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Natural, chimeric and hybrid glycosaminoglycan polymers and methods of making and using sameRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Preparing Compound Containing Saccharide Radical, N-glycosideNatural, chimeric and hybrid glycosaminoglycan polymers and methods of making and using same description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060188966, Natural, chimeric and hybrid glycosaminoglycan polymers and methods of making and using same. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit under 35 U.S.C. 119(e) of U.S. Provisional No. 60/305,263, filed Jul. 13, 2001, entitled "NANO HYALURONIC ACID AND METHODS OF MAKING AND USING SAME," the contents of which is expressly incorporated herein in its entirety by reference. [0002] This application is a continuation-in-part of copending U.S. Ser. No. 09/437,277, filed Nov. 11, 1999, entitled "POLYMER GRAFTING BY POLYSACCHARIDE SYNTHASES," which claims benefit under 35 U.S.C. 119(e) of U.S. Provisional No. 60/107,929, filed Nov. 11, 1998, entitled "POLYMER GRAFTING BY POLYSACCHARIDE SYNTHASES," the contents of both of which are expressly incorporated herein in their entirety by reference. [0003] This application is also a continuation-in-part of copending U.S. Ser. No. 09/283,402, filed Apr. 1, 1999, entitled "DNA ENCODING HYALURONAN SYNTHASE FROM PASTEURELLA MULTOCIDA AND METHODS;" which claims benefit under 35 U.S.C. 119(e) of U.S. Provisional No. 60/080,414, filed Apr. 2, 1998, entitled "UNIQUE HYALURONAN SYNTHASE FROM PASTEURELLA MULTOCIDA," the contents of both of which are expressly incorporated herein in their entirety by reference. [0004] This application is also a continuation-in-part of copending U.S. Serial No. 09/842,484, filed Apr. 25, 2001, entitled "CHONDROITIN SYNTHASE GENE AND METHODS OF MAKING AND USING SAME;" which claims benefit under 35 U.S.C. 119(e) of U.S. Provisional No. 60/199,538, filed Apr. 25, 2000, entitled "POLYMER FORMATION AND RECOGNITION MECHANISMS AND METHODS OF MAKING AND USING SAME," the contents of both of which are expressly incorporated herein in their entirety by reference. [0005] This application is also a continuation-in-part of copending U.S. Ser. No. 10/142,143, filed May 8, 2002, entitled "HEPARIN/HEPAROSAN SYNTHASE FROM P. MULTOCIDA AND METHODS OF MAKING AND USING SAME;" which claims benefit under 35 U.S.C. 119(e) of U.S. Provisional No. 60/289,554, filed May 8, 2001, entitled "PASTEURELLA MULTOCIDA HEPARIN SYNTHASE GENE AND METHODS OF MAKING AND USING SAME," the contents of both of which are expressly incorporated herein in their entirety by reference. [0006] This application also claims benefit under 35 U.S.C. 119(e) of U.S. Provisional No. 60/350,642, filed Jan. 22, 2002, entitled "HEPARIN POLYMER GRAFTING," the contents of which is expressly incorporated herein in its entirety by reference. BACKGROUND [0008] 1. Field of the Invention [0009] The present invention relates to methodology for the production of polymers, such as oligosaccharides, by a glycosaminoglycan synthase and, more particularly, polymer production utilizing glycosaminoglycan synthases from Pasteurella multocida. [0010] Various oligosaccharides (small sugar chains) show potential as non-toxic therapeutic agents to modulate blood coagulation, cancer metastasis, or cell growth. Complex sugars cause biological effects by binding to target proteins including enzymes and receptors. Methodologies to synthesize many compounds, however, and to test for potency and selectivity are limiting steps in drug discovery. As such, the presently claimed and disclosed invention also relates to a chemoenzymatic synthesis methodology to create both pure, chimeric, and hybrid polymers, such as oligosaccharides, composed of hyaluronan, chondroitin, keratan, dermatan, heparin units, and combinations thereof (e.g. "chimeric or hybrid" polymers). A library of distinct oligosaccharides are used in parallel using a microarray format or a microtiter plate, for example. Target proteins implicated in diseases are thereafter tested for their ability to bind to microarrays or microtiter plates and thus have potential as therapeutic agents. [0011] 2. Description of the Related Art [0012] Polysaccharides are large carbohydrate molecules comprising from about 25 sugar units to thousands of sugar units. Oligosaccharides are smaller carbohydrate molecules comprising less than about 25 sugar units. Animals, plants, fungi and bacteria produce an enormous variety of polysaccharide structures that are involved in numerous important biological functions such as structural elements, energy storage, and cellular interaction mediation. Often, the polysaccharide's biological function is due to the interaction of the polysaccharide with proteins such as receptors and growth factors. The glycosaminoglycan class of polysaccharides and oligosaccharides, which includes heparin, chondroitin, dermatan, keratan, and hyaluronic acid, plays major roles in determining cellular behavior (e.g. migration, adhesion) as well as the rate of cell proliferation in mammals. These polysaccharides and oligosaccharides are, therefore, essential for the correct formation and maintenance of the organs of the human body. [0013] Several species of pathogenic bacteria and fungi also take advantage of the polysaccharide's role in cellular communication. These pathogenic microbes form polysaccharide surface coatings or capsules that are identical or chemically similar to host molecules. For instance, Group A & C Streptococcus and Type A Pasteurella multocida produce authentic hyaluronic acid capsules, and other Pasteurella multocida (Type F and D) and pathogenic Escherichia coli (K4 and K5) are known to make capsules composed of polymers very similar to chondroitin and heparin. The pathogenic microbes form the polysaccharide surface coatings or capsules because such a coating is nonimmunogenic and protects the bacteria from host defenses, thereby providing the equivalent of molecular camouflage. [0014] Enzymes alternatively called syntheses, synthetases, or transferases, catalyze the polymerization of polysaccharides found in living organisms. Many of the known enzymes also polymerize activated sugar nucleotides. The most prevalent sugar donors contain UDP, but ADP, GDP, and CMP are also used depending on (1) the particular sugar to be transferred and (2) the organism. Many types of polysaccharides are found at, or outside of, the cell surface. Accordingly, most of the synthase activity is typically associated with either the plasma membrane on the cell periphery or the Golgi apparatus membranes that are involved in secretion. In general, these membrane-bound synthase proteins are difficult to manipulate by typical procedures, and only a few enzymes have been identified after biochemical purification. [0015] A larger number of synthases have been cloned and sequenced at the nucleotide level using "reverse genetic" approaches in which the gene or the complementary DNA (CDNA) was obtained before the protein was characterized. Despite this sequence information, the molecular details concerning the three-dimensional native structures, the active sites, and the mechanisms of catalytic action of the polysaccharide syntheses, in general, are very limited or absent. For example, the catalytic mechanism for glycogen synthesis is not yet known in detail even though the enzyme was discovered decades ago. In another example, it is still a matter of debate whether most of the enzymes that produce heteropolysaccharides utilize one UDP-sugar binding site to transfer both precursors, or alternatively, if there exists two dedicated regions for each substrate. [0016] A wide variety of polysaccharides are commercially harvested from many sources, such as xanthan from bacteria, carrageenans from seaweed, and gums from trees. This substantial industry supplies thousands of tons of these raw materials for a multitude of consumer products ranging from ice cream desserts to skin cream cosmetics. Vertebrate tissues and pathogenic bacteria are the sources of more exotic polysaccharides utilized in the medical field--e.g. as surgical aids, vaccines, and anticoagulants. For example, two glycosaminoglycan polysaccharides, heparin from pig intestinal mucosa and hyaluronic acid from rooster combs, are employed in several applications including clot prevention and eye surgery, respectively. Polysaccharides extracted from bacterial capsules (e.g. various Streptococcus pneumoniae strains) are utilized to vaccinate both children and adults against disease with varying levels of success. However, for the most part, one must use the existing structures found in the raw materials as obtained from nature. In many of the older industrial processes, chemical modification (e.g. hydrolysis, sulfation, deacetylation) is used to alter the structure and properties of the native polysaccharide. However, the synthetic control and the reproducibility of large-scale reactions are not always successful. Additionally, such polysaccharides are only available having a large molecular weight distribution, and oligosaccharides of the same repeat units are not available. [0017] Some of the current methods for designing and constructing carbohydrate polymers in vitro utilize: (i) difficult, multistep sugar chemistry, or (ii) reactions driven by transferase enzymes involved in biosynthesis, or (iii) reactions harnessing carbohydrate degrading enzymes catalyzing transglycosylation or hydrolysis. The latter two methods are often restricted by the specificity and the properties of the available naturally occurring enzymes. Many of these enzymes are neither particularly abundant nor stable but are almost always expensive. Overall, the procedures currently employed yield polymers containing between 2 and about 12 sugars. Unfortunately, many of the physical and biological properties of polysaccharides do not become apparent until the polymer contains 25, 100, or even thousands of monomers. [0018] As stated above, polysaccharides are the most abundant biomaterials on earth, yet many of the molecular details of their biosynthesis and function are not clear. Hyaluronic acid or "HA" is a linear polysaccharide of the glycosaminoglycan class and is composed of up to thousands of .beta.(1,4)GlcUA-.beta.(1,3)GlcNAc repeats. In vertebrates, HA is a major structural element of the extracellular matrix and plays roles in adhesion and recognition. HA has a high negative charge density and numerous hydroxyl groups, therefore, the molecule assumes an extended and hydrated conformation in solution. The viscoelastic properties of cartilage and synovial fluid are, in part, the result of the physical properties of the HA polysaccharide. HA also interacts with proteins such as CD44, RHAMM, and fibrinogen thereby influencing many natural processes such as angiogenesis, cancer, cell motility, wound healing, and cell adhesion. [0019] There are numerous medical applications of HA. For example, HA has been widely used as a viscoelastic replacement for the vitreous humor of the eye in ophthalmic surgery during implantation of intraocular lenses in cataract patients. HA injection directly into joints is also used to alleviate pain associated with arthritis. Chemically cross-linked gels and films are also utilized to prevent deleterious adhesions after abdominal surgery. Other researchers using other methods have demonstrated that adsorbed HA coatings also improve the biocompatibility of medical devices such as catheters and sensors by reducing fouling and tissue abrasion. [0020] HA is also made by certain microbes that cause disease in humans and animals. Some bacterial pathogens, namely Gram-negative Pasteurella multocida Type A and Gram-positive Streptococcus Group A and C, produce an extracellular HA capsule which protects the microbes from host defenses such as phagocytosis. Mutant bacteria that do not produce HA capsules are 10.sup.2- and 10.sup.3-fold less virulent in comparison to the encapsulated strains. Furthermore, the Paramecium bursaria Chlorella virus (PBCV-1) directs the algal host cells to produce a HA surface coating early in infection. [0021] The various HA syntheses ("HAS"), the enzymes that polymerize HA, utilize UDP-GlcUA and UDP-GlcNAc sugar nucleotide precursors in the presence of a divalent Mn, Mg, or Co ion to polymerize long chains of HA. The HA chains can be quite large (n=10.sup.2 to 10.sup.4). In particular, the HASs are membrane proteins localized to the lipid bilayer at the cell surface. During HA biosynthesis, the HA polymer is transported across the bilayer into the extracellular space. In all HASs, a single species of polypeptide catalyzes the transfer of two distinct sugars. In contrast, the vast majority of other known glycosyltransferases transfer only one monosaccharide. [0022] HasA (or spHAS) from Group A Streptococcus pyogenes was the first HA synthase to be described at the molecular level. The various vertebrate homologs (Xenopus DG42 or XIHAS1; murine and human HAS1, HAS2, and HAS3) and the viral enzyme, A98R, are quite similar at the amino acid level to certain regions of the HasA polypeptide chain (.about.30% identity overall) and were discovered only after the sequence of spHAS was disclosed in 1994 . At least 7 short motifs (5-9 residues) interspersed throughout these Class I enzymes are identical or quite conserved. The evolutionary relationship among these HA synthases from such dissimilar sources is not clear at present. The enzymes are predicted to have a similar overall topology in the bilayer: membrane-associated regions at the amino and the carboxyl termini flank a large cytoplasmic central domain (.about.200 amino acids). The amino terminal region appears to contain two transmembrane segments, while the carboxyl terminal region appears to contain three to five membrane-associated or transmembrane segments, depending on the species. Very little of these HAS polypeptide chains are expected to be exposed to the outside of the cell. [0023] With respect to the reaction pathway utilized by this group of enzymes, mixed findings have been reported from indirect experiments. The Group A streptococcal enzyme was reported to add sugars to the nonreducing terminus of the growing chain as determined by selective labeling and degradation studies. Using a similar approach, however, two laboratories working with the enzyme preparations from mammalian cells concluded that the new sugars were added to the reducing end of the nascent chain. In comparing these various studies, the analysis of the enzymatically-released sugars from the streptococcal system added more rigorous support for their interpretation. In another type of experiment, HA made in mammalian cells was reported to have a covalently attached UDP group as measured by an incorporation of low amounts of radioactivity derived from .sup.32P-labeled UDP-sugar into an anionic polymer. This data implied that the last sugar was transferred to the reducing end of the polymer. Thus, it remains unclear if these rather similar HAS polypeptides from vertebrates and streptococci actually utilize different reaction pathways. Continue reading about Natural, chimeric and hybrid glycosaminoglycan polymers and methods of making and using same... 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