrations. Km and kcat values were extrapolated from V0 vs. [S] curves fit to the Michaelis Menten equation by a non-linear, least squares regression.
For experiments measuring the relative effect of ionic strength on glycuronidase activity, the NaCl concentration was varied from 0.05 to 1 M in 0.1 M sodium phosphate buffer (pH 6.4), 200 μM ΔUHNS,6S and 100 μM enzyme under otherwise standard reaction conditions. The effect of pH on catalytic activity was kinetically determined at varying ΔUHNS,6S concentrations in 0.1 M sodium phosphate buffer at pH 5.2, 5.6, 6.0, 6.4, 6.8, 7.2 and 7.8. Data were fit to Michaelis-Menten kinetics as described above and the relative kcat/Km ratios plotted as a function of pH.
Detection of Δ4, 5 glycuronidase activity by capillary electrophoresis. 200 μg of heparin (Celsus Laboratories) was subject to exhaustive heparinase digestion as described [Venkataraman, G., Shriver, Z., Raman, R., and Sasisekharan, R., 1999, Science 286, 537-42] with certain modifications that included a 50 mM PIPES buffer, pH 6.5 with 100 mM NaCl in a 100 μl reaction volume. Following heparinase treatment, 25 picomoles of Δ4, 5 glycuronidase was added to one-half of the original reaction (pre-equilibrated at 30° C.). 20 μl aliquots were removed at 1 minute and 30 minutes and activity quenched by heating at 95° C. for 10 minutes. 20 μl of the minus Δ4, 5 control (also heated for 10 minutes) was used as the 0 time point. Disaccharide products were resolved by capillary electrophoresis run for 25 minutes under positive polarity mode as previously described [Rhomberg, A. J., Ernst, S., Sasisekharan, R., and Biemann, K., 1998, Proc Natl Acad Sci USA 95, 4176-81].
Molecular mass determinations. Molecular mass determinations were carried out by MALDI-MS as described [Rhomberg, A. J., Ernst, S., Sasisekharan, R., and Biemann, K., 1998, Proc Natl Acad Sci USA 95, 4176-81].
To clone the Δ4, 5 glycuronidase gene, we isolated a series of Δ4, 5 glycuronidase-derived peptides after protease treatment of the purified enzyme. The native enzyme was directly purified from fermentation cultures of F. heparinum using a 5-step chromatography scheme as previously described [McLean, M. W., Bruce, J. S., Long, W. F., and Williamson, F. B., 1984, Eur J Biochem 145, 607-15]. The extent of purity was ultimately characterized by reverse phase chromatography, which indicated a single major peak (
Based on this information, we designed degenerate primers corresponding to peaks 19, 24, and 26. These primers were used to PCR amplify Δ4, 5 specific sequences to be used as a suitable DNA hybridization probes for screening the Flavobacterial genomic library. A combination of two primer pairs in particular (peak 19 forward and peak 24 reverse) gave a discrete PCR product of approximately 450 base pairs. The translation of the corresponding DNA sequence indicated that it contained the expected amino acid sequence corresponding to peaks 19 and 24. The peak 12 peptide also mapped to this region. We used this discrete PCR amplified DNA fragment (designated as probe 1,
Alternate approaches were taken in an attempt to obtain the 5′ terminus of the glycuronidase gene. The size of this missing region was estimated, based on the molecular weight of the native protein, to be approximately 45 amino acids (or 135 base pairs). We completed DNA southern analyses to identify potentially useful DNA restriction sites flanking the 5′ end of the Δ4, 5 gene (
A summary of the full-length gene obtained from the two overlapping cloning methods is depicted in
A search for related sequences in the NCBI protein database led to several functionally related enzymes. In a multiple sequence alignment of our cloned enzyme with select glucuronyl hydrolases, we found a homology that generally corresponded to upwards of 30% identity and nearly 50% similarity at the primary sequence level (
Using PCR, we cloned from the F. heparinum genome both the full-length enzyme and the “mature” enzyme lacking the N-terminal 20 amino acid signal sequence (Δ4,5Δ20) into a T7-based expression plasmid. Cloning into pET28a permitted the expression of the glycuronidase as an N-terminal 6×His-tag fusion protein. Pilot expression studies focused on the full-length enzyme. In these initial experiments, we examined several different induction conditions such as temperature, time and length of induction, and even IPTG concentrations. In every case, the full-length enzyme was present nearly exclusively as an insoluble fraction. Attempts to purify the enzyme directly from inclusion bodies and then refold the apparently mis-folded protein were initially not successful; while solubility was partially achieved by a combined use of detergents (e.g., CHAPS), increasing salt concentrations, and the presence of glycerol, the partially purified enzyme was largely inactive.
A possible explanation for this insolubility points to the presence of a very hydrophobic region within the wild-type protein sequence spanning the first 20 amino acids. The N-terminal sequence is also predicted to comprise a cleavable prokaryotic signal sequence with the most likely cleavage site occurring between position G20 and M21 (
A summary of the expression and purification of Δ4,5Δ20 is summarized in
A molecular mass of 44,209 daltons was determined for the recombinant enzyme (i.e., Δ4,5Δ20) by MALDI-MS. The amino acid and nucleotide sequence for the enzyme which lacks the N-terminal 20 amino acid signal sequence is given as SEQ ID Nos.: 1 and 2, respectively. This empirically-established molecular weight is consistent with its theoretical value of 43,956 Daltons based on its amino acid composition. This value physically differs by 1357 Daltons in comparison to a molecular weight of 45,566 daltons likewise measured for the native enzyme. This mass differential is largely accounted for by the engineered removal in the recombinant protein of the 20 amino acid signal sequence. However, we cannot exclude the possibility of differential posttranslational modifications such as glycosylation largely accounting for the observed differences between the two enzyme populations. Unfortunately, chemical blocking precluded us from determining the N-terminal sequence of the native protein.
To determine the optimal reaction conditions for Δ4, 5 glycuronidase activity, we analyzed initial reaction rates as a function of buffer, pH, temperature, and ionic strength (
Under these conditions, we observed a NaCl concentration-activity dependence that was optimal between 50 and 100 mM. NaCl concentrations exceeding 100 mM demonstrated a significant and relatively sharply negative effect on specific activity (
The observed pH profile for the glycuronidase is bell-shaped (
As a final variable for optimizing Δ4, 5 glycuronidase in vitro reaction conditions, we also considered any requirement for divalent metal ions. We found no evidence that metals are either required for catalysis or activate the enzyme to any appreciable extent.
Having established the reaction conditions for optimal Δ4, 5 glycuronidase activity, we next compared the specific activity of the recombinant enzyme (Δ4,5Δ20) relative to the native enzyme purified directly from F. heparinum (
The specificity of the Δ4, 5 glycuronidase acting on various glycosaminoglycan disaccharide substrates was investigated. The various substrates examined included both heparin and chondroitin disaccharides as well as an hyaluronadate. In particular, we considered the possibility of any structural discriminations pertaining to glycosidic linkage position (1→4 vs. 1→3) and relative sulfation state within the disaccharide. For each substrate, kinetic parameters were determined based on substrate saturation profiles that fit Michaelis-Menten assumptions (
The heparin disaccharide ΔU2SHNS was not a substrate at any of the concentrations studied, even following an extended incubation time spanning several hours. For those heparin disaccharides that were hydrolyzed under the conditions tested and for which kinetic parameters could be determined, an interesting substrate preference was apparent. In this hierarchy and under these conditions, the two disaccharides ΔUHNAc and ΔUHNac6S were the best substrates; in comparison, ΔUHNH26S and ΔUHNS were less good as substrates. The kinetic values for ΔUHNS,6S fell roughly in the middle between these two boundaries.
The data show that heparin is a better substrate than chondroitin/dermatan and/or hyaluronan, although these compounds are also substrates. None of the non-heparin disaccharides were hydrolyzed under the conditions for measuring substrate kinetics. This result indicates an unequivocal discrimination of the Δ4, 5 in regard to linkage position, with a strong preference for 1→4 versus 1→3 linkages. At the same time, these disaccharides were slowly hydrolyzed to varying degrees when the enzyme reactions were conducted over a much longer timecourse (>12 hours) and at considerably higher enzyme concentrations. After approximately 18 hours, greater than 80% of monosulfated chondroitin disaccharide (ΔUGalNac6S) was hydrolyzed, whereas the non-sulfated chondroitin (ΔUGalNAc) and the hyaluronan disaccharide (ΔUH) were still present at approximately 40% and 65%, respectively. The importance of the linkage position is, therefore, not absolute. The apparently positive effect of chondroitin 6-O-sulfation within the galactosamine is consistent with our results for the heparin substrates and provides further evidence for the influence of this position in dictating substrate specificity.
Based on the kinetically defined substrate specificity for the disaccharides, we set out to validate these results while, at the same time, to investigate the utility of the Δ4, 5 glycuronidase as an enzymatic tool for HSGAG compositional analyses. In this manner, the Δ4, 5 should be very useful in assessing the composition of disaccharides resulting from prior heparinase treatment of heparin/heparan sulfate. For this particular experiment, we pre-treated 200 μg of heparin with a heparinase cocktail. This exhaustive digestion was then directly followed by a relatively short (1 minute) or long (30 minute) Δ4, 5 glycuronidase treatment carried out under optimal reaction conditions. The disaccharide products were then resolved by capillary electrophoresis. The electrophoretic mobility profile for the Δ4, 5 treated saccharides were then directly compared to the untreated control (i.e., heparinase treatment only) run under identical conditions (
In addition to the assigned disaccharides, the Δ4, 5 glycuronidase also acted on a heparinase-generated tetrasaccharide (identified as peak 2 in
Each of the foregoing patents, patent applications and references that are recited in this application are herein incorporated in their entirety by reference. Having described the presently preferred embodiments, and in accordance with the present invention, it is believed that other modifications, variations and changes will be suggested to those skilled in the art in view of the teachings set forth herein. It is, therefore, to be understood that all such variations, modifications, and changes are believed to fall within the scope of the present invention as defined by the appended claims.