US 20030104601A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0104601 A1 DeAngelis (43) Pub. Date: Jun. 5, 2003 (54) CHONDROITINSYNTHASE GENE AND Publication Classification METHODS OF MAKING AND USING SAME (51) Int. Cl." ............................ C12P 21/02; C12N 1/21; (76) Inventor: Paul L. DeAngelis, Edmond, OK (US) C07H 21/04; CO8B 37/00; C12N 9/24; C12N 15/74; Correspondence Address: C12O 1/68 DUNLAP, CODDING & ROGERS PC. (52) U.S. Cl. ..................... 435/200; 435/69.1; 435/320.1; PO BOX 16370 435/252.3; 536/23.2; 536/54; OKLAHOMA CITY, OK 73114 (US) 435/6 (21) Appl. No.: 09/842,484 (57) ABSTRACT (22) Filed: Apr. 25, 2001 Related U.S. Application Data The present invention relates to a chondroitin Synthase gene and methods of making and using Same. In more particular, (63) Continuation-in-part of application No. 09/283,402, but not by way of limitation, the present invention relates to filed on Apr. 1, 1999, now abandoned, and which is a a chondroitin Synthase gene from Pasteurella multocida and continuation-in-part of application No. 09/437.277, methods of isolating and using Same. Additionally, the filed on Nov. 10, 1999, now Pat. No. 6,444447. present invention relates to the use of unsulfated chondroitin and its preparation, as well as conversion into modified (60) Provisional application No. 60/199,538, filed on Apr. versions Such as dermatan Sulfate and chondroitin Sulfate 25, 2000. polymers. Patent Application Publication Jun. 5, 2003. Sheet 5 of 10 US 2003/0104601 A1 Std CS V HAS 2. S . w y - as Y 8 as is a s sis s O A4 C - - - 12 14 16 18 20 22 Time (min) Fig. 3 Patent Application Publication US 2003/0104601 A1 r bib a vil ?t Patent Application Publication Jun. 5, 2003. Sheet 7 of 10 US 2003/0104601 A1 DEANGELIS STD. SNMOL 10/15/00 Standard 1000 800 600 400 200 O 10.0 20.0 30.0 40.0 50.0 60.0 SAMPLEF REPEAT 5NMOL. 10/15 Pasteurella 1000 Chondroitin 800 600 400 200 O 0 200 300 40.0 50.0 60.0 Patent Application Publication Jun. 5, 2003 Sheet 8 of 10 US 2003/0104601 A1 100 75 É 50 25 9 10 11 12 13 14 5 6 Time (min) Fig. 6 200 150 É 100 50 Time (min) Fig. 7 Patent Application Publication Jun. 5, 2003 Sheet 9 of 10 US 2003/0104601 A1 NMR of Typc F Polymer HOD 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm Fig. 8 Patent Application Publication Jun. 5, 2003. Sheet 10 of 10 US 2003/0104601 A1 ABCase Treatment 0.012 0.010 0.008 0.006 e 0.004 0.002 O.OOO ADiOS -0.002 -0.004 25 50 75 100 minutes Fig. 9 ACase Treatment 0.012 0.010 0.008 t (). OO6 0.004 ADiOS OOO2 0.000 5() 75 minutes Fig. 10 US 2003/0104601 A1 Jun. 5, 2003 CHONIDROITN SYNTHASE GENE AND a GAG intermediate. Also GAGS may sequester or bind METHODS OF MAKING AND USING SAME certain proteins (e.g. growth or coagulation factors) to cell Surfaces. CROSS-REFERENCE TO RELATED 0008 Certain pathogenic bacteria produce an extracellu APPLICATIONS lar polysaccharide coating, called a capsule, which Serves as 0001. This application is related to U.S. provisional a virulence factor. In a few cases, the capsule is composed application Serial No. 60/199,538 filed Apr. 25, 2000 and of GAG or GAG-like polymers. As the microbial polysac entitled “POLYMER FORMATION AND RECOGNITION charide is identical or very similar to the host GAG, the MECHANISMS AND METHODS OF MAKING AND antibody response is either very limited or non-existent. The USING SAME'; is a continuation-in-part of pending U.S. capsule is thought to assist in the evasion of host defenses patent application Ser. No. 09/283,402, filed Apr. 4, 1999 Such as phagocytosis and complement. Examples of this and entitled “DNA ENCODING HYALURONAN SYN clever Strategy of molecular camouflage are the production THASE FROM PASTEURELLA MULTOCIDA AND of an authentic HA polysaccharide by Gram-negative Type METHODS OF USE"; and is a continuation-in-part of A Pasteurella multocida and Gram-positive Group A and C pending U.S. patent application Ser. No. 09/437,277, filed StreptococcuS. The HA capsule of these microbes increases Nov. 10, 1999 and entitled “POLYMER GRAFTING BY virulence by 10° to 10-fold as measured by LDsovalues, the POLYSACCHARIDE SYNTHASES. number of colony forming units that will kill 50% of the test animals after bacterial challenge. STATEMENT REGARDING FEDERALLY 0009. The invasiveness and pathogenicity of certain E. FUNDED RESEARCH coli Strains has also been attributed to their polysaccharide 0002 The government owns certain rights in the present capsules. Two Escherichia coli capsular types, K4 and K5, invention pursuant to a grant from the National Institutes of make polymers composed of GAG-like polymers. The E. Health (GM56497) and a grant from the National Science coli K4 polymer is an unsulfated chondroitin backbone Foundation (MCB-9876193). decorated with fructose side-branches on the C3 position of the GlcUA residues. The K5 capsular material is a polysac BACKGROUND OF THE INVENTION charide, called heparosan, identical to mammalian heparin except that the bacterial polymer is unsulfated and there is 0003) 1. Field of the Invention no epimerization of GlcUA to iduronic acid. 0004. The present invention relates to a chondroitin syn 0010. The studies of GAG biosynthesis have been instru thase gene and methods of making and using Same. In more mental in understanding polysaccharide production in gen particular, but not by way of limitation, the present invention eral. The HA synthases were the first GAG glycosyltrans relates to a chondroitin Synthase gene from Pasteurella ferases to be identified at the molecular level. These multocida and methods of isolating and using Same. Addi enzymes utilize UDP-Sugar nucleotide Substrates to produce tionally, the present invention relates to the use of unsulfated large polymers containing thousands of disaccharide chondroitin and its preparation, as well as conversion into repeats. The genes encoding bacterial, Vertebrate, and viral modified versions Such as dermatan Sulfate and chondroitin HAS enzymes have been cloned. In all these cases, expres Sulfate polymers. Sion studies demonstrated that transformation with DNA 0005 2. Background Information Relating to the Inven encoding a single HAS polypeptide conferred the ability of tion foreign hosts to Synthesize HA. Except for the most recent HAS to be identified, P. multocida pmHAS, these proteins 0006 Glycosaminoglycans GAGs are long linear have similar amino acid Sequences and predicted topology in polysaccharides consisting of disaccharide repeats that con the membrane. Two classes of HASs have been proposed to tain an amino Sugar and are found in most animals. Chon exist based on these structural differences as well as poten droitin B(1, 4)GlcUA-B(1, 3)GalNAc), heparin/heparan tial differences in reaction mechanism C.1 4)GlcUA-B(1, 4)GlcNAc, and hyaluronan IB(1, 4)GlcUA-B(1, 3)GlcNAc) are the three most prevalent 0011. The biochemical study of chondroitin biosynthesis GAGS found in humans. Chondroitin and heparin typically in vertebrates was initiated in the 1960s. Only recently have have n=20 to 100, while hyaluronan typically has n=10. the mammalian enzymes for elongation of the polysaccha Chondroitin and heparin are Synthesized as glycoproteins ride backbone of chondroitin been tentatively identified by and are Sulfated at various positions in vertebrates. Hyalu biochemical means. An 80-kDa GlcUA transferase found in roman is not Sulfated in Vertebrates. A Substantial fraction of vertebrate cartilage and liver was implicated in the biosyn the GlcUA residues of heparin and chondroitin are epimer thesis of the chondroitin backbone by photoaffinity labeling ized to form iduronic acid. with an azidoUDP-GlcUA probe. A preparation from bovine 0007 Many lower animals possess these same GAGs or serum with the appropriate GalNAc- and GlcUA-transferase very Similar molecules. GAGS play both Structural and activities in vitro was obtained by conventional chromatog recognition roles on the cell Surface and in the extracellular raphy, but Several bands on SDS polyacrylamide gels matrix. By virtue of their physical characteristics, namely a (including a few migrating ~80 kDa) were observed. high negative charge density and a multitude of polar 0012 Chondroitin polysaccharide (beta-1,3-GalNAc hydroxyl groups, GAGS help hydrate and expand tissues. beta-1,4-GlcUA); n=~10-2000) has use as a hyaluronan Numerous proteins bind selectively to one or more of the (HA) polysaccharide Substitute in medical or cosmetic appli GAGs. Thus the proteins found on cell surfaces or the cations. Both chondroitin and hyaluronan form Viscoelastic associated extracellular matrices (e.g. CD44, collagen, gels (Suitable for eye or joint applications) or hydrophilic, fibronectin) of different cell types may adhere or interact via hygroscopic materials (Suitable for moisturizer or wound US 2003/0104601 A1 Jun. 5, 2003 dressings). Unmodified or underivatized chondroitin is not multocida and methods of isolating and using Same. Addi known to exist or, if present, in Very Small quantities in the tionally, the present invention relates to the use of unsulfated human body. The main advantage is that byproducts of chondroitin and its preparation, as well as conversion into natural HA degradation (by shear, enzyme, radical or oxi modified versions Such as dermatan Sulfate and chondroitin dation processes) have certain biological activities with Sulfate polymers. respect to Vascularization, angiogenesis, cancer, tissue modulation, but similar byproducts of chondroitin (in the BRIEF DESCRIPTION OF THE SEVERAL proposed unsulfated, unmodified State) may not have the VIEWS OF THE DRAWINGS Same biological activity.