Identification and Molecular Cloning of a Chondroitin Synthase from Pasteurella multocida Type F
2000; Elsevier BV; Volume: 275; Issue: 31 Linguagem: Inglês
10.1074/jbc.m003385200
ISSN1083-351X
AutoresPaul L. DeAngelis, Amy J. Padgett-McCue,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoPasteurella multocida Type F, the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported that the capsule was removed by treating microbes with chondroitin AC lyase. We found by acid hydrolysis that the polysaccharide contained galactosamine and glucuronic acid. We molecularly cloned a Type F polysaccharide synthase and characterized its enzymatic activity. The 965-residue enzyme, called P. multocida chondroitin synthase (pmCS), is 87% identical at the nucleotide and the amino acid level to the hyaluronan synthase, pmHAS, from P. multocidaType A. A recombinant Escherichia coli-derived truncated, soluble version of pmCS (residues 1–704) was shown to catalyze the repetitive addition of sugars from UDP-GalNAc and UDP-GlcUA to chondroitin oligosaccharide acceptors in vitro. Other structurally related sugar nucleotide precursors did not substitute in the elongation reaction. Polymer molecules composed of ∼103 sugar residues were produced, as measured by gel filtration chromatography. The polysaccharide synthesized in vitro was sensitive to the action of chondroitin AC lyase but resistant to the action of hyaluronan lyase. This is the first report identifying a glycosyltransferase that forms a polysaccharide composed of chondroitin disaccharide repeats, [β(1,4)GlcUA-β(1,3)GalNAc]n. In analogy to known hyaluronan synthases, a single polypeptide species, pmCS, possesses both transferase activities. Pasteurella multocida Type F, the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported that the capsule was removed by treating microbes with chondroitin AC lyase. We found by acid hydrolysis that the polysaccharide contained galactosamine and glucuronic acid. We molecularly cloned a Type F polysaccharide synthase and characterized its enzymatic activity. The 965-residue enzyme, called P. multocida chondroitin synthase (pmCS), is 87% identical at the nucleotide and the amino acid level to the hyaluronan synthase, pmHAS, from P. multocidaType A. A recombinant Escherichia coli-derived truncated, soluble version of pmCS (residues 1–704) was shown to catalyze the repetitive addition of sugars from UDP-GalNAc and UDP-GlcUA to chondroitin oligosaccharide acceptors in vitro. Other structurally related sugar nucleotide precursors did not substitute in the elongation reaction. Polymer molecules composed of ∼103 sugar residues were produced, as measured by gel filtration chromatography. The polysaccharide synthesized in vitro was sensitive to the action of chondroitin AC lyase but resistant to the action of hyaluronan lyase. This is the first report identifying a glycosyltransferase that forms a polysaccharide composed of chondroitin disaccharide repeats, [β(1,4)GlcUA-β(1,3)GalNAc]n. In analogy to known hyaluronan synthases, a single polypeptide species, pmCS, possesses both transferase activities. glycosaminoglycan P. multocida chondroitin synthase hyaluronan, hyaluronate, or hyaluronic acid HA synthase P. multocida HAS glucuronic acid N-acetylgalactosamine N-acetylglucosamine galacturonic acid polymerase chain reaction Glycosaminoglycans (GAGs),1 long linear polysaccharides consisting of disaccharide repeats that contain an amino sugar, are found in most animals (1Roden L. Lennarz W.J. The Biochemistry of Glycoproteins and Proteoglycans. Plenum Publishing Corp., New York1980: 267-371Crossref Google Scholar, 2Hascall V.C. Hascall G.K. Hay E.D. Cell Biology of Extracellular Matrix. Plenum Publishing Corp., New York1981: 39-78Crossref Google Scholar, 3Lidholt K. Biochem. Soc. Trans. 1997; 25: 866-870Crossref PubMed Scopus (12) Google Scholar, 4Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2052) Google Scholar). Chondroitin (β(1,4)GlcUA-β(1,3)GalNAc)n, heparin/heparan (α(1,4)GlcUA-β(1,4)GlcNAc)n, and hyaluronan (β(1,4)GlcUA-β(1,3)GlcNAc)n are the three most prevalent GAGs found in humans. In the former two polymers, usuallyn = 20 to 100, whereas in the case of HA,n = 103–4. Chondroitin and heparin/heparan, but not HA, are synthesized as glycoproteins and are sulfated at various positions in vertebrates. A substantial fraction of the GlcUA residues of heparin are epimerized to form iduronic acid. Many lower animals possess these same GAGs or very similar molecules (5Rahemtulla F. Lovtrup S. Comp. Biochem. Physiol. 1975; 50B: 631-635Google Scholar). GAGs play both structural and recognition roles on the cell surface and in the extracellular matrix. By virtue of their physical characteristics, namely a high negative charge density and a multitude of polar hydroxyl groups, GAGs help hydrate and expand tissues. A plethora of proteins bind selectively to one or more of the GAGs (6Lindahl U. Hook M. Annu. Rev. Biochem. 1978; 47: 385-417Crossref PubMed Scopus (570) Google Scholar,7Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1004) Google Scholar). Thus the proteins found on cell surfaces or the associated extracellular matrices (e.g. CD44, collagen, fibronectin) of different cell types may adhere or interact via a GAG intermediate. Also GAGs may sequester or bind certain proteins (e.g.growth or coagulation factors) to cell surfaces.Certain pathogenic bacteria produce an extracellular polysaccharide coating called a capsule that serves as a virulence factor (8Roberts I.S. Annu. Rev. Microbiol. 1996; 50: 285-315Crossref PubMed Scopus (517) Google Scholar). In a few cases, the capsule is composed of GAG or GAG-like polymers. As the microbial polysaccharide is identical or very similar to the host GAG, the antibody response is either very limited or non-existent. The capsule is thought to assist in the evasion of host defenses such as phagocytosis and complement. Examples of this clever strategy of molecular camouflage are the production of authentic HA polysaccharide by Gram-negative Type A Pasteurella multocida (9Carter G.R. Annau E. Am. J. Vet. Res. 1953; 14: 475-478PubMed Google Scholar) and Gram-positive Group A and C Streptococcus (10Kass E.H. Seastone C.V. J. Exp. Med. 1944; 79: 319-330Crossref PubMed Scopus (75) Google Scholar). The HA capsule of these microbes increases virulence by 102–103-fold as measured by LD50values, the number of colony-forming units that will kill 50% of the test animals after bacterial challenge (11Harmon B.G. Glisson J.R. Latimer K.S. Steffens W.L. Nunnally J.C. Am. J. Vet. Res. 1991; 52: 1507-1511PubMed Google Scholar, 12Wessels M.R. Moses A.E. Goldberg J.B. DiCesare T.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8317-8321Crossref PubMed Scopus (269) Google Scholar). The invasiveness and pathogenicity of certain Escherichia coli strains has also been attributed to their polysaccharide capsule (8Roberts I.S. Annu. Rev. Microbiol. 1996; 50: 285-315Crossref PubMed Scopus (517) Google Scholar). Two E. coli capsular types, K4 and K5, make polymers composed of GAG-like polymers. The E. coli K4 polymer is an unsulfated chondroitin backbone decorated with fructose side branches on the C3 position of the GlcUA residues (13Rodriguez M. Jann B. Jann K. Eur. J. Biochem. 1988; 177: 117-124Crossref PubMed Scopus (106) Google Scholar). The K5 capsular material is a polysaccharide, called heparosan, identical to mammalian heparin, except that the bacterial polymer is unsulfated, and there is no epimerization of GlcUA to iduronic acid (14Vann W.F. Schmidt M.A. Jann B. Jann K. Eur. J. Biochem. 1981; 116: 359-364Crossref PubMed Scopus (210) Google Scholar).The studies of GAG biosynthesis have been instrumental in understanding polysaccharide production in general. The HA synthases were the first GAG glycosyltransferases to be identified at the molecular level (15DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar, 16Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 17DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 18DeAngelis P.L. Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (160) Google Scholar). These enzymes utilize UDP-sugar nucleotide substrates to produce large polymers containing thousands of disaccharide repeats. The genes encoding bacterial, vertebrate, and viral HAS enzymes have been cloned. In all these cases, expression studies demonstrated that transformation with DNA encoding a single HAS polypeptide conferred the ability of foreign hosts to synthesize HA. Except for the most recent HAS to be identified, P. multocida pmHAS (17DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), these proteins have similar amino acid sequences and predicted topology in the membrane (18DeAngelis P.L. Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (160) Google Scholar). Two classes of HASs have been proposed to exist based on these structural differences as well as potential differences in reaction mechanism (18DeAngelis P.L. Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (160) Google Scholar, 19DeAngelis P.L. J. Biol. Chem. 1999; 274: 26557-26562Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar).The biochemical study of chondroitin biosynthesis in vertebrates was initiated in the 1960s (20Telser A. Robinson H.C. Dorfman A. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 912-919Crossref PubMed Scopus (69) Google Scholar, 21Silbert J.E. DeLuca S. Biochem. Biophys. Res. Commun. 1968; 31: 990-995Crossref PubMed Scopus (4) Google Scholar, 22DeLuca S. Silbert J.E. J. Biol. Chem. 1968; 243: 2725-2729Abstract Full Text PDF PubMed Google Scholar). Only recently have the mammalian enzymes for elongation of the polysaccharide backbone of chondroitin been tentatively identified by biochemical means. An 80-kDa GlcUA-transferase found in vertebrate cartilage and liver was implicated in the biosynthesis of the chondroitin backbone by photoaffinity labeling with an azido-UDP-GlcUA probe (23Sugumaran G. Katsman M. Sunthankar P. Drake R.R. J. Biol. Chem. 1997; 272: 14399-14403Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). A preparation from bovine serum with the appropriate GalNAc- and GlcUA-transferase activities in vitro was obtained by conventional chromatography, but several bands on SDS-polyacrylamide gels (including a few migrating ∼80 kDa) were observed (24Tsuchida K. Lind T. Kitagawa H. Lindahl U. Sugahara K. Lidholt K. Eur. J. Biochem. 1999; 264: 461-467Crossref PubMed Scopus (17) Google Scholar). The situation will probably be unclear in vertebrates until a chondroitin synthase has been sequenced at the gene level and functionally expressed.With respect to related microbial GAG synthases other than the HASs, only the E. coli K5 glycosyltransferase, KfiC, that synthesizes heparosan has been identified by genetic and biochemical means (25Griffiths G. Cook N.J. Gottfridson E. Lind T. Lidholt K. Roberts I.S. J. Biol. Chem. 1998; 273: 11752-11757Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In analogy to the HASs, it appears that KfiC is capable of transferring both sugars of the disaccharide repeat to the growing polymer chain. The chondroitin backbone-synthesizing enzyme of E. coli K4 has been enzymatically characterized (26Lidholt K. Fjelstad M. J. Biol. Chem. 1997; 272: 2682-2687Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), but the genes encoding the relevant glycosyltransferases have not yet been identified.Many P. multocida isolates produce GAG or GAG-like molecules as assessed by enzymatic degradation and removal of the capsule of living bacterial cells (27Rimler R.B. Vet. Rec. 1994; 134: 191-192Crossref PubMed Scopus (53) Google Scholar, 28Rimler R.B. Register K.B. Magyar T. Ackermann M.R. Vet. Microbiol. 1995; 47: 287-294Crossref PubMed Scopus (13) Google Scholar). Type A P. multocida, the major fowl cholera pathogen, makes a capsule that is sensitive to hyaluronidase. Subsequent NMR structural studies of capsular extracts confirmed that HA was the major polysaccharide present (29Rosner H. Grimmecke H.D. Knirel Y.A. Shashkov A.S. Carbohydr. Res. 1992; 223: 329-333Crossref PubMed Scopus (36) Google Scholar). A specific HA-binding protein, aggrecan, also interacts with HA from Type AP. multocida (17DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Two other distinct P. multocida types, a swine pathogen, Type D, and a minor fowl cholera pathogen, Type F, produce polymers that are chondroitin or chondroitin-like based on the observation that their capsules are degraded by Flavobacterium chondroitin AC lyase (27Rimler R.B. Vet. Rec. 1994; 134: 191-192Crossref PubMed Scopus (53) Google Scholar, 28Rimler R.B. Register K.B. Magyar T. Ackermann M.R. Vet. Microbiol. 1995; 47: 287-294Crossref PubMed Scopus (13) Google Scholar). After enzymatic removal of the capsule, both types were more readily phagocytosed by neutrophils in vitro (28Rimler R.B. Register K.B. Magyar T. Ackermann M.R. Vet. Microbiol. 1995; 47: 287-294Crossref PubMed Scopus (13) Google Scholar). The capsule of Type D cells, but not Type F cells, is also reported to be degraded by heparinase III (27Rimler R.B. Vet. Rec. 1994; 134: 191-192Crossref PubMed Scopus (53) Google Scholar).In this report, we have analyzed the monosaccharide composition of the P. multocida Type F polysaccharide and used the DNA sequence information of the Type A HA biosynthesis locus (17DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) to obtain the homologous region from the Type F chromosome. We describe pmCS, the first chondroitin synthase to be identified and molecularly cloned from any source.DISCUSSIONWe verify that P. multocida Type F produces a chondroitin or chondroitin-like capsule. We have also molecularly cloned the glycosyltransferase responsible for polymerizing the chondroitin backbone component of the capsular polysaccharide. This enzyme seems to be a close homolog of the pmHAS enzyme. Recently, we determined that the pmHAS enzyme contains two active sites in a single polypeptide by generating mutants that transfer only GlcUA or only GlcNAc (33Jing, W., and DeAngelis, P. L. (2000)Glycobiology, in pressGoogle Scholar). Interestingly, mixing the two different mutant proteins reconstituted the HA synthase activity. We hypothesized that one domain, called A1, is responsible for GlcNAc transfer, and the other domain, called A2, is responsible for GlcUA transfer (33Jing, W., and DeAngelis, P. L. (2000)Glycobiology, in pressGoogle Scholar). Comparison of the pmHAS and the pmCS sequences reveals that the majority of the sequence differences exist in the vicinity of the Al domain. The pmCS enzyme transfers a different hexosamine, GalNAc; thus, this observation is consistent with our proposed two-domain structure for pmHAS. Future experiments will be directed at identifying which residues of the Al domain are responsible for recognition and/or transfer of GlcNAc or GalNAc sugars.The pmHAS protein was also hypothesized to interact with a putative polysaccharide transporter system or a membrane-bound partner via its carboxyl terminus because deletion of residues 704 to 972 from the native-length enzyme resulted in the formation of a soluble enzyme (33Jing, W., and DeAngelis, P. L. (2000)Glycobiology, in pressGoogle Scholar). However, no substantial membrane-associated or hydrophobic regions are predicted to reside in this sequence. Because pmHAS and pmCS are highly homologous in this region, which is not essential for their glycosyltransferase activities, it is quite likely that the carboxyl terminus contains domains or motifs required for interacting with the polysaccharide transport machinery or a membrane-bound partnerin vivo.The evolutionary relationship between Type A and Type F P. multocida strains has not yet been delineated. Both organisms are widespread causative agents of fowl cholera, but many more isolates from diseased birds in North America are Type A microbes with HA capsules (30Rimler R.B. Rhoades K.R. J. Clin. Microbiol. 1987; 25: 615-618Crossref PubMed Google Scholar). It is likely that the progenitor of the two distinct capsular types had either a chondroitin synthase-like or a HAS-like gene. The specificity of this ancestral enzyme may have changed after a few mutations, resulting in the appearance of another capsular type. Apparently, the sugar transfer specificity is rather selective, since neither recombinant pmCS nor pmHAS misincorporate the inappropriate hexosamine into polymer in vitro. Some Gram-negative bacteria (e.g. E. coli) possess an UDP-GlcNAc/UDP-GalNAc epimerase; therefore, the hexosamine precursor either for HA or for chondroitin could have been available for polysaccharide biosynthesis without the need to gain an auxiliary metabolic enzyme simultaneously. Typically the UDP-glucose dehydrogenase, the enzyme that forms the UDP-GlcUA precursor, is found in Gram-negative bacteria only if the microbe possesses a GlcUA-containing polymer or glycoconjugate. In both Type A and Type FP. multocida, the UDP-glucose dehydrogenase gene is directly downstream of the GAG synthase (17DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar).The relationship between the bacterial chondroitin synthase and the putative mammalian counterpart is unclear. No similar vertebrate proteins are deposited in the data base as yet. Both bacterial pmCS and the vertebrate chondroitin synthase utilize UDP-sugars to extend acceptor carbohydrates in vitro. In most cases, the mammalian enzyme in cell-free extracts, however, does not produce long chondroitin chains, and only the half-reaction (e.g. adding a single GlcUA to a GalNAc-terminated oligosaccharide or vice versa) is readily observed in vitro (23Sugumaran G. Katsman M. Sunthankar P. Drake R.R. J. Biol. Chem. 1997; 272: 14399-14403Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 24Tsuchida K. Lind T. Kitagawa H. Lindahl U. Sugahara K. Lidholt K. Eur. J. Biochem. 1999; 264: 461-467Crossref PubMed Scopus (17) Google Scholar). In vertebrate tissues, other enzymes modify chondroitin extensively by sulfation and/or epimerization (1Roden L. Lennarz W.J. The Biochemistry of Glycoproteins and Proteoglycans. Plenum Publishing Corp., New York1980: 267-371Crossref Google Scholar). Hopefully, the discovery and the characterization of pmCS will assist the further study of the rather recalcitrant mammalian chondroitin synthase enzymes.Note Added in ProofRecently it was reported (Nadanka, S., Kitagawa, H.., Goto, F., Tamura, J., Neumann, K. W., Ogawa, T., and Sugahara, K. (1999) Biochem. J. 340,353–357) that a partially purified chondroitin synthase preparation from a melanoma cell line catalyzed the repetitive addition of chondroitin disaccharide repeats in vitroto the linkage tetrasaccharide of an α-thrombomodulin acceptor but not to the free tetrasaccharide, suggesting a role for the core protein in mammalian GAG biosynthesis. Glycosaminoglycans (GAGs),1 long linear polysaccharides consisting of disaccharide repeats that contain an amino sugar, are found in most animals (1Roden L. Lennarz W.J. The Biochemistry of Glycoproteins and Proteoglycans. Plenum Publishing Corp., New York1980: 267-371Crossref Google Scholar, 2Hascall V.C. Hascall G.K. Hay E.D. Cell Biology of Extracellular Matrix. Plenum Publishing Corp., New York1981: 39-78Crossref Google Scholar, 3Lidholt K. Biochem. Soc. Trans. 1997; 25: 866-870Crossref PubMed Scopus (12) Google Scholar, 4Laurent T.C. Fraser J.R.E. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2052) Google Scholar). Chondroitin (β(1,4)GlcUA-β(1,3)GalNAc)n, heparin/heparan (α(1,4)GlcUA-β(1,4)GlcNAc)n, and hyaluronan (β(1,4)GlcUA-β(1,3)GlcNAc)n are the three most prevalent GAGs found in humans. In the former two polymers, usuallyn = 20 to 100, whereas in the case of HA,n = 103–4. Chondroitin and heparin/heparan, but not HA, are synthesized as glycoproteins and are sulfated at various positions in vertebrates. A substantial fraction of the GlcUA residues of heparin are epimerized to form iduronic acid. Many lower animals possess these same GAGs or very similar molecules (5Rahemtulla F. Lovtrup S. Comp. Biochem. Physiol. 1975; 50B: 631-635Google Scholar). GAGs play both structural and recognition roles on the cell surface and in the extracellular matrix. By virtue of their physical characteristics, namely a high negative charge density and a multitude of polar hydroxyl groups, GAGs help hydrate and expand tissues. A plethora of proteins bind selectively to one or more of the GAGs (6Lindahl U. Hook M. Annu. Rev. Biochem. 1978; 47: 385-417Crossref PubMed Scopus (570) Google Scholar,7Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1004) Google Scholar). Thus the proteins found on cell surfaces or the associated extracellular matrices (e.g. CD44, collagen, fibronectin) of different cell types may adhere or interact via a GAG intermediate. Also GAGs may sequester or bind certain proteins (e.g.growth or coagulation factors) to cell surfaces. Certain pathogenic bacteria produce an extracellular polysaccharide coating called a capsule that serves as a virulence factor (8Roberts I.S. Annu. Rev. Microbiol. 1996; 50: 285-315Crossref PubMed Scopus (517) Google Scholar). In a few cases, the capsule is composed of GAG or GAG-like polymers. As the microbial polysaccharide is identical or very similar to the host GAG, the antibody response is either very limited or non-existent. The capsule is thought to assist in the evasion of host defenses such as phagocytosis and complement. Examples of this clever strategy of molecular camouflage are the production of authentic HA polysaccharide by Gram-negative Type A Pasteurella multocida (9Carter G.R. Annau E. Am. J. Vet. Res. 1953; 14: 475-478PubMed Google Scholar) and Gram-positive Group A and C Streptococcus (10Kass E.H. Seastone C.V. J. Exp. Med. 1944; 79: 319-330Crossref PubMed Scopus (75) Google Scholar). The HA capsule of these microbes increases virulence by 102–103-fold as measured by LD50values, the number of colony-forming units that will kill 50% of the test animals after bacterial challenge (11Harmon B.G. Glisson J.R. Latimer K.S. Steffens W.L. Nunnally J.C. Am. J. Vet. Res. 1991; 52: 1507-1511PubMed Google Scholar, 12Wessels M.R. Moses A.E. Goldberg J.B. DiCesare T.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8317-8321Crossref PubMed Scopus (269) Google Scholar). The invasiveness and pathogenicity of certain Escherichia coli strains has also been attributed to their polysaccharide capsule (8Roberts I.S. Annu. Rev. Microbiol. 1996; 50: 285-315Crossref PubMed Scopus (517) Google Scholar). Two E. coli capsular types, K4 and K5, make polymers composed of GAG-like polymers. The E. coli K4 polymer is an unsulfated chondroitin backbone decorated with fructose side branches on the C3 position of the GlcUA residues (13Rodriguez M. Jann B. Jann K. Eur. J. Biochem. 1988; 177: 117-124Crossref PubMed Scopus (106) Google Scholar). The K5 capsular material is a polysaccharide, called heparosan, identical to mammalian heparin, except that the bacterial polymer is unsulfated, and there is no epimerization of GlcUA to iduronic acid (14Vann W.F. Schmidt M.A. Jann B. Jann K. Eur. J. Biochem. 1981; 116: 359-364Crossref PubMed Scopus (210) Google Scholar). The studies of GAG biosynthesis have been instrumental in understanding polysaccharide production in general. The HA synthases were the first GAG glycosyltransferases to be identified at the molecular level (15DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar, 16Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 17DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 18DeAngelis P.L. Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (160) Google Scholar). These enzymes utilize UDP-sugar nucleotide substrates to produce large polymers containing thousands of disaccharide repeats. The genes encoding bacterial, vertebrate, and viral HAS enzymes have been cloned. In all these cases, expression studies demonstrated that transformation with DNA encoding a single HAS polypeptide conferred the ability of foreign hosts to synthesize HA. Except for the most recent HAS to be identified, P. multocida pmHAS (17DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), these proteins have similar amino acid sequences and predicted topology in the membrane (18DeAngelis P.L. Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (160) Google Scholar). Two classes of HASs have been proposed to exist based on these structural differences as well as potential differences in reaction mechanism (18DeAngelis P.L. Cell. Mol. Life Sci. 1999; 56: 670-682Crossref PubMed Scopus (160) Google Scholar, 19DeAngelis P.L. J. Biol. Chem. 1999; 274: 26557-26562Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The biochemical study of chondroitin biosynthesis in vertebrates was initiated in the 1960s (20Telser A. Robinson H.C. Dorfman A. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 912-919Crossref PubMed Scopus (69) Google Scholar, 21Silbert J.E. DeLuca S. Biochem. Biophys. Res. Commun. 1968; 31: 990-995Crossref PubMed Scopus (4) Google Scholar, 22DeLuca S. Silbert J.E. J. Biol. Chem. 1968; 243: 2725-2729Abstract Full Text PDF PubMed Google Scholar). Only recently have the mammalian enzymes for elongation of the polysaccharide backbone of chondroitin been tentatively identified by biochemical means. An 80-kDa GlcUA-transferase found in vertebrate cartilage and liver was implicated in the biosynthesis of the chondroitin backbone by photoaffinity labeling with an azido-UDP-GlcUA probe (23Sugumaran G. Katsman M. Sunthankar P. Drake R.R. J. Biol. Chem. 1997; 272: 14399-14403Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). A preparation from bovine serum with the appropriate GalNAc- and GlcUA-transferase activities in vitro was obtained by conventional chromatography, but several bands on SDS-polyacrylamide gels (including a few migrating ∼80 kDa) were observed (24Tsuchida K. Lind T. Kitagawa H. Lindahl U. Sugahara K. Lidholt K. Eur. J. Biochem. 1999; 264: 461-467Crossref PubMed Scopus (17) Google Scholar). The situation will probably be unclear in vertebrates until a chondroitin synthase has been sequenced at the gene level and functionally expressed. With respect to related microbial GAG synthases other than the HASs, only the E. coli K5 glycosyltransferase, KfiC, that synthesizes heparosan has been identified by genetic and biochemical means (25Griffiths G. Cook N.J. Gottfridson E. Lind T. Lidholt K. Roberts I.S. J. Biol. Chem. 1998; 273: 11752-11757Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In analogy to the HASs, it appears that KfiC is capable of transferring both sugars of the disaccharide repeat to the growing polymer chain. The chondroitin backbone-synthesizing enzyme of E. coli K4 has been enzymatically characterized (26Lidholt K. Fjelstad M. J. Biol. Chem. 1997; 272: 2682-2687Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), but the genes encoding the relevant glycosyltransferases have not yet been identified. Many P. multocida isolates produce GAG or GAG-like molecules as assessed by enzymatic degradation and removal of the capsule of living bacterial cells (27Rimler R.B. Vet. Rec. 1994; 134: 191-192Crossref PubMed Scopus (53) Google Scholar, 28Rimler R.B. Register K.B. Magyar T. Ackermann M.R. Vet. Microbiol. 1995; 47: 287-294Crossref PubMed Scopus (13) Google Scholar). Type A P. multocida, the major fowl cholera pathogen, makes a capsule that is sensitive to hyaluronidase. Subsequent NMR structural studies of capsular extracts confirmed that HA was the major polysaccharide present (29Rosner H. Grimmecke H.D. Knirel Y.A. Shashkov A.S. Carbohydr. Res. 1992; 223: 329-333Crossref PubMed Scopus (36) Google Scholar). A specific HA-binding protein, aggrecan, also interacts with HA from Type AP. multocida (17DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Two other distinct P. multocida types, a swine pathogen, Type D, and a minor fowl cholera pathogen, Type F, produce polymers that are chondroitin or chondroitin-like based on the observation that their capsules are degraded by Flavobacterium chondroitin AC lyase (27Rimler R.B. Vet. Rec. 1994; 134: 191-192Crossref PubMed Scopus (53) Google Scholar, 28Rimler R.B. Register K.B. Magyar T. Ackermann M.R. Vet. Microbiol. 1995; 47: 287-294Crossref PubMed Scopus (13) Google Scholar). After enzymatic removal of the capsule, both types were more readily phagocytosed by neutrophils in vitro (28Rimler R.B. Register K.B. Magyar T. Ackermann M.R. Vet. Microbiol. 1995; 47: 287-294Crossref PubMed Scopus (13) Google Scholar). The capsule of Type D cells, but not Type F cells, is also reported to be degraded by heparinase III (27Rimler R.B. Vet. Rec. 1994; 134: 191-192Crossref PubMed Scopus (53) Google Scholar). In this report, we have analyzed the monosaccharide composition of the P. multocida Type F polysaccharide and used the DNA sequence information of the Type A HA biosynthesis locus (17DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) to obtain the homologous region from the Type F chromosome. We describe pmCS, the first chondroitin synthase to be identified and molecularly cloned from any source. DISCUSSIONWe verify that P. multocida Type F produces a chondroitin or chondroitin-like capsule. We have also molecularly cloned the glycosyltransferase responsible for polymerizing the chondroitin backbone component of the capsular polysaccharide. This enzyme seems to be a close homolog of the pmHAS enzyme. Recently, we determined that the pmHAS enzyme contains two active sites in a single polypeptide by generating mutants that transfer only GlcUA or only GlcNAc (33Jing, W., and DeAngelis, P. L. (2000)Glycobiology, in pressGoogle Scholar). Interestingly, mixing the two different mutant proteins reconstituted the HA synthase activity. We hypothesized that one domain, called A1, is responsible for GlcNAc transfer, and the other domain, called A2, is responsible for GlcUA transfer (33Jing, W., and DeAngelis, P. L. (2000)Glycobiology, in pressGoogle Scholar). Comparison of the pmHAS and the pmCS sequences reveals that the majority of the sequence differences exist in the vicinity of the Al domain. The pmCS enzyme transfers a different hexosamine, GalNAc; thus, this observation is consistent with our proposed two-domain structure for pmHAS. Future experiments will be directed at identifying which residues of the Al domain are responsible for recognition and/or transfer of GlcNAc or GalNAc sugars.The pmHAS protein was also hypothesized to interact with a putative polysaccharide transporter system or a membrane-bound partner via its carboxyl terminus because deletion of residues 704 to 972 from the native-length enzyme resulted in the formation of a soluble enzyme (33Jing, W., and DeAngelis, P. L. (2000)Glycobiology, in pressGoogle Scholar). However, no substantial membrane-associated or hydrophobic regions are predicted to reside in this sequence. Because pmHAS and pmCS are highly homologous in this region, which is not essential for their glycosyltransferase activities, it is quite likely that the carboxyl terminus contains domains or motifs required for interacting with the polysaccharide transport machinery or a membrane-bound partnerin vivo.The evolutionary relationship between Type A and Type F P. multocida strains has not yet been delineated. Both organisms are widespread causative agents of fowl cholera, but many more isolates from diseased birds in North America are Type A microbes with HA capsules (30Rimler R.B. Rhoades K.R. J. Clin. Microbiol. 1987; 25: 615-618Crossref PubMed Google Scholar). It is likely that the progenitor of the two distinct capsular types had either a chondroitin synthase-like or a HAS-like gene. The specificity of this ancestral enzyme may have changed after a few mutations, resulting in the appearance of another capsular type. Apparently, the sugar transfer specificity is rather selective, since neither recombinant pmCS nor pmHAS misincorporate the inappropriate hexosamine into polymer in vitro. Some Gram-negative bacteria (e.g. E. coli) possess an UDP-GlcNAc/UDP-GalNAc epimerase; therefore, the hexosamine precursor either for HA or for chondroitin could have been available for polysaccharide biosynthesis without the need to gain an auxiliary metabolic enzyme simultaneously. Typically the UDP-glucose dehydrogenase, the enzyme that forms the UDP-GlcUA precursor, is found in Gram-negative bacteria only if the microbe possesses a GlcUA-containing polymer or glycoconjugate. In both Type A and Type FP. multocida, the UDP-glucose dehydrogenase gene is directly downstream of the GAG synthase (17DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar).The relationship between the bacterial chondroitin synthase and the putative mammalian counterpart is unclear. No similar vertebrate proteins are deposited in the data base as yet. Both bacterial pmCS and the vertebrate chondroitin synthase utilize UDP-sugars to extend acceptor carbohydrates in vitro. In most cases, the mammalian enzyme in cell-free extracts, however, does not produce long chondroitin chains, and only the half-reaction (e.g. adding a single GlcUA to a GalNAc-terminated oligosaccharide or vice versa) is readily observed in vitro (23Sugumaran G. Katsman M. Sunthankar P. Drake R.R. J. Biol. Chem. 1997; 272: 14399-14403Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 24Tsuchida K. Lind T. Kitagawa H. Lindahl U. Sugahara K. Lidholt K. Eur. J. Biochem. 1999; 264: 461-467Crossref PubMed Scopus (17) Google Scholar). In vertebrate tissues, other enzymes modify chondroitin extensively by sulfation and/or epimerization (1Roden L. Lennarz W.J. The Biochemistry of Glycoproteins and Proteoglycans. Plenum Publishing Corp., New York1980: 267-371Crossref Google Scholar). Hopefully, the discovery and the characterization of pmCS will assist the further study of the rather recalcitrant mammalian chondroitin synthase enzymes. We verify that P. multocida Type F produces a chondroitin or chondroitin-like capsule. We have also molecularly cloned the glycosyltransferase responsible for polymerizing the chondroitin backbone component of the capsular polysaccharide. This enzyme seems to be a close homolog of the pmHAS enzyme. Recently, we determined that the pmHAS enzyme contains two active sites in a single polypeptide by generating mutants that transfer only GlcUA or only GlcNAc (33Jing, W., and DeAngelis, P. L. (2000)Glycobiology, in pressGoogle Scholar). Interestingly, mixing the two different mutant proteins reconstituted the HA synthase activity. We hypothesized that one domain, called A1, is responsible for GlcNAc transfer, and the other domain, called A2, is responsible for GlcUA transfer (33Jing, W., and DeAngelis, P. L. (2000)Glycobiology, in pressGoogle Scholar). Comparison of the pmHAS and the pmCS sequences reveals that the majority of the sequence differences exist in the vicinity of the Al domain. The pmCS enzyme transfers a different hexosamine, GalNAc; thus, this observation is consistent with our proposed two-domain structure for pmHAS. Future experiments will be directed at identifying which residues of the Al domain are responsible for recognition and/or transfer of GlcNAc or GalNAc sugars. The pmHAS protein was also hypothesized to interact with a putative polysaccharide transporter system or a membrane-bound partner via its carboxyl terminus because deletion of residues 704 to 972 from the native-length enzyme resulted in the formation of a soluble enzyme (33Jing, W., and DeAngelis, P. L. (2000)Glycobiology, in pressGoogle Scholar). However, no substantial membrane-associated or hydrophobic regions are predicted to reside in this sequence. Because pmHAS and pmCS are highly homologous in this region, which is not essential for their glycosyltransferase activities, it is quite likely that the carboxyl terminus contains domains or motifs required for interacting with the polysaccharide transport machinery or a membrane-bound partnerin vivo. The evolutionary relationship between Type A and Type F P. multocida strains has not yet been delineated. Both organisms are widespread causative agents of fowl cholera, but many more isolates from diseased birds in North America are Type A microbes with HA capsules (30Rimler R.B. Rhoades K.R. J. Clin. Microbiol. 1987; 25: 615-618Crossref PubMed Google Scholar). It is likely that the progenitor of the two distinct capsular types had either a chondroitin synthase-like or a HAS-like gene. The specificity of this ancestral enzyme may have changed after a few mutations, resulting in the appearance of another capsular type. Apparently, the sugar transfer specificity is rather selective, since neither recombinant pmCS nor pmHAS misincorporate the inappropriate hexosamine into polymer in vitro. Some Gram-negative bacteria (e.g. E. coli) possess an UDP-GlcNAc/UDP-GalNAc epimerase; therefore, the hexosamine precursor either for HA or for chondroitin could have been available for polysaccharide biosynthesis without the need to gain an auxiliary metabolic enzyme simultaneously. Typically the UDP-glucose dehydrogenase, the enzyme that forms the UDP-GlcUA precursor, is found in Gram-negative bacteria only if the microbe possesses a GlcUA-containing polymer or glycoconjugate. In both Type A and Type FP. multocida, the UDP-glucose dehydrogenase gene is directly downstream of the GAG synthase (17DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The relationship between the bacterial chondroitin synthase and the putative mammalian counterpart is unclear. No similar vertebrate proteins are deposited in the data base as yet. Both bacterial pmCS and the vertebrate chondroitin synthase utilize UDP-sugars to extend acceptor carbohydrates in vitro. In most cases, the mammalian enzyme in cell-free extracts, however, does not produce long chondroitin chains, and only the half-reaction (e.g. adding a single GlcUA to a GalNAc-terminated oligosaccharide or vice versa) is readily observed in vitro (23Sugumaran G. Katsman M. Sunthankar P. Drake R.R. J. Biol. Chem. 1997; 272: 14399-14403Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 24Tsuchida K. Lind T. Kitagawa H. Lindahl U. Sugahara K. Lidholt K. Eur. J. Biochem. 1999; 264: 461-467Crossref PubMed Scopus (17) Google Scholar). In vertebrate tissues, other enzymes modify chondroitin extensively by sulfation and/or epimerization (1Roden L. Lennarz W.J. The Biochemistry of Glycoproteins and Proteoglycans. Plenum Publishing Corp., New York1980: 267-371Crossref Google Scholar). Hopefully, the discovery and the characterization of pmCS will assist the further study of the rather recalcitrant mammalian chondroitin synthase enzymes. Note Added in ProofRecently it was reported (Nadanka, S., Kitagawa, H.., Goto, F., Tamura, J., Neumann, K. W., Ogawa, T., and Sugahara, K. (1999) Biochem. J. 340,353–357) that a partially purified chondroitin synthase preparation from a melanoma cell line catalyzed the repetitive addition of chondroitin disaccharide repeats in vitroto the linkage tetrasaccharide of an α-thrombomodulin acceptor but not to the free tetrasaccharide, suggesting a role for the core protein in mammalian GAG biosynthesis. Recently it was reported (Nadanka, S., Kitagawa, H.., Goto, F., Tamura, J., Neumann, K. W., Ogawa, T., and Sugahara, K. (1999) Biochem. J. 340,353–357) that a partially purified chondroitin synthase preparation from a melanoma cell line catalyzed the repetitive addition of chondroitin disaccharide repeats in vitroto the linkage tetrasaccharide of an α-thrombomodulin acceptor but not to the free tetrasaccharide, suggesting a role for the core protein in mammalian GAG biosynthesis. We thank Dr. Ann Mary Achyuthan and Wei Jing for technical assistance, Drs. Anne Leppanen and Richard Cummings for assistance with and the use of the Dionex chromatograph, Dr. Richard Rimler for providing the Type F P. multocida strains, Dr. Geetha Sugumaran for supplying the chondroitin sugar acceptor, and Drs. Robert Linhardt, Jeremiah Silbert, and Paul Weigel for helpful suggestions.
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