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Characterization of a Heparan Sulfate Octasaccharide That Binds to Herpes Simplex Virus Type 1 Glycoprotein D

2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês

10.1074/jbc.m202034200

1083-351X

Jian Liu, Zach Shriver, R. Marshall Pope, Suzanne C. Thorp, Michael B. Duncan, Ronald J. Copeland, Christina S. Raska, Keiichi Yoshida, Roselyn J. Eisenberg, Gary H. Cohen, Robert J. Linhardt, Ram Sasisekharan,

Carbohydrate Chemistry and Synthesis

Herpes simplex virus type 1 utilizes cell surface heparan sulfate as receptors to infect target cells. The unique heparan sulfate saccharide sequence offers the binding site for viral envelope proteins and plays critical roles in assisting viral infections. A specific 3-O-sulfated heparan sulfate is known to facilitate the entry of herpes simplex virus 1 into cells. The 3-O-sulfated heparan sulfate is generated by the heparan sulfate d-glucosaminyl-3-O-sulfotransferase isoform 3 (3-OST-3), and it provides binding sites for viral glycoprotein D (gD). Here, we report the purification and structural characterization of an oligosaccharide that binds to gD. The isolated gD-binding site is an octasaccharide, and has a binding affinity to gD around 18 μm, as determined by affinity coelectrophoresis. The octasaccharide was prepared and purified from a heparan sulfate oligosaccharide library that was modified by purified 3-OST-3 enzyme. The molecular mass of the isolated octasaccharide was determined using both nanoelectrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The results from the sequence analysis suggest that the structure of the octasaccharide is a heptasulfated octasaccharide. The proposed structure of the octasaccharide is ΔUA-GlcNS-IdoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S-GlcNH23S6S. Given that the binding of 3-O-sulfated heparan sulfate to gD can mediate viral entry, our results provide structural information about heparan sulfate-assisted viral entry. Herpes simplex virus type 1 utilizes cell surface heparan sulfate as receptors to infect target cells. The unique heparan sulfate saccharide sequence offers the binding site for viral envelope proteins and plays critical roles in assisting viral infections. A specific 3-O-sulfated heparan sulfate is known to facilitate the entry of herpes simplex virus 1 into cells. The 3-O-sulfated heparan sulfate is generated by the heparan sulfate d-glucosaminyl-3-O-sulfotransferase isoform 3 (3-OST-3), and it provides binding sites for viral glycoprotein D (gD). Here, we report the purification and structural characterization of an oligosaccharide that binds to gD. The isolated gD-binding site is an octasaccharide, and has a binding affinity to gD around 18 μm, as determined by affinity coelectrophoresis. The octasaccharide was prepared and purified from a heparan sulfate oligosaccharide library that was modified by purified 3-OST-3 enzyme. The molecular mass of the isolated octasaccharide was determined using both nanoelectrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The results from the sequence analysis suggest that the structure of the octasaccharide is a heptasulfated octasaccharide. The proposed structure of the octasaccharide is ΔUA-GlcNS-IdoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S-GlcNH23S6S. Given that the binding of 3-O-sulfated heparan sulfate to gD can mediate viral entry, our results provide structural information about heparan sulfate-assisted viral entry. heparan sulfate 3′-phosphoadenosine 5′-phosphosulfate matrix-assisted laser desorption/ionization mass spectrometry nano-electrospray ionization mass spectrometry herpes simplex virus type 1 gC, and gD, herpes envelope glycoprotein B, glycoprotein C, and glycoprotein D, respectively heparan sulfate d-glucosaminyl-3-O-sulfotransferase Δ4,5-unsaturated uronic acid D-glucuronic acid α-iduronic acid N-unsubstituted glucosamine molecular weight cut-off 2,5-anhydromannitol 2-(N-morpholino)ethanesulfonic acid high performance liquid chromatography Heparan sulfates (HS),1highly sulfated polysaccharides, are present on the surface of mammalian cells and in the extracellular matrix in large quantities. HS play critical roles in a variety of biological interactions, including assisting viral infection, regulating blood coagulation and embryonic development, suppressing tumor growth, and controlling the eating behavior of mice by interacting with specific regulatory proteins (1Liu J. Thorp S.C. Med. Res. Rev. 2002; 22: 1-25Crossref PubMed Scopus (246) Google Scholar, 2Rosenberg R.D. Showrak N.W. Liu J. Schwartz J.J. Zhang L. J. Clin. Invest. 1997; 99: 2062-2070Crossref PubMed Scopus (258) Google Scholar, 3Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2332) Google Scholar, 4Alexander C.M. Reichsman F. Hinkes M.T. Lincecum J. Becker K.A. Cumberledge S. Bernfield M. Nat. Genet. 2000; 25: 329-332Crossref PubMed Scopus (311) Google Scholar, 5Reizes O. Lincecum J. Wang Z. Goldberger O. Huang L. Kaksonen M. Ahima R. Hinkes M.T. Barsh G.S. Rauvala H. Bernfield M. Cell. 2001; 106: 105-116Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). HS is initially synthesized as a copolymer of glucuronic acid and N-acetylated glucosamine by d-glucuronyl andN-acetyl-d-glucosaminyl transferase, followed by various modifications (6Lindahl U. Kusche-Gullberg M. Kjellen L. J. Biol. Chem. 1998; 273: 24979-24982Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). These modifications include C5-epimerization of glucuronic acid to form iduronic acid residues, 2-O-sulfation of iduronic and glucuronic acid,N-deacetylation and N-sulfation of glucosamine, as well as 6-O-sulfation and 3-O-sulfation of glucosamine. Numerous HS biosynthetic enzymes have been cloned and characterized (for review, see Esko and Lindahl (7Esko J.D. Lindahl U. J. Clin. Invest. 2001; 108: 169-173Crossref PubMed Scopus (791) Google Scholar)). The specific sulfated saccharide sequences play critical roles in determining the functions of HS. A recent report suggests that the expression levels of various isoforms of each class of HS biosynthetic enzyme contribute to the synthesis of specific saccharide sequences in specific tissues (8Liu J. Shworak N.W. Sinay¨ P. Schwartz J.J. Zhang L. Fritze L.M.S. Rosenberg R.D. J. Biol. Chem. 1999; 274: 5185-5192Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). HSN-deacetylase/N-sulfotransferase, 3-O-sulfotransferase, and 6-O-sulfotransferase are present in multiple isoforms, and each isoform is believed to recognize the saccharide sequence around the modification site to generate a specific sulfated saccharide sequence (8Liu J. Shworak N.W. Sinay¨ P. Schwartz J.J. Zhang L. Fritze L.M.S. Rosenberg R.D. J. Biol. Chem. 1999; 274: 5185-5192Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 9Aikawa J.-i. Grobe K. Tsujimoto M. Esko J.D. J. Biol. Chem. 2001; 276: 5876-5882Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 10Habuchi H. Tanaka M. Habuchi O. Yoshida K. Suzuki H. Ban K. Kimata K. J. Biol. Chem. 2000; 275: 2859-2868Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). For instance, HS d-glucosaminyl-3-O-sulfotransferase (3-OST) isoforms generate 3-O-sulfated glucosamine that is linked to different sulfated uronic acid residues. 3-OST-1 transfers sulfate to the 3-OH position of the N-sulfated glucosamine residue that is linked to a glucuronic acid residue at the nonreducing end (GlcUA-GlcNS ± 6S), whereas, 3-OST-3 transfers sulfate to the 3-OH position of the N-unsubstituted glucosamine residue that is linked to a 2-O-sulfated iduronic acid at the nonreducing end (IdoUA2S-GlcNH2 ± 6S) (11Liu J. Shriver Z. Blaiklock P. Yoshida K. Sasisekharan R. Rosenberg R.D. J. Biol. Chem. 1999; 274: 38155-38162Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The difference in substrate specificity of 3-OSTs results in distinct biological functions of the HS modified by 3-OSTs. For example, HS modified by 3-OST-1 binds to antithrombin and has anticoagulant activity (12Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), whereas the HS modified by 3-OST-3 binds to herpes simplex 1 envelope glycoprotein D (gD) and assists in viral entry (13Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar). Herpes simplex virus type 1 (HSV-1) is a member of the herpesvirus family, and infection in humans is prevalent. HSV-1 infection requires a two-step process that can be separated experimentally: attachment to cells and entry into cells (14Spear P.G. Eisenberg R.J. Cohen G.H. Virology. 2000; 275: 1-8Crossref PubMed Scopus (419) Google Scholar). It is now known that HS is involved in assisting viral binding as well as viral entry (15Shukla D. Spear P.G. J. Clin. Invest. 2001; 108: 503-510Crossref PubMed Scopus (449) Google Scholar). HSV-1 binds to host cells through an interaction of virion envelope glycoprotein C (gC), or in some cases of glycoprotein B (gB), with HS (16Mettenleiter T.C. Virology. 1989; 171: 623-625Crossref PubMed Scopus (116) Google Scholar, 17Herold B.C. WuDunn D. Soltys N. Spear P.G. J. Virol. 1991; 65: 1090-1098Crossref PubMed Google Scholar, 18Trybala E. Bergstrom T. Svennerholm B. Jeansson S. Glorioso J.C. Olofsson S. J. Gen. Virol. 1994; 75: 743-752Crossref PubMed Scopus (91) Google Scholar). Structural analysis of gC-binding HS revealed that a minimum of 10–12 sugar residues containing IdoUA2S and GlcNS(or Ac)6S are necessary (19Feyzi E. Trybala E. Bergstrom T. Lindahl U. Spillmann D. J. Biol. Chem. 1997; 272: 24850-24857Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), and this conclusion was confirmed by another study (20Herold B. Gerber S.I. Belval B.J. Siston A.M. Shulman N. J. Virol. 1996; 70: 3461-3469Crossref PubMed Google Scholar). A recent report suggests that a specific 3-O-sulfated HS is involved in assisting HSV-1 entry (13Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar). The 3-O-sulfated HS is generated by 3-OST-3, but not by 3-OST-1. It should be noted that 3-OST-3-modified HS is rarely found in HS from natural sources, suggesting that HSV-1 recognizes a unique saccharide structure (11Liu J. Shriver Z. Blaiklock P. Yoshida K. Sasisekharan R. Rosenberg R.D. J. Biol. Chem. 1999; 274: 38155-38162Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In addition, a biochemical study revealed that 3-O-sulfated HS provides binding sites for HSV-1 envelope glycoprotein gD, which is a key viral protein involved in entry of HSV-1. It is believed that the interaction between gD and the 3-O-sulfated HS triggers the fusion between the virus and the cell in the presence of other viral envelope proteins, including gB, gH, and gL, via an uncharacterized mechanism. The study of the crystal structure of gD and herpes entry receptor HveA suggest that the binding of HveA to gD induces conformational changes in gD (21Carfi A. Willis S.H. Whitbeck J.C. Krummenacher C. Cohen G.H. Eisenberg R.J. Wiley D.C. Mol. Cell. 2001; 8: 169-179Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). This study also predicts a 3-O-sulfated HS-binding pocket on gD near the HveA-binding site (21Carfi A. Willis S.H. Whitbeck J.C. Krummenacher C. Cohen G.H. Eisenberg R.J. Wiley D.C. Mol. Cell. 2001; 8: 169-179Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The exact carbohydrate sequence of the gD-binding site in 3-O-sulfated HS remains to be investigated. In this article, we report the characterization of the structure of a gD-binding octasaccharide. The results from extensive sequencing analysis suggest that the structure of the gD-binding octasaccharide is ΔUA-GlcNS-IdoUA2S-GlcNAc-UA2S- GlcNS-IdoUA2S-GlcNH23S6S (residue 1 is GlcNH23S6S and residue 8 is ΔUA). The octasaccharide apparently has two motifs: a relatively low sulfation domain (residues 5–8) that contains two sulfate groups and a high sulfation domain (residue 1 to residue 4) that contains five sulfate groups. Although we still do not know the contribution of each sulfate group to the binding affinity of the octasaccharide and gD, the results from this study will provide the structural information to understand HS-assisted viral infection mechanisms. Recombinant 3-OST-3A and 3-OST-1 enzymes were expressed in Sf9 cells using baculovirus expression system. The enzymes were purified by using heparin-Toyopearl and 3′,5′-ADP-agarose chromatographies as described previously (11Liu J. Shriver Z. Blaiklock P. Yoshida K. Sasisekharan R. Rosenberg R.D. J. Biol. Chem. 1999; 274: 38155-38162Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 22Hernaiz M. Liu J. Rosenberg R.D. Linhardt R.J. Biochem. Biophys. Res. Commun. 2000; 276: 292-297Crossref PubMed Scopus (52) Google Scholar). [35S]PAPS was prepared by incubating 0.4 mCi/ml [35S]Na2SO4 (carrier-free, ICN) and 16 mm ATP with 5 mg/ml dialyzed yeast extract (Sigma) (12Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Iduronate-2-sulfatase, α-iduronidase, α-N-acetylglucosaminidase, glucosamine-6-sulfatase, and sulfamidase were obtained from Glyko. Recombinant heparin lyase I (EC4.2.2.7), II (no EC number), and III (E.C. 4.2.2.8) were prepared as described previously (23Godavarti R. Sasisekharan R. J. Biol. Chem. 1998; 273: 248-255Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Δ4,5-Glycuronidase was isolated from Flavobacterium heparinum (24McLean M.W. Bruce J.S. Long W.F. Williamson F.B. Eur. J. Biochem. 1984; 145: 607-615Crossref PubMed Scopus (51) Google Scholar). HS from bovine kidney was obtained from ICN. A truncated form of herpes simplex virus 1 glycoprotein D, gD-1-(306t), and monoclonal anti-gD-(DL6) were prepared as previously described (25Nicola A.V. Willis S.H. Naidoo N.N. Eisenberg R.J. Cohen G.H. J. Virol. 1996; 70: 3815-3822Crossref PubMed Google Scholar). The library was prepared by incubating HS with limited amounts of heparin lyase III followed by size fractionation on a Bio-Gel P-6 (Bio-Rad) column as described by Pye et al. (26Pye D.A. Vives R.R. Turnbull J.E. Hyde P. Gallagher J.T. J. Biol. Chem. 1998; 273: 22936-22942Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). In a typical preparation, HS from bovine kidney (1 mg) was incubated with 2 milliunits of heparin lyase III in 1 ml of buffer containing 50 mm sodium phosphate and 100 μg/ml bovine serum albumin, pH 7.0, at 37 °C overnight. The digestion was terminated by heating at 100 °C for 15 min. The sample was then loaded on a Bio-Gel P-6 (0.75 × 200 cm) equilibrated with 0.5 m ammonium bicarbonate at a flow rate of 5 ml/h and 0.5-ml fractions were collected. The absorbance at 232 nm was measured for each fraction. Peaks corresponding to tetra- to greater than dodecasaccharides were pooled individually, and dialyzed against 50 mm ammonium bicarbonate using MWCO 3,500 membrane. Each pool was dried on a Speed-Vac concentrator (Labconco) and reconstituted in 50 μl of water. The optical density (232 nm) of the resultant solution was about 3. We processed a total of 40 mg of HS to obtain a sufficient amount of gD-binding octasaccharide for the structural analysis. To prepare 3-OST-3A-modified HS oligosaccharide, 20 μl of the oligosaccharide library or intact HS (1 μg) was mixed with 240 ng of purified 3-OST-3A enzyme and 10 μm [35S]PAPS (14,000 dpm/pmol) in a buffer containing 50 mm MES, 1% Triton X-100, 1 mm MgCl2, 2 mmMnCl2, 150 mm NaCl, and 168 μg/ml bovine serum albumin, pH 7, in a final volume of 50 μl. The reaction was incubated at 37 °C for 2 h and was then heated at 100 °C for 2 min. The resultant solution was centrifuged at 14,000 rpm for 1 min to remove insoluble materials. The supernatant was dialyzed against 50 mm ammonium bicarbonate using MWCO 3,500 membrane and dried. To prepare 3-OST-1-modified oligosaccharides, we followed nearly identical procedures except for omitting the 150 mmNaCl and using 70 ng of 3-OST-1 enzyme during the enzymatic modification reaction. The 3-OST-3-modified oligosaccharides were applied to a silica-based polyamine (PAMN) HPLC column (0.46 × 25 cm, Waters). The column was eluted with a linear gradient of KH2PO4 from 350 mm to 1m for 60 min followed by an additional wash with 1m KH2PO4 for 20 min at a flow rate of 1 ml/min (11Liu J. Shriver Z. Blaiklock P. Yoshida K. Sasisekharan R. Rosenberg R.D. J. Biol. Chem. 1999; 274: 38155-38162Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The fractions containing35S-radioactivity were pooled separately and resolved on Bio-Gel P-6. The fractions were dialyzed against 25 mmammonium acetate using MWCO 3,500 membrane and dried. They were further purified by DEAE-NPR HPLC chromatography (0.46 × 7.5 cm, Tosohaas). The DEAE-NPR column was eluted with a linear gradient of NaCl in 50 mm Tris-HCl, pH 7, from 100 to 500 mm in 60 min followed by an additional wash for 20 min with 1 m NaCl in 50 mm Tris-HCl, pH 7, at a flow rate of 0.5 ml/min. The eluted oligosaccharides were monitored by35S-radioactivity and the absorbance at 232 nm. We obtained about 200 to 300 pmol of purified 35S-labeled oligosaccharides from 20 mg of HS that was partially digested with heparin lyase III. We did observe a UV peak that overlapped with the35S-radioactive peak when a large amount of 3-O-35S-sulfated oligosaccharides (>200 pmol) were injected on DEAE-NPR-HPLC. The assay for determining the binding of 3-O-sulfated HS oligosaccharides to gD was carried out by an immunoprecipitation procedure using gD and anti-gD monoclonal antibody as described previously but at a lower pH (13Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar). Briefly, 3-O-sulfated HS (1–10 pmol) was incubated in 50 μl of buffer containing 50 mm MES and 0.01% Triton, pH 6 (binding buffer), and 2 mg/ml gD at room temperature for 30 min. The anti-gD monoclonal antibody DL6 (5 μl) was added and incubated at 4 °C for 1 h followed by addition of the protein A-agarose gel (80 μl of 1:1 slurry) and agitated at 4 °C for an additional hour. The protein A-agarose gel (Pierce) was then washed with 0, 50, 150, and 500 mm NaCl in the above binding buffer. The binding affinity between 3-O-sulfated oligosaccharides and gD was determined using affinity co-electrophoresis, as previously described (13Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar). The gel was dried and analyzed on a PhosphorImager (Amersham Biosciences, Storm 860) to determine the migration of [35S]oligosaccharides. The 35S-intensity was plotted against the migration distance through the separation zone to define the distance migrated in the presence or absence of gD. The conditions for digestion with Δ4,5-glycuronidase and HS glycuronate-2-sulfatase were described elsewhere (27Zhang L. Yoshida K. Liu J. Rosenberg R.D. J. Biol. Chem. 1999; 274: 5681-5691Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The conditions for the nitrous acid degradations under pH 1.5 and 4.5 were described in a prior publication (8Liu J. Shworak N.W. Sinay¨ P. Schwartz J.J. Zhang L. Fritze L.M.S. Rosenberg R.D. J. Biol. Chem. 1999; 274: 5185-5192Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The degraded octasaccharide was analyzed by DEAE-NPR-HPLC. The oligosaccharide (5 × 105 to 1 × 106 cpm, 36–72 pmol) was dissolved in 20 μl of a solvent containingN′,N′-dimethylformamide and triethylamine (1:1, v/v) and 5 μl of acetic anhydride, and incubated on ice for 1 h. Tris (20 μl of 50 mm) was then added and the reaction mixture was incubated on ice for an additional hour. The sample was then diluted with 10 volumes of water and dialyzed against 50 mmammonium bicarbonate using a MWCO 3,500 membrane. Derivatizations were carried out by reacting 5 μl of oligosaccharide solution with 5 μl of 50 mm semicarbazide and 60 mm Tris acetic acid (pH 7.0, prepared fresh daily) for 16 h at 30 °C. Two mass spectrometry techniques, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nanoelectrospray ionization mass spectrometry (nESI-MS), were employed. The MALDI-MS spectra were acquired in the linear mode using a Perseptive Biosystems Voyager Elite reflectron time-of-flight instrument fitted with a 337-nm laser as described elsewhere (28Shriver Z. Raman R. Venkataraman G. Drummond K. Turnbull J. Toida T. Linhardt R. Biemann K. Sasisekharan R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10359-10364Crossref PubMed Scopus (93) Google Scholar). The nESI-MS analysis was carried out using a Micromass Quattro II with QhQ geometry, a Z-spray source, and pulled borosilicate glass nanovials (29Pope M. Raska C. Thorp S.C. Liu J. Glycobiology. 2001; 11: 505-513Crossref PubMed Scopus (53) Google Scholar). In the neutral loss scan, MS/MS spectra were obtained by scanning Q1 and Q3 with an offset of 26.7 or 20 m/z in their scan cycles, corresponding to the loss of sulfate from the triple or quadruple charged octasaccharide, respectively (29Pope M. Raska C. Thorp S.C. Liu J. Glycobiology. 2001; 11: 505-513Crossref PubMed Scopus (53) Google Scholar). To obtain a high quality nESI-MS spectrum, the purified octasaccharide was further dialyzed against 25 mm ammonium acetate (purity of ammonium acetate is 99.9999%, Aldrich) using MWCO 13,000 hollow fiber dialysis tubing (Spectrum). Control studies showed that 80–95% of 3-O-[35S]pentasaccharide (Mr = 1507) could be recovered using this dialysis tubing. The approach for the analysis of oligosaccharides followed a previously described method with modifications (30Rhomberg A.J. Ernst S. Sasisekharan R. Biemann K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4176-4181Crossref PubMed Scopus (86) Google Scholar). Briefly, the analysis was carried out on a Beckman P/ACE MDQ unit using an uncoated fused silica capillary (inner diameter = 75 μm;Ltot = 106 cm). Hydrodynamic injection was employed under 9.5 p.s.i. for 5 s. About 274 nl of the sample was calculated to be injected by CE Expert software. The electrolyte was a solution of 10 μm dextran sulfate and 50 mm Tris-phosphoric acid, pH 2.5. Separation was carried out at 25 kV. A gD-binding octasaccharide was purified from a 3-OST-3A-modified HS oligosaccharide library. The HS oligosaccharide library was prepared by incubating HS with a limited amount of heparin lyase III. The resultant material was fractionated by a Bio-Gel P-6 column based upon the size of the oligosaccharides, obtaining di-, tetra,-, … , dodeca-, and >dodecasaccharides (data not shown), and a similar approach to prepare a HS oligosaccharide library was reported by Pye and colleagues (26Pye D.A. Vives R.R. Turnbull J.E. Hyde P. Gallagher J.T. J. Biol. Chem. 1998; 273: 22936-22942Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Because of the limited resolution capability of Bio-Gel P-6, each oligosaccharide library undoubtedly contained the oligosaccharides with different sizes. These fractions were then subjected to 3-OST-3A modification and assayed for gD binding (TableI). Because 3-OST-1-modified HS does not bind to gD, we utilized the 3-OST-1-modified oligiosaccharides as a negative control (13Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar). As shown in Table I, the gD-binding percentage of 3-OST-3A-modified oligosaccharides was about 3-fold higher than that of the 3-OST-1-modified counterparts. We chose to purify a gD-binding oligosaccharide from the 3-OST-3A-modified hexasaccharide pool based upon the following two reasons: 1) the purification of hexasaccharides or octasaccharides can be achieved by anion exchange HPLC; 2) sequencing analysis for hexa- or octasaccharide is significantly less complex than larger oligosaccharides.Table IThe binding of 3-O-sulfated oligosaccharides to gDSize of the oligosaccharidesgD-binding3-OST-1-modified3-OST-3A-modified%Intact HS8.822.9>Dodecasaccharides2.07.2Dodecasaccharides2.47.6Decasaccharides5.37.0Octasaccharides1.35.4Hexasaccharides1-aBecause 3-OST-1 sulfated hexasaccharides very poorly, we could not obtain sufficient amount of 3-OST-1-modified hexasaccharides for the binding experiment. Therefore, we were unable to compare the binding of 3-OST-1-modified hexasaccharides and 3-OST-3A-modified hexasaccharides. Nevertheless, a gD-binding octasaccharide was isolated from the 3-OST-3 modified-hexasaccharide library as described in the text.Not determined3.4The binding of HS and oligosaccharides to gD was carried out at pH 6 by using an immunoprecipitation approach as described under "Experimental Procedures."1-a Because 3-OST-1 sulfated hexasaccharides very poorly, we could not obtain sufficient amount of 3-OST-1-modified hexasaccharides for the binding experiment. Therefore, we were unable to compare the binding of 3-OST-1-modified hexasaccharides and 3-OST-3A-modified hexasaccharides. Nevertheless, a gD-binding octasaccharide was isolated from the 3-OST-3 modified-hexasaccharide library as described in the text. Open table in a new tab The binding of HS and oligosaccharides to gD was carried out at pH 6 by using an immunoprecipitation approach as described under "Experimental Procedures." We purified a gD-binding oligosaccharide by successive anion-exchange HPLC, PAMN-, and DEAE-NPR-HPLC. Five major 3-O-35S-sulfated oligosaccharides were resolved by PAMN-HPLC (data not shown). To identify which [35S]oligosaccharide has the highest binding affinity for gD, the 3-OST-3A-modified oligosaccharides were fractionated by an immunoprecipitation approach as described under "Experimental Procedures." The eluents were analyzed by PAMN-HPLC (Fig.1A). Comparing the chromatograms of the 3-O-35S-sulfated oligosaccharides eluted from protein A-agarose under different concentrations of sodium chloride, we found that fraction D was present when the protein A-agarose was eluted with 150 mm NaCl (Fig. 1A, bottom chromatogram). In contrast, fraction A was present when the protein A-agarose was eluted with the buffer without NaCl (Fig. 1A, top chromatogram). This result suggests that fraction D has higher affinity for gD than fraction A. As indicated, it was observed that 32% of fraction D binds gD, whereas only 9% of fraction A binds to gD (Fig. 1B). Thus, we designated fraction D as a gD-binding oligosaccharide and fraction A as a gD-nonbinding oligosaccharide. Fraction B and fraction C were considered gD-nonbinding oligosaccharides, and were not subject to further structural study as their binding percentages to gD are similar to that of fraction A (Fig. 1B). Additional35S-labeled molecules were eluted from the protein A-agarose column with 500 mm sodium chloride. However, those molecules did not give sharp peaks on PAMN-HPLC. In addition, those molecules migrated as the oligosaccharides that were much larger than octasaccharides on Bio-Gel P-6. It is possible that these molecules represented the 35S-labeled oligosaccharide contaminants that were larger than octasaccharides in the oligosaccharide library. Fraction D was further purified on DEAE-NPR-HPLC. To confirm the purity, fraction D was analyzed by capillary electrophoresis using a UV 230 nm on-line detector. As shown in Fig. 2 (bottom electrophoretogram), the octasaccharide library was well resolved by capillary electrophoresis, suggesting that the resolution of the oligosaccharides on capillary electrophoresis is high. Fraction D migrated predominantly as a single peak under such conditions (Fig. 2, top electrophoretogram). In addition, the area of the major UV peak is consistent with the estimated concentration of the octasaccharide based upon the specific35S-radioactivity. Having considered the minor UV peaks resulting from contaminants, we calculated the purity of fraction D to be greater than 80%. We also determined the binding affinity (Kd) between fraction D and gD using affinity coelectrophoresis as described by Lee and Lander (31Lee M.K. Lander A.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2768-2772Crossref PubMed Scopus (155) Google Scholar). Fraction D was separated under electrophoresis in an agarose gel through zones containing gD at various concentrations (Fig.3A). From these data, theKd for fraction D and gD was determined to be 18 μm (Fig. 3C), which is somewhat higher than the Kd of intact 3-O-sulfated HS and gD (2 μm) (13Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar). We also attempted to determine theKd between fraction A (gD-nonbinding oligosaccharide) and gD using this method. We failed to observe any obvious retarded migration of fraction A, suggesting that the binding affinity between fraction A and gD is low (Fig. 3B). We estimated that the Kd for fraction A and gD is greater than 200 μm. The molecular mass of fraction D was determined by both nESI-MS and MALDI-MS. The nESI-MS spectrum of fraction D is shown in Fig.4. The sample shows a triple charged ion, [M-3H]3−, at m/z 648.8 and a strong quadruple charged ion, [M-4H]4−, atm/z 486.4 (Fig. 4A). We confirmed that the signals at m/z 648.8 and 486.4 contain sulfate groups by using neutral loss experiments as described in a prior publication (29Pope M. Raska C. T