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  Delta 4,5 glycuronidase and methods of analyzing therewithUSPTO Application #: 20060177885Title: Delta 4,5 glycuronidase and methods of analyzing therewith Abstract: The invention relates to Δ4,5 glycuronidase, related compositions, and methods of use thereof. (end of abstract)
Agent: Wolf Greenfield & Sacks, PC - Boston, MA, US Inventors: James R. Myette, Zachary Shriver, Ganesh Venkataraman, Ram Sasisekharan, Maitland W. McLean USPTO Applicaton #: 20060177885 - Class: 435018000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Hydrolase The Patent Description & Claims data below is from USPTO Patent Application 20060177885. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application is a divisional of U.S. patent application Ser. No. 10/429921, filed on May 5, 2003 and currently pending, which claims priority under 35 U.S.C. .sctn.119 from U.S. provisional application Ser. No. 60/377,488 filed May 3, 2002, the entire contents of each of which are incorporated herein by reference. FIELD OF THE INVENTION [0003] The invention relates to .DELTA.4,5 glycuronidase and uses thereof. In particular, the invention relates to substantially pure .DELTA.4,5 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 .DELTA.4,5 glycuronidase and/or GAG fragments produced by enzymatic cleavage with .DELTA.4,5 glycuronidase. BACKGROUND OF THE INVENTION [0004] 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 Couchman, J. R. (2000) Int J Biochem Cell Biol 32, 269-88] requisite for cell growth and differentiation, cell adhesion and migration, and tissue morphogenesis. [0005] 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 .beta.-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). [0006] 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, H.sub.NAc/S,6SGH.sub.NS,3S,6SI.sub.2SH.sub.NS,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]. [0007] 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. [0008] 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 applications Ser. Nos. 09/557,997 and 09/558,137, both filed on Apr. 24, 2000, having common inventorship). SUMMARY OF THE INVENTION [0009] .DELTA.4,5 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 .DELTA.4,5 glycuronidase. In one embodiment of the invention the substantially pure .DELTA.4,5 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 .DELTA.4,5 glycuronidase is produced using a host cell comprising the expression vector. In another embodiment the substantially pure .DELTA.4,5 glycuronidase is a synthetic glycuronidase. [0010] 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. [0011] In yet another aspect of the invention the polypeptide of the .DELTA.4,5 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. [0012] In one aspect, the invention is a composition comprising, an isolated .DELTA.4,5 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 .DELTA.4,5 glycuronidase has a specific activity that is about 2 fold higher than the native enzyme. In another embodiment the .DELTA.4,5 glycuronidase has a specific activity that is about 3 fold higher. The specific activity of the .DELTA.4,5 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. [0013] 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 .DELTA.4,5 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. [0014] Pharmaceutical compositions of any of the compositions or vectors described herein are also encompassed in the invention. [0015] In other aspects the invention relates to a method of cleaving a glycosaminoglycan with a .DELTA.4,5 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. [0016] 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 2-0 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 .DELTA.UH.sub.NAc; .DELTA.UH.sub.NAc,6S; .DELTA.UH.sub.NS,6S; or .DELTA.UH.sub.NS. In another embodiment the invention also provides for the products of the cleavage of a glycosaminoglycan with the .DELTA.4,5 glycuronidase. In some embodiments the glycuronidase is used to generate a LMWH. [0017] 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. [0018] 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. [0019] 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. [0020] 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. [0021] 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. Continue reading... Full patent description for Delta 4,5 glycuronidase and methods of analyzing therewith Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Delta 4,5 glycuronidase and methods of analyzing therewith patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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