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Acceptor Specificity of the Pasteurella Hyaluronan and Chondroitin Synthases and Production of Chimeric Glycosaminoglycans

2006; Elsevier BV; Volume: 282; Issue: 1 Linguagem: Inglês

10.1074/jbc.m607569200

ISSN

1083-351X

Autores

Breca S. Tracy, Fikri Y. Avci, Robert J. Linhardt, Paul L. DeAngelis,

Tópico(s)

Microbial infections and disease research

Resumo

The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (∼102-4 sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes. The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (∼102-4 sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes. Glycosaminoglycans (GAGs), 2The abbreviations used are: GAG, glycosaminoglycan; PmHAS, P. multocida hyaluronan synthase; HA, hyaluronan, hyaluronate, or hyaluronic acid; C, chondroitin; PmCS, P. multocida chondroitin synthase; HexNAc, N-acetylhexosamine; SEC, size exclusion chromatography; SAX-HPLC, strong anion exchange-high performance liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; Me2SO, dimethyl sulfoxide; Hep, heparin; MALLS, multiangle laser light scattering. 2The abbreviations used are: GAG, glycosaminoglycan; PmHAS, P. multocida hyaluronan synthase; HA, hyaluronan, hyaluronate, or hyaluronic acid; C, chondroitin; PmCS, P. multocida chondroitin synthase; HexNAc, N-acetylhexosamine; SEC, size exclusion chromatography; SAX-HPLC, strong anion exchange-high performance liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; Me2SO, dimethyl sulfoxide; Hep, heparin; MALLS, multiangle laser light scattering. polysaccharides containing a hexosamine as part of their repeat unit, serve essential biological functions in vertebrates, including adhesion, modulation of motility and proliferation, and coagulation (1Capila I. Linhardt R.J. Angew. Chem. Int. Ed. Engl. 2002; 41: 391-412Crossref PubMed Scopus (1514) Google Scholar, 2Sugahara K. Kitagawa H. IUBMB Life. 2002; 54: 163-175Crossref PubMed Scopus (214) Google Scholar, 3Silbert J.E. Sugumaran G. IUBMB Life. 2002; 54: 177-186Crossref PubMed Scopus (246) Google Scholar, 4Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1003) Google Scholar, 5Oohira A. Matsui F. Tokita Y. Yamauchi S. Aono S. Arch. Biochem. Biophys. 2000; 374: 24-34Crossref PubMed Scopus (151) Google Scholar, 6Fraser J.R. Laurent T.C. Laurent U.B. J. Intern. Med. 1997; 242: 27-33Crossref PubMed Scopus (1431) Google Scholar, 7Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (598) Google Scholar, 8Noble P.W. Matrix Biol. 2002; 21: 25-29Crossref PubMed Scopus (458) Google Scholar). Certain pathogenic bacteria camouflage themselves with GAGs to increase virulence and enhance infection in their animal hosts. These capsular polysaccharides serve as molecular camouflage as well as a means to potentially hijack host functions (9DeAngelis P.L. Glycobiology. 2002; 12: R9-R16Crossref PubMed Google Scholar). The backbones of the vertebrate GAGs, HA (-β1,4-GlcUA-β1,3-Glc-NAc-), chondroitin sulfate (-β1,4-GlcUA-β1,3-GalNAc-), heparan sulfate (α1,4-GlcUA-β1,4-GlcNAc-), heparin (-α1,4-IdoUA-α 1,4-GlcNAc-), and keratan sulfate (-β1,3-Gal-β1,4-GlcNAc-), are sulfated except for HA. The bacterial GAG polymers are not known to be sulfated. The enzymes involved in the biosynthesis of all the GAG backbones, except for keratan, have been molecularly cloned.Certain bacterial enzymes, including the Gram-negative Pasteurella multocida type A hyaluronan synthase, PmHAS, and the type F chondroitin synthase, PmCS, are particularly amenable to study because of their two active center architecture (10Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (95) Google Scholar, 11Jing W. DeAngelis P.L. Glycobiology. 2003; 13: R661-R671Crossref PubMed Scopus (63) Google Scholar), their ability to polymerize long chains in vitro (12Jing W. DeAngelis P.L. J. Biol. Chem. 2004; 279: 42345-42349Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), and their ability to elongate exogenously supplied acceptor oligosaccharides in vitro (13DeAngelis P.L. J. Biol. Chem. 1999; 274: 26557-26562Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 14DeAngelis P.L. Oatman L.C. Gay D.F. J. Biol. Chem. 2003; 278: 35199-35203Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The Escherichia coli K4 chondroitin polymerase, KfoC (15Ninomiya T. Sugiura N. Tawada A. Sugimoto K. Watanabe H. Kimata K. J. Biol. Chem. 2002; 277: 21567-21575Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), is ∼60% identical to the PmHAS and PmCS; therefore, this system probably operates in a similar fashion. The streptococcal and animal HA synthase enzymes, however, do not readily utilize exogenously supplied acceptors and are more difficult to study (13DeAngelis P.L. J. Biol. Chem. 1999; 274: 26557-26562Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Heparosan synthases from Pasteurella type D, PmHS1 (16DeAngelis P.L. White C.L. J. Biol. Chem. 2002; 277: 7209-7213Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), and Pasteurella types A, D, and F, PmHS2 (17DeAngelis P.L. White C.L. J. Bacteriol. 2004; 186: 8529-8532Crossref PubMed Scopus (41) Google Scholar), promise to be interesting experimental models as well, but their overall primary structure differs substantially from PmHAS and PmCS.The PmHAS and PmCS enzymes each possess independent hexosamine and glucuronic acid transfer sites as assessed by mutating various sequence motifs (10Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (95) Google Scholar, 11Jing W. DeAngelis P.L. Glycobiology. 2003; 13: R661-R671Crossref PubMed Scopus (63) Google Scholar). Kinetic studies suggest that there is a separate acceptor binding pocket for each of these glycosyltransferase activities that apparently interacts with three or four saccharide units of the nascent HA chain (18Williams K.J. Halkes K.M. Kamerling J.P. DeAngelis P.L. J. Biol. Chem. 2006; 281: 5391-5397Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The sugar nucleotide specificity of native sequence enzymes has been evaluated with naturally occurring UDP-sugar donors; only the authentic monosaccharide molecules are transferred efficiently by the native enzymes (e.g. C-4 epimers will not substitute). All known Pasteurella enzymes add single sugars in a repetitive stepwise fashion to the nonreducing terminus of their cognate acceptor oligosaccharides (13DeAngelis P.L. J. Biol. Chem. 1999; 274: 26557-26562Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 17DeAngelis P.L. White C.L. J. Bacteriol. 2004; 186: 8529-8532Crossref PubMed Scopus (41) Google Scholar, 19DeAngelis P.L. Padgett-McCue A.J. J. Biol. Chem. 2000; 275: 24124-24129Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Here we report that PmHAS and PmCS will elongate a range of acceptor molecules in addition to their cognate sugars; this finding sheds light on the nature of the synthase active sites as well as significantly expanding the potential repertoire of oligosaccharide and polysaccharide targets that may now be synthesized.EXPERIMENTAL PROCEDURESNatural GAGs—All GAG polysaccharides, except for HA, are heterogeneous in nature with respect to composition and sulfation pattern. It is currently impossible to isolate GAG polysaccharides from animal sources in one pure isomeric form. Therefore, throughout the text all polysaccharides are defined with respect to their source and are given a simple nomenclature code (Table 1).TABLE 1Carbohydrate nomenclature, code and structure Due to the presence of multiple sulfation isomers within any one commercial chondroitin sulfate preparation (e.g. CSA and CSB, etc.), the simple identification code designates the animal source of polymer (the source is underlined). The manufacturer assay values are presented for polysaccharides. The oligosaccharide structures were verified by mass spectrometry and/or NMR. R indicates functional group on hexosamine.CodeOligosaccharide (source)StructureHA4Hyaluronan tetrasaccharide (S. zooepidemicus)(β1,3-GlcNAc-β1,4-GlcUA)2CSbt3Chondroitin 4-sulfate trisaccharide (bovine trachea)(GalNAc-4SO4)-β1,4-GlcUA-β1,3-(GalNAc-4SO4)CSs3Chondroitin 6-sulfate trisaccharide (shark cartilage)(GalNAc-6SO4)-β1,4-GlcUA-β1,3-(GalNAc-6SO4)CSs5Chondroitin 6-sulfate pentasaccharide (shark cartilage)(Cs3)-β1,4-GlcUA-β1,3-(GalNAc-6SO4)CSpII3C-5-epimerized chondroitin 4-sulfate trisaccharide (porcine intestinal mucosa, Prep II; Celsus)(GalNAc-4SO4)-β1,4-IdoUA-α1,3-(GalNAc-4SO4)CSpII5C-5-epimerized chondroitin 4-sulfate pentasaccharide (porcine intestinal mucosa, Prep II; Celsus)(CSpII3)-β1,4-IdoUA- α1,3-(GalNAc-4SO4)dCSpII3Desulfated C-5-epimerized chondroitin sulfate trisaccharide (porcine intestinal mucosa, Prep II; Celsus)GalNAc-β1,4-IdoUA-α1,3-GalNAcHep5Synthetic heparin pentasaccharide (Arixtra®)(GlcNR′-6OR)-α1,4-(GlcUA)-β1,4-(GlcNR′-3OR-6OR)-α1,4-(Ido UA-2OR)-α1,4-(GlcNR′-6OR)) methyl glycoside, where R, R′ = SO3He5Heparosan pentasaccharide (E. coli K5)GlcNAc-(α1,4-GlcUA-β1,4-GlcNAc)2CodePolysaccharide (source)StructureHAHyaluronic acid (S. zooepidemicus)(β1,3-GlcNAc-β1,4-GlcUA)∼200CUnsulfated chondroitin (Pasteurella type F)(β1,3-GalNAc-β1,4-GlcUA)∼40CSbtChondroitin sulfate (bovine trachea)(β1,3-(GalNAc-4SO4 or -6SO4)-β1,4-GlcUA)∼50, where 70% CSA = 4-SO4, 30% CSC = 6-SO4CSpIC-5-epimerized chondroitin sulfate (porcine intestinal mucosa, Prep I; Sigma)(β1,3-(GalNAc-4SO4)-α1,4-IdoUA)∼50, where 90% CSA = 4-SO4; ? % epimerizedCSpIIC-5-epimerized chondroitin 4-sulfate (porcine intestinal mucosa, Prep II; Celsus)(β1,3-(GalNAc-4SO4)-α1,4-IdoUA)∼60, ∼95% epimerizeddCSpIIDesulfated C-5-epimerized chondroitin sulfate (porcine intestinal mucosa, Prep II; Celsus)(β1,3-GalNAc-α1,4-IdoUA)∼25, ∼95% epimerizedCSsChondroitin sulfate (shark cartilage)(β1,3-(GalNAc-4SO4 or -6SO4)-β1,4-GlcUA)∼130, where 90% CSC = 6-SO4, 10% CSA = 4-SO4CSsfChondroitin sulfate (shark fin)((β1,3-GalNAc-6SO4)-(β1,4-GlcUA/IdoUA-2SO4))∼100 aThe major disaccharide unit of the polysaccharide. Percentages for purity and/or contaminants were not assessedCSqChondroitin sulfate (squid cartilage)((β1,3-GalNAc-4,6-diSO4)-β1,4-GlcUA/IdoUA)∼350aThe major disaccharide unit of the polysaccharide. Percentages for purity and/or contaminants were not assessedHepHeparin (porcine intestinal mucosa)(α(β)1,4-GlcNAc-α1,4-IdoUA/GlcUA)∼45aThe major disaccharide unit of the polysaccharide. Percentages for purity and/or contaminants were not assessed,bMolecular weight was based on an estimated typical literature value of ∼18 kDa with various O- and N-sulfation patternsHHeparosan (P. multocida type D)(β1,4-GlcNAc-α1,4-GlcUA)∼500a The major disaccharide unit of the polysaccharide. Percentages for purity and/or contaminants were not assessedb Molecular weight was based on an estimated typical literature value of ∼18 kDa Open table in a new tab A variety of GAGs were obtained from Sigma, including Streptococcus zooepidemicus hyaluronan (HA), bovine trachea chondroitin sulfate (CSbt) (sold as "chondroitin sulfate A"; mainly composed of chondroitin 4-sulfate), C-5 epimerized porcine intestinal mucosa chondroitin sulfate (CSpI) (also referred to as dermatan sulfate and sold as "chondroitin sulfate B"), shark cartilage chondroitin sulfate (CSs) (sold as "chondroitin sulfate C"; mainly composed of chondroitin 6-sulfate), and porcine intestinal heparin (Hep). Shark fin chondroitin sulfate (CSsf) (sold as "chondroitin sulfate D") and squid cartilage chondroitin sulfate (CSq) (sold as "chondroitin sulfate E") were obtained from Associates of Cape Cod/Seikagaku America (Falmouth, MA). In addition, a highly C-5 epimerized (>95% 4S-GalNAc-IdoUA repeats) chondroitin sulfate (CSpII; a high purity dermatan sulfate from porcine intestinal mucosa similar to CSpI above, but better characterized) and heparan sulfate (porcine intestinal mucosa) were obtained from Celsus Laboratories (Cincinnati, OH). Unsulfated chondroitin polysaccharide (C) and unsulfated heparosan polysaccharide was prepared from cultures of either type F or type D P. multocida, respectively (20DeAngelis P.L. Gunay N.S. Toida T. Mao W.J. Linhardt R.J. Carbohydr. Res. 2002; 337: 1547-1552Crossref PubMed Scopus (86) Google Scholar). The degree of polymerization of the HA and the unsulfated chondroitin was reduced to ∼80 and ∼20 kDa, respectively, by autohydrolysis (121 °C, 20 p.s.i., 20 min) to be more comparable with the smaller molecular weight chondroitin sulfates (∼15-40 kDa) and heparin (∼17-19 kDa). In addition, 80-kDa monodisperse HA was prepared by synchronized stoichiometrically controlled chemoenzymatic reactions (12Jing W. DeAngelis P.L. J. Biol. Chem. 2004; 279: 42345-42349Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar).To avoid the heterogeneity problem intrinsic to natural GAG polysaccharides, a series of defined oligosaccharides of known structure and length (Table 1) was prepared as described below and outlined in Table 2.TABLE 2Oligosaccharide derivative preparation strategy The various polysaccharides were cleaved by GAG digestive enzymes (HAase, testicular hyaluronidase; ABCase, chondroitin ABC lyase; HepIIIase, heparin lyase III) and/or desulfated by solvolysis. Each oligosaccharide was purified by gel filtration and/or anion exchange chromatography (chrom).HACSbtCSpIICSsHeparosan↓ HAase↓ ABCase↓ H+/MeOH↓ ABCase↓ ABCase↓ HepIIIase↓ chrom↓ Hg2+↓ NaOH↓ Hg2+↓ Hg2+↓ Hg2+↓ chrom↓ chrom↓ chrom↓ chromHA4CSbt3dCSpIICSpII3 CSpII5CSs3 CSs5He5↓ H+/MeOH↓ NaOHdCSpII3 Open table in a new tab Hyaluronic Acid Tetrasaccharide (HA4)—The tetrasaccharide HA4 was generated by exhaustive degradation of streptococcal HA polymer with ovine testicular hyaluronidase (type V; Sigma) and purified by extensive chloroform extraction, ultrafiltration, and SEC on Bio-Gel P2 resin (Bio-Rad) (14DeAngelis P.L. Oatman L.C. Gay D.F. J. Biol. Chem. 2003; 278: 35199-35203Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).Chondroitin 4-Sulfate Trisaccharide (CSbt3)—Chondroitin 4-sulfate from bovine trachea, CSbt (1.0 g), was treated with chondroitin ABC lyase (EC 4.2.2.4, Proteus vulgaris; Sigma; 1 unit) in 50 mm Tris-HCl/sodium acetate buffer at 37 °C to obtain a mixture of unsaturated oligosaccharides. The resulting oligosaccharide products were fractionated on a Bio-Gel P6 (Bio-Rad) column eluted with 100 mm sodium chloride. Sized oligosaccharides were then desalted by SEC on a Bio-Gel P2 column. Charge separation of the tetrasaccharide fraction was carried out by semi-preparative strong anion exchange-high performance liquid chromatography (SAX-HPLC) (21Yang H.O. Gunay N.S. Toida T. Kuberan B. Yu G. Kim Y.S. Linhardt R.J. Glycobiology. 2000; 10: 1033-1039Crossref PubMed Scopus (61) Google Scholar). The major peaks were pooled, lyophilized, and desalted on a Bio-Gel P2 column. The unsaturated chondroitin sulfate tetrasaccharide containing two 4-O-sulfonated GlcNAc residues (1 mg) was dissolved in distilled water (1 mg/ml). To remove the nonreducing terminal unsaturated GlcUA residue, equal volumes of the oligosaccharide solution and mercuric acetate reagent (35 mm, adjusted to pH 5 using acetic acid in distilled water) were added and stirred for 15 min at room temperature. The reaction mixture was then passed through a pre-washed Dowex 50W-X8 H+ column, neutralized with saturated sodium bicarbonate solution, and then applied to a Bio-Gel P2 column to obtain the pure trisaccharide CSb3. The structure of this trisaccharide was confirmed by ESI-MS analysis (Table 3) (21Yang H.O. Gunay N.S. Toida T. Kuberan B. Yu G. Kim Y.S. Linhardt R.J. Glycobiology. 2000; 10: 1033-1039Crossref PubMed Scopus (61) Google Scholar).TABLE 3Electrospray ionization mass spectral data for chondroitin sulfate derived oligosaccharidesCSbt3, CSs3, CSpII3CSs5, CSpII5dCSpII3Parent ionaHighest abundance ion is shown[M – 3Na + H]2– = 379.1 (calculated, 379.1)[M – 2Na]2– = 641.5 (calculated, 641.5)[M – Na]– = 599.1 (calculated, 599.2)Molecular ion[M – Na]– = 803.0 (calculated, 803.1)[M – Na]– = 1306.0 (calculated, 1306.1)[M – Na]– = 599.1 (calculated, 599.2)Mass826.11329.1622.2a Highest abundance ion is shown Open table in a new tab Chondroitin 6-Sulfate Trisaccharide (CSs3) and Pentasaccharide (CSs5)—Chondroitin 6-sulfate from shark cartilage, CSs (50 mg), was treated with chondroitin ABC lyase (0.05 units) as above. The resulting oligosaccharide products were fractionated on a Bio-Gel P6 column, and each oligosaccharide was desalted on a Bio-Gel P2 column. The unsaturated chondroitin tetrasaccharide (1 mg) and hexasaccharide (1 mg) were treated with mercuric acetate reagent as described earlier to obtain the trisaccharide CSs3 and pentasaccharide CSs5. The structures of these products were determined by ESI-MS analysis (Table 3).C-5-epimerized Chondroitin 4-Sulfate Trisaccharide and Pentasaccharide (CSpII3 and CSpII5)—C-5-epimerized chondroitin sulfate from porcine intestinal mucosa, CSpII (Celsus; 10 g), was treated with chondroitin ABC lyase (20 units) as above. The resulting oligosaccharide products were fractionated on a Bio-Gel P6 column, and each oligosaccharide was desalted by SEC on a Bio-Gel P2 column. Charge separation of sized oligosaccharide fractions was carried out by SAX-HPLC. The major peaks were pooled, lyophilized, and desalted on a Bio-Gel P2 column. The unsaturated C-5-epimerized chondroitin sulfate tetrasaccharide (6 mg) and hexasaccharide (2 mg) were treated with mercuric acetate reagent to obtain the pure C-5-epimerized chondroitin sulfate trisaccharide, CSpII3, and pentasaccharide, CSpII5. The structures of these oligosaccharides were confirmed by 1H NMR and ESI-MS analysis (Table 3) (21Yang H.O. Gunay N.S. Toida T. Kuberan B. Yu G. Kim Y.S. Linhardt R.J. Glycobiology. 2000; 10: 1033-1039Crossref PubMed Scopus (61) Google Scholar).Desulfated C-5-epimerized Chondroitin Sulfate (dCSpII) Polysaccharide—The sodium salt of C-5-epimerized chondroitin sulfate (high purity dermatan sulfate from porcine intestinal mucosa; Celsus), CSpII (250 mg), was treated with 250 ml of acidic methanol (1.25 ml of acetyl chloride in 250 ml of methanol) for 3 days at room temperature. The recovered solid product (methyl ester of desulfated epimerized chondroitin sulfate) was treated with 10 ml of 0.1 m sodium hydroxide for 24 h at room temperature to obtain free carboxylate, O-desulfated epimerized chondroitin sulfate dCSpII (22Avci F.Y. Toida T. Linhardt R.J. Carbohydr. Res. 2003; 338: 2101-2104Crossref PubMed Scopus (7) Google Scholar). This structure was confirmed by 1H NMR spectroscopy (23Sudo M. Sato K. Chaidedgumjorn A. Toyoda H. Toida T. Imanari T. Anal. Biochem. 2001; 297: 42-51Crossref PubMed Scopus (36) Google Scholar).Desulfated C-5-epimerized Chondroitin Sulfate Trisaccharide (dCSpII3)—The dCSpII polymer (100 mg) was treated with chondroitin ABC lyase (1 unit) as described earlier. The resulting oligosaccharide products were fractionated on a Bio-Gel P6 column, and each oligosaccharide was desalted on a Bio-Gel P2 column. Charge separation of sized oligosaccharide fractions was carried out by SAX-HPLC. The major peaks were pooled, lyophilized, and desalted on a Bio-Gel P2 column. C-5-epimerized chondroitin tetrasaccharide without sulfate groups at Glc-NAc residues (1 mg) was treated with mercuric acetate reagent to obtain the trisaccharide. The structure of this trisaccharide was confirmed by ESI-MS analysis (Table 3).Synthetic Heparin Pentasaccharide (Hep5)—The synthetic analog of the anti-thrombin III binding pentasaccharide, fondaparinux sodium (Arixtra®), was obtained from the pharmacy. The contents of 10 syringes (25 mg in 5 ml of isotonic saline total) were collected and dialyzed (1000-Da cutoff) against water for 2 days at 4 °C and lyophilized to obtain the pentasaccharide Hep5 in quantitative yield.Heparosan Pentasaccharide (He5)—Heparosan polysaccharide was isolated from E. coli K5 and purified according to the published procedure (24Vann W.F. Schmidt M.A. Jann B. Jann K. Eur. J. Biochem. 1981; 116: 359-364Crossref PubMed Scopus (210) Google Scholar). Using heparin lyase III from Flavobacterium heparinum, a controlled partial depolymerization was performed. The oligosaccharide mixture was size-fractionated using a Bio-Gel P6 column. Each fraction was concentrated and desalted on a Bio-Gel P2 SEC column. The unsaturated hexasaccharide was treated with mercuric acetate reagent to obtain heparosan pentasaccharide He5. Analysis by ESI-MS confirmed the structure of this product (data not shown).Sugar Quantitation—All carbohydrates were assayed by carbazole method (25Bitter T. Muir H.M. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5132) Google Scholar) for uronic acid using GlcUA as a standard.Synthase Purification—The recombinant E. coli cells expressing the truncated, soluble dual-action catalysts PmHAS-(1-703) or PmCS-(46-695) were extracted with the permeabilization agent octylthioglucoside (1% w/v) in 1 m ethylene glycol, 50 mm Tris, pH 7.2, containing protease inhibitors (14DeAngelis P.L. Oatman L.C. Gay D.F. J. Biol. Chem. 2003; 278: 35199-35203Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The extract was clarified by centrifugation and applied to a pseudoaffinity column (Tosoh Toyopearl AF-Red-650) equilibrated in the same buffer except 50 mm Hepes was substituted for Tris. The protein eluted with a NaCl gradient (0-1.5 m NaCl for 120 min), and the enzyme peak was determined by Coomassie Blue staining of SDS-polyacrylamide gels. The pooled enzyme (∼95% pure PmHAS or PmCS) was concentrated with an ultrafiltration device (Amicon Ultra 15 ml, 50-kDa cutoff). The protein was quantitated by the Bradford assay with a bovine serum albumin standard (Pierce).Synthase Assays and Acceptor Utilization Tests—PmHAS or PmCS-catalyzed polymerization was measured with radiolabeled sugar incorporation assays. UDP-GlcUA and UDP-Hex-NAc polymerization was recorded by monitoring incorporation of UDP-[3H]GlcUA as denoted in Equation 1, acceptor+nUDP-[H3]GlcUA+nUDP-HexNAc→2nUDP+([H3]GlcUA-HexNAc)n-acceptor(Eq. 1) Typically, 25-μl reactions containing ∼1 μg (∼13 pmol) of enzyme, PmHAS-(1-703) or PmCS-(46-695), and ∼0.01 nmol to 3.75 nmol of a given acceptor were employed. A "no acceptor" control was used for all analyses; this background value corresponding to de novo polymer initiation was subtracted from the value obtained from the assay with acceptor. Assays with an HA polymer acceptor were employed in every data set to allow for normalization. Polymerization reactions contained 50 mm Tris, pH 7.2, 1 m ethylene glycol, 2 mm MnCl2, 0.05 mm UDP-[3H]GlcUA (1 μCi; PerkinElmer Life Sciences), and 0.2 mm UDP-HexNAc (UDP-GlcNAc for PmHAS or UDP-Gal-NAc for PmCS) and were incubated at 30 °C for either 3 min (polysaccharide acceptors) or 30 min (oligosaccharide acceptors). Reactions were stopped by placing on ice and adding SDS (2% final w/v). Descending paper chromatography was used to separate the unincorporated radiolabel from the elongated acceptors (10Jing W. DeAngelis P.L. Glycobiology. 2000; 10: 883-889Crossref PubMed Scopus (95) Google Scholar). The assays were performed in duplicate and were linear with respect to enzyme concentration and time; less than 5% UDP-sugar substrate was consumed.Single sugar additions were also monitored by reverse phase HPLC ESI-MS (26Thanawiroon C. Linhardt R.J. J. Chromatogr. A. 2003; 1014: 215-223Crossref PubMed Scopus (64) Google Scholar). Briefly, similar reaction conditions were utilized as described above, except only single unlabeled UDP-sugar (2-6 mm final) was employed. The enzyme adds a monosaccharide unit onto the nonreducing end of various acceptors as shown in Equation 2 or 3. acceptor+UDP-GlcUA→UDP+(GlcUA-acceptor)(Eq. 2) acceptor+UDP-HexNAc→UDP+(HexNAc-acceptor)(Eq. 3) Chimeric GAG Synthesis—The purified enzymes PmHAS-(1-703) or PmCS-(46-695) (∼1 μg) were used to add unlabeled HA or chondroitin chains, respectively, to various GAG polysaccharide acceptors (∼8-225 μg). Reactions (25 μl) contained the same reaction buffer as above except that 2-4 mm UDP-GlcUA and 2-4 mm UDP-HexNAc (UDP-GlcNAc for PmHAS or UDP-GalNAc for PmCS) were utilized at 30 °C for varying times (see Equation 1, but without the 3H label).Size Analysis of Polysaccharides—Polymers were analyzed using 1-1.2% 1× TAE-agarose gels (30 V, 5 h, Stains-All detection) (27Lee H.G. Cowman M.K. Anal. Biochem. 1994; 219: 278-287Crossref PubMed Scopus (259) Google Scholar). The specific Streptomyces hyaluronate lyase from Sigma was employed to destroy and thus identify authentic HA chains. Defined HA molecular weight standards were from (Hyalose L.L.C., Oklahoma City, OK) (12Jing W. DeAngelis P.L. J. Biol. Chem. 2004; 279: 42345-42349Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Kilobase DNA standards were from Stratagene (La Jolla, CA).Analytical high performance SEC was performed with PLaquagel-OH 60, -OH 50, -OH 40 columns in tandem (15 μm, 7.5 × 300 mm, Polymer Laboratories Amherst, MA) eluted with 50 mm NaPO4, 150 mm NaCl, pH 7, at 0.4-0.5 ml/min. Multiangle laser light scattering (MALLS) detection was performed to quantify absolute molecular weights (12Jing W. DeAngelis P.L. J. Biol. Chem. 2004; 279: 42345-42349Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar).RESULTS AND DISCUSSIONThe finding that recombinant PmHAS, and later PmCS, was able to elongate certain small oligosaccharides, including HA4 and chondroitin sulfate hexasaccharides, respectively (13DeAngelis P.L. J. Biol. Chem. 1999; 274: 26557-26562Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 19DeAngelis P.L. Padgett-McCue A.J. J. Biol. Chem. 2000; 275: 24124-24129Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), led to a broader investigation of the potential range of acceptor usage (Tables 4 and 5). In contrast, the other distinct bacterial HA synthase enzymes from group A and C Streptococcus cannot be tested at this time because of their inability to elongate exogenously added acceptors.TABLE 4PmHAS and PmCS polysaccharide acceptor specificity Polymerization assays with each acceptor (at least three experiments in duplicate) were performed. The value of no acceptor control (∼300 – 600 dpm) was subtracted from each point. The averaged values for PmHAS or PmCS are presented. The ratio (nanomoles of test acceptor/nmol of HA standard) was employed to normalize to the HA signal (30,000 dpm for 0.02 nmol of HA), which was set to 100%. For all results, * = p < 0.05 and ** = p < 0.01 in a Student's one-tailed t test for comparison of a given test acceptor to the no acceptor control assay. ND indicates not determined. Both enzymes may utilize either HA or chondroitin polymers, bu

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