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Structural Basis for the Adherence of Plasmodium falciparum-infected Erythrocytes to Chondroitin 4-Sulfate and Design of Novel Photoactivable Reagents for the Identification of Parasite Adhesive Proteins

2006; Elsevier BV; Volume: 282; Issue: 2 Linguagem: Inglês

10.1074/jbc.m604741200

ISSN

1083-351X

Autores

A. S. Prakasha Gowda, SubbaRao V. Madhunapantula, Rajeshwara N. Achur, Manojkumar Valiyaveettil, V.P. Bhavanandan, D. Channe Gowda,

Tópico(s)

Research on Leishmaniasis Studies

Resumo

A dodecasaccharide motif of the low-sulfated chondroitin 4-sulfate (C4S) mediate the binding of Plasmodium falciparum-infected red blood cells (IRBCs) in human placenta. Here we studied the detailed C4S structural requirements by assessing the ability of chemically modified C4S to inhibit IRBC binding to the placental chondroitin sulfate proteoglycan. Replacement of the N-acetyl groups with bulky N-acyl or N-benzoyl substituents had no effect on the inhibitory activity of C4S, whereas reduction of the carboxyl groups abrogated the activity. Dermatan sulfates showed ∼50% inhibitory activity when compared with C4Ss with similar sulfate contents. These data demonstrate that the C4S carboxyl groups and their equatorial orientation but not the N-acetyl groups are critical for IRBC binding. Conjugation of bulky substituents to the reducing end N-acetylgalactosamine residues of C4S dodecasaccharide had no effect on its inhibitory activity. Based on these results, we prepared photoaffinity reagents for the identification of the parasite proteins involved in C4S binding. Cross-linking of the IRBCs with a radioiodinated photoactivable C4S dodecasaccharide labeled a ∼22-kDa novel parasite protein, suggesting strongly for the first time that a low molecular weight IRBC surface protein rather than a 200–400-kDa PfEMP1 is involved in C4S binding. Conjugation of biotin to the C4S dodecasaccharide photoaffinity probe afforded a strategy for the isolation of the labeled protein by avidin affinity precipitation, facilitating efforts to identify the C4S-adherent IRBC protein(s). Our results also have broader implications for designing oligosaccharide-based photoaffinity probes for the identification of proteins involved in glycosaminoglycan-dependent attachment of microbes to hosts. A dodecasaccharide motif of the low-sulfated chondroitin 4-sulfate (C4S) mediate the binding of Plasmodium falciparum-infected red blood cells (IRBCs) in human placenta. Here we studied the detailed C4S structural requirements by assessing the ability of chemically modified C4S to inhibit IRBC binding to the placental chondroitin sulfate proteoglycan. Replacement of the N-acetyl groups with bulky N-acyl or N-benzoyl substituents had no effect on the inhibitory activity of C4S, whereas reduction of the carboxyl groups abrogated the activity. Dermatan sulfates showed ∼50% inhibitory activity when compared with C4Ss with similar sulfate contents. These data demonstrate that the C4S carboxyl groups and their equatorial orientation but not the N-acetyl groups are critical for IRBC binding. Conjugation of bulky substituents to the reducing end N-acetylgalactosamine residues of C4S dodecasaccharide had no effect on its inhibitory activity. Based on these results, we prepared photoaffinity reagents for the identification of the parasite proteins involved in C4S binding. Cross-linking of the IRBCs with a radioiodinated photoactivable C4S dodecasaccharide labeled a ∼22-kDa novel parasite protein, suggesting strongly for the first time that a low molecular weight IRBC surface protein rather than a 200–400-kDa PfEMP1 is involved in C4S binding. Conjugation of biotin to the C4S dodecasaccharide photoaffinity probe afforded a strategy for the isolation of the labeled protein by avidin affinity precipitation, facilitating efforts to identify the C4S-adherent IRBC protein(s). Our results also have broader implications for designing oligosaccharide-based photoaffinity probes for the identification of proteins involved in glycosaminoglycan-dependent attachment of microbes to hosts. An unusual feature of Plasmodium falciparum infection compared with the three other human malarial parasites, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, is that only the former parasite is able to sequester in the microvascular capillaries of various organs (1Pasloske B.L. Howard R.J. Annu. Rev. Med. 1994; 45: 283-295Crossref PubMed Scopus (81) Google Scholar, 2Weatherall D.J. Miller L.H. Baruch D.I. Marsh K. Doumbo O.K. Casals-Pascual C. Roberts D.J. Hematol. Am. Soc. Hematol. Educ. Program. 2002; : 35-57Crossref PubMed Scopus (144) Google Scholar). In P. falciparum-infected individuals, the infected red blood cells (IRBCs) 5The abbreviations used are: IRBC, infected red blood cell; C4S, chondroitin 4-sulfate; CSD, chondroitin sulfate D (chondroitin 2,6-disulfate); DS, dermatan sulfate; CSPG, chondroitin sulfate proteoglycan; bCSA, bovine tracheal cartilage chondroitin sulfate A; BSA, bovine serum albumin; TBS, Tris-buffered saline, pH 6.8; EDAC, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt; FACE, fluorophore-assisted carbohydrate electrophoresis; ASA, N-4-azidosalicylic acid; NHS-ASA, N-hydroxysuccinimidyl-4-azidosalicylic acid; ASBA, 4-azidosalicylamidobutylamine; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography. adhere to the vascular endothelial surface by binding to the cell adhesion molecules. This adherence property is believed to play an important role in the development of cerebral and other severe malaria syndromes (2Weatherall D.J. Miller L.H. Baruch D.I. Marsh K. Doumbo O.K. Casals-Pascual C. Roberts D.J. Hematol. Am. Soc. Hematol. Educ. Program. 2002; : 35-57Crossref PubMed Scopus (144) Google Scholar, 3Sherman I.W. Eda S. Winograd E. Microbes Infect. 2003; 5: 897-909Crossref PubMed Scopus (159) Google Scholar, 4Mackintosh C.L. Beeson J.G. Marsh K. Trends Parasitol. 2004; 20: 597-603Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Several host molecules have been implicated in the IRBC adherence, including thrombospondin, CD36, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, P-selectin, platelet endothelial cell adhesion molecule/CD31, and chondroitin 4-sulfate (1Pasloske B.L. Howard R.J. Annu. Rev. Med. 1994; 45: 283-295Crossref PubMed Scopus (81) Google Scholar, 2Weatherall D.J. Miller L.H. Baruch D.I. Marsh K. Doumbo O.K. Casals-Pascual C. Roberts D.J. Hematol. Am. Soc. Hematol. Educ. Program. 2002; : 35-57Crossref PubMed Scopus (144) Google Scholar, 3Sherman I.W. Eda S. Winograd E. Microbes Infect. 2003; 5: 897-909Crossref PubMed Scopus (159) Google Scholar, 4Mackintosh C.L. Beeson J.G. Marsh K. Trends Parasitol. 2004; 20: 597-603Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 5Cooke B. Coppel R. Wahlgren M. Parasitol. Today. 2000; 16: 416-420Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 6Heddini A. Pettersson F. Kai O. Shafi J. Obiero J. Chen Q. Barragan A. Wahlgren M. Marsh K. Infect. Immun. 2001; 69: 5849-5856Crossref PubMed Scopus (121) Google Scholar). In response to the development of adherent phenotype-specific immunity by hosts, parasites with different adherent receptor specificity evolve to efficiently survive in the host. However, eventually, when immunity to various adhesive parasite phenotypes is developed, the host effectively controls infection and avoids pathogenesis. This is generally true for malaria-immune people regardless of gender. However, in the case of pregnant women, placenta presents a new receptor for IRBC adherence allowing selection and propagation of parasites with different adherent specificities to which the host was not previously exposed. Accumulation of red blood cells infected with these phenotypically distinct parasites and subsequent monocyte/macrophage infiltration in the placenta lead to pregnancy-associated malaria characterized by poor pregnancy outcomes and maternal fatalities (7McGregor I.A. Wilson M.E. Billewicz W.Z. Trans. R. Soc. Trop. Med. Hyg. 1983; 77: 232-244Abstract Full Text PDF PubMed Scopus (313) Google Scholar, 8Menendez C. Ordi J. Ismail M.R. Ventura P.J. Aponte J.J. Kahigwa E. Font F. Alonso P.L. J. Infect. Dis. 2000; 181: 1740-1745Crossref PubMed Scopus (341) Google Scholar, 9Beeson J.G. Duffy P.E. Curr. Top. Microbiol. Immunol. 2005; 297: 187-227PubMed Google Scholar). The discovery by Fried and Duffy (10Fried M. Duffy P.E. Science. 1996; 272: 1502-1504Crossref PubMed Scopus (946) Google Scholar) in 1996 that placental adherent IRBCs bind specifically to C4S but not to other glycosaminoglycans was confirmed by us and others (11Rogerson S.J. Brown G.V. Parasitol. Today. 1997; 13: 70-75Abstract Full Text PDF PubMed Scopus (57) Google Scholar, 12Gowda D.C. Ockenhouse C.F. Biosci. Rep. 1999; 19: 261-271Crossref PubMed Scopus (6) Google Scholar, 13Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Particularly we showed that an unusually low sulfated CSPG localized predominantly in the intervillous space is the natural receptor for the placental IRBC adherence (14Muthusamy A. Achur R.N. Bhavanandan V.P. Fouda G.G. Taylor D.W. Gowda D.C. Am. J. Pathol. 2004; 164: 2013-2025Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Further we and others demonstrated that optimal IRBC binding requires the participation of both 4-sulfated and 4-nonsulfated disaccharide moieties (15Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 16Achur R.N. Valiyaveettil M. Gowda D.C. J. Biol. Chem. 2003; 278: 11705-11713Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 17Fried M. Lauder R.M. Duffy P.E. Exp. Parasitol. 2000; 95: 75-78Crossref PubMed Scopus (53) Google Scholar, 18Chai W. Beeson J.G. Lawson A.M. J. Biol. Chem. 2002; 277: 22438-22446Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We have also shown that a dodecasaccharide motif of the C4S with at least two 4-sulfated disaccharides is the minimum requirement for the efficient IRBC binding (15Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Whereas C4S has been conclusively shown to be the receptor for placental IRBC adherence, the identity of the parasite adhesive ligand expressed on the surface of the IRBCs remains unclear (19Rowe J.A. Kyes S.A. Mol. Microbiol. 2004; 53: 1011-1019Crossref PubMed Scopus (58) Google Scholar). It is widely thought that a subset of PfEMP1s containing domains analogous to the erythrocyte Duffy-binding ligands of P. vivax, which are known to be expressed on the IRBC surface, mediate the IRBC adherence to various host receptors, including CD36, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and C4S (20Baruch D.I. Gormely J.A. Ma C. Howard R.J. Pasloske B.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3497-3502Crossref PubMed Scopus (339) Google Scholar, 21Gardner J.P. Pinches R.A. Roberts D.J. Newbold C.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3503-3508Crossref PubMed Scopus (157) Google Scholar, 22Gamain B. Gratepanche S. Miller L.H. Baruch D.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10020-10024Crossref PubMed Scopus (48) Google Scholar). PfEMP1s comprise a family of 200–400-kDa, antigenically variant proteins encoded by ∼60 var genes. It appears that the primary function of PfEMP1 is to prevent IRBCs from being readily recognized by the host immune system (23Recker M. Nee S. Bull P.C. Kinyanjui S. Marsh K. Newbold C. Gupta S. Nature. 2004; 429: 555-558Crossref PubMed Scopus (135) Google Scholar, 24Bull P.C. Pain A. Ndungu F.M. Kinyanjui S.M. Roberts D.J. Newbold C.I. Marsh K. J. Infect. Dis. 2005; 192: 1119-1126Crossref PubMed Scopus (34) Google Scholar). In 1999, two groups reported that two distinct PfEMP1 variants, one termed FCR3var1CSA (from FCR-3 parasite strain) and the other termed CS2var1CSA (from CS2 parasite strain), mediate IRBC binding to C4S in a strain-dependent manner (25Buffet P.A. Gamain B. Scheidig C. Baruch D. Smith J.D. Hernandez-Rivas R. Pouvelle B. Oishi S. Fujii N. Fusai T. Parzy D. Miller L.H. Gysin J. Scherf A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12743-12748Crossref PubMed Scopus (217) Google Scholar, 26Reeder J.C. Cowman A.F. Davern K.M. Beeson J.G. Thompson J.K. Rogerson S.J. Brown G.V. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5198-5202Crossref PubMed Scopus (204) Google Scholar). In both cases, a specific domain called DBL-3γ was reported to be involved in the IRBC binding. However, it remains to be authenticated whether PfEMP1 is the ligand for placental IRBC adherence because although some subsequent studies support the above observations others do not (19Rowe J.A. Kyes S.A. Mol. Microbiol. 2004; 53: 1011-1019Crossref PubMed Scopus (58) Google Scholar). Recently a well conserved var gene subfamily PfEMP1 named var2CSA has been proposed to be the parasite ligand involved in C4S binding (27Salanti A. Staalsoe T. Lavstsen T. Jensen A.T. Sowa M.P. Arnot D.E. Hviid L. Theander T.G. Mol. Microbiol. 2003; 49: 179-191Crossref PubMed Scopus (575) Google Scholar, 28Salanti A. Dahlback M. Turner L. Nielsen M.A. Barfod L. Magistrado P. Jensen A.T. Lavstsen T. Ofori M.F. Marsh K. Hviid L. Theander T.G. J. Exp. Med. 2004; 200: 1197-1203Crossref PubMed Scopus (445) Google Scholar, 29Gamain B. Trimnell A.R. Scheidig C. Scherf A. Miller L.H. Smith J.D. J. Infect. Dis. 2005; 191: 1010-1013Crossref PubMed Scopus (129) Google Scholar). The var2CSA gene has been shown to transcribe at high levels in several C4S-binding parasite strains, and antibodies raised against a recombinant var2CSA can bind to IRBC surface in a gender-specific and parity-dependent manner (27Salanti A. Staalsoe T. Lavstsen T. Jensen A.T. Sowa M.P. Arnot D.E. Hviid L. Theander T.G. Mol. Microbiol. 2003; 49: 179-191Crossref PubMed Scopus (575) Google Scholar, 28Salanti A. Dahlback M. Turner L. Nielsen M.A. Barfod L. Magistrado P. Jensen A.T. Lavstsen T. Ofori M.F. Marsh K. Hviid L. Theander T.G. J. Exp. Med. 2004; 200: 1197-1203Crossref PubMed Scopus (445) Google Scholar, 30Tuikue Ndam N.G. Salanti A. Bertin G. Dahlback M. Fievet N. Turner L. Gaye A. Theander T. Deloron P. J. Infect. Dis. 2005; 192: 331-335Crossref PubMed Scopus (143) Google Scholar, 31Barfod L. Nielsen M.A. Turner L. Dahlback M. Jensen A.T. Hviid L. Theander T.G. Salanti A. Infect. Immun. 2006; 74: 4357-4360Crossref PubMed Scopus (51) Google Scholar). However, the ability of the recombinant var2CSA or anti-var2CSA antibodies to inhibit IRBC binding to C4S has not been demonstrated. Finally direct biochemical evidence in support of either DBL-3γ domain of var1or var2 PfEMP1 binding to C4S is lacking, and the possible involvement of other proteins cannot be ruled out. A major reason hindering the efforts to definitely identify the P. falciparum C4S-adhesive protein(s) is that the very low affinity of C4S-IRBC interaction precludes affinity purification. One approach to circumvent the problem will be to photoaffinity label the protein prior to affinity purification. A prerequisite for this approach is the elucidation of the role of functional groups in the C4S motif involved in binding to IRBC. Accordingly in this study, we investigated the potential of chemically modified C4S to inhibit the binding of IRBC to the placental CSPG. Based on the results, we prepared 125I-labeled photoactivable derivatives of C4S dodecasaccharide by partially replacing the acetyl groups of GalNAc by 4-azidosalicylyl groups. The C4S-IRBC adhesion-inhibitory activities of these derivatives were almost comparable to that of the unmodified oligosaccharide. Cross-linking of the photoaffinity probes to the C4S-adherent IRBC surface specifically labeled a ∼22-kDa parasite protein. In another approach, photoaffinity cross-linking of a probe containing a biotin residue at the C-1 of the reducing end of C4S dodecasaccharide to IRBC surface proteins enabled the isolation of the proteins by avidin affinity precipitation. Thus, our approaches offer, for the first time, strategies for the purification and biochemical characterization of the parasite adhesive protein(s). Moreover these procedures have important implications for designing photoactivable probes for the identification of adhesive protein(s) involved in the glycosaminoglycan-dependent adherence of various pathogens to host cells. Materials—Proteus vulgaris chondroitinase ABC (110 units/mg), sturgeon notochord C4S (98% 4-sulfated, 1.5% 6-sulfated, and 0.5% nonsulfated disaccharides), and chondroitin sulfate D (28% 4-sulfated, 46% 6-sulfated, 23% 4,6-disulfated, and 3% nonsulfated disaccharides) were from Seikagaku America (Falmouth, MA). Ovine testicular hyaluronidase (2160 units/mg) was from ICN Biomedicals (Costa Mesa, CA). N,O-Bis(trimethylsilyl)acetamide; bCSA (52% 4-sulfated, 39% 6-sulfated, and 9% nonsulfated disaccharides); EDAC; DS (pig intestinal mucosa); sodium cyanoborohydride; anhydrous hydrazine; hydrazine sulfate; and acetic, propionic, butyric, hexanoic, and benzoic anhydrides were from Sigma. Percoll, Na125I (100 mCi/ml) Sephadex G-15, Sepharose CL-4B, Sepharose CL-6B, DEAE-Sepharose, and blue dextran were from Amersham Biosciences. Bio-Gel P-6, Bio-Gel P-10, 4–15% polyacrylamide gradient minigels, and protein molecular mass markers (10–250 kDa) were from Bio-Rad. Protease Inhibitor Mixture set I was from Calbiochem. ASBA, NHS-ASA, biotin hydrazide, chloramine T-immobilized polystyrene beads (Iodobeads), biotin-immobilized avidin-agarose beads (NeutrAvidin beads), HPLC grade 6 m HCl, and micro-BCA protein assay kit were from Pierce. SYPRO Ruby protein gel stain was from Molecular Probes (Eugene, OR). Glyko® FACE ANTS labeling reagent kit was from Prozyme (San Leandro, CA). Placental CSPG—The low sulfated, P. falciparum-IRBC-binding CSPG of the human placenta was isolated and purified as reported previously (13Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 16Achur R.N. Valiyaveettil M. Gowda D.C. J. Biol. Chem. 2003; 278: 11705-11713Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Parasites—C4S-adherent P. falciparum were selected from a 3D7 parasite clone (derived from NF-54 strain) on plastic plates coated with the low sulfated CSPG purified from human placenta (13Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). These parasites are the same as those used in our previous studies (13Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 15Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 16Achur R.N. Valiyaveettil M. Gowda D.C. J. Biol. Chem. 2003; 278: 11705-11713Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). They bind efficiently to the placental CSPG and show exclusive specificity to C4S. The parasites were cultured in RPMI 1640 medium using human O-positive erythrocytes and O-positive serum as described previously (15Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). IRBC Adhesion Inhibition Assay—Adhesion/inhibition assays were carried out according to Alkhalil et al. (15Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Briefly a solution of the placental CSPG (200 ng/ml) in PBS, pH 7.2, was coated onto polystyrene Petri dishes (Falcon 1058) as circular spots (∼4-mm diameter) for 4 h at room temperature and blocked with 1% BSA in PBS for 1 h. Suspensions (2%) of parasite culture (20–35% parasitemia) cell pellet in PBS, pH 7.2, were preincubated with C4S or the test C4S derivatives at room temperature for 30 min with occasional shaking and then overlaid onto the CSPG-coated spots. After 30 min at room temperature, the unbound cells were removed by washing with PBS, pH 7.2. The bound cells were fixed with 2% glutaraldehyde, stained with 1% Giemsa, and counted under a light microscope at 40× magnifications in five to six different fields. The number of cells in one-fourth (one grid) of these fields was counted. The average number of cells per grid was typically 65–110. These values were used to calculate the number of cells (4000–7000) bound per 1 mm2 of CSPG-coated plates. In inhibition studies, the number of cells bound per 1 mm2 in the presence of the inhibitor was calculated as a percentage of that bound in the absence of inhibitors. All assays were performed in duplicates and repeated two to three times. Regioselective 6-O-Desulfation of bCSA—A partially 4-sulfated C4S that efficiently inhibits IRBC binding to the placental CSPG was prepared as reported previously (15Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). bCSA (0.5 g) was converted into its pyridinium salt by chromatography on Dowex 50W-X 8 (H+) followed by neutralization with pyridine. The lyophilized material was dissolved in anhydrous pyridine (100 ml), and N,O-bis(trimethylsilyl)acetamide (20 ml) was added and heated at 80 °C for 4 h under anhydrous condition. After cooling in an ice bath and dilution to 250 ml with ice-cold water to decompose excess sialylating reagent, the reaction mixture was dialyzed against water and lyophilized. The 6-O-desulfated chondroitin sulfate was applied onto a DEAE-Sepharose column (1.5 × 10 cm) in 50 mm NaOAc, pH 5.5, containing 100 mm NaCl, washed with the above buffer containing 0.2 m NaCl, and eluted with a gradient of 0.2–1 m NaCl. Fractions (3 ml) were collected, and aliquots were assayed for uronic acid (13Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The fractions were pooled as reported previously (32Muthusamy A. Achur R.N. Valiyaveettil M. Madhunapantula S.V. Kakizaki I. Bhavanandan V.P. Gowda D.C. Glycobiology. 2004; 14: 635-645Crossref PubMed Scopus (21) Google Scholar), and the disaccharide composition of the fractions was determined. A pooled fraction consisting of 40% 4-sulfated, 59% nonsulfated, and 1% 6-sulfated disaccharides was used in this study. Solvolytic Desulfation of Fully Sulfated C4S and DS—The sodium salts of sturgeon notochord C4S or dermatan sulfate (20 mg each) were converted to the pyridinium salt as above (15Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The pyridinium salts were dissolved separately in 10% aqueous Me2SO (10 ml), and each was divided into four equal parts and placed in screw-capped glass vials. The vials were heated at 80 °C, and one each was removed at 15, 40, 50, and 60 min and cooled in an ice bath. The content of each vial was diluted to 5.0 ml with water, adjusted to pH 9.0 with 0.1 m NaOH, dialyzed against water, and lyophilized. The sulfate contents of the partially desulfated C4S and DS ranged from 28 to 78% (see Table 2).TABLE 2Disaccharide composition of partially sulfated C4S and DS used for evaluating IRBC adhesion-inhibitory activity shown in Fig. 5GAGsDisaccharidesaDetermined by HPLC analysis of disaccharide released by digestion with chondroitinase ABC (38).ΔDi-0SΔDi-4SΔDi-6S% mol proportionC4S-121781.0C4S-248520C4S-362380C4S-470300DS-125750DS-245550DS-358420DS-472280a Determined by HPLC analysis of disaccharide released by digestion with chondroitinase ABC (38Sugahara K. Shigeno K. Masuda M. Fujii N. Kurosaka A. Takeda K. Carbohydr. Res. 1994; 255: 145-163Crossref PubMed Scopus (82) Google Scholar). Open table in a new tab Preparation of C4S Oligosaccharides—Oligosaccharides of varying sizes were prepared by the testicular hyaluronidase digestion of C4S (10 mg) followed by chromatography on a Bio-Gel P-6 column as described previously (15Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). N-Deacetylation of C4S—The C4S was N-deacetylated by hydrazinolysis according to Shaklee and Conrad (33Shaklee P.N. Conrad H.E. Biochem. J. 1984; 217: 187-197Crossref PubMed Scopus (78) Google Scholar). C4S (70 mg) was dissolved in anhydrous hydrazine (1.2 ml) containing 1% hydrazine sulfate in a reaction ampoule and flushed with nitrogen. The ampoule was sealed and heated in an oil bath at 105 °C. After 5 h, the reaction mixture was cooled and freeze-dried, and the residue was washed three times with 0.5 ml of toluene. The solid residue was dissolved in 0.25 m iodic acid (1 ml) and extracted with chloroform to remove the released iodine. The aqueous solution was neutralized to pH 7.4 with 0.1 m NaOH, dialyzed, and lyophilized. N-Acylation—N-Deacetylated C4S (2 mg) in 500 μl of PBS was dialyzed against saturated aqueous NaHCO3. To this solution, 3 × 50-μl aliquots of 10% solutions of anhydrides (acetic, propionic, butyric, hexanoic, or benzoic) in acetone were added at 20-min intervals (34Finne J. Krusius T. Methods Enzymol. 1982; 83: 269-277Crossref PubMed Scopus (98) Google Scholar). The reaction mixtures were allowed to stand at room temperature for 1 h, dialyzed against water, and lyophilized. The extent of N-acylation or N-benzoylation was determined by nitrous acid treatment followed by analysis of the products on Bio-Gel P-10 columns (35Cifonelli J.A. Methods Carbohydr. Chem. 1976; 8: 139-141Google Scholar). With hexanoic and benzoic anhydrides, the N-acylation procedure was repeated three times to achieve near complete acylation of free -NH2 groups, and any remaining -NH2 groups were acetylated with acetic anhydride. Reduction of Carboxyl Groups—To a solution of C4S (100 mg) in 15 ml of water solid EDAC (380 mg) was added, and the pH was maintained at 4.75 with 0.1 m HCl. After 2 h at room temperature, solid sodium borohydride (0.5–150 mg) was added over a period of 1 h, and the pH of the reaction mixture was maintained at 7.0 with the addition of 1 m HCl (36Taylor R.L. Shively J.E. Conrad H.E. Methods Carbohydr. Chem. 1976; 8: 149-151Google Scholar). The solution was then acidified to pH 5 with 0.1 m HCl to decompose the excess of borohydride. The carboxyl-reduced C4Ss were treated with 50 mm NaOH at 37 °C for 10–12 h to hydrolyze any EDAC ester of unreduced carboxylic acid groups, neutralized with 2 m acetic acid, dialyzed, and lyophilized. 2-O-Desulfation—Chondroitin sulfate D (5 mg) was dissolved in 10 ml of 0.05 m sodium hydroxide, and pH of the solution was adjusted to 12.5 by the addition of 1 m sodium hydroxide to a final concentration of 0.2 m (37Jaseja M. Rej R.N. Sauriol F. Perlin A.S. Can. J. Chem. 1989; 67: 1449-1456Crossref Scopus (139) Google Scholar). The solution was lyophilized, dissolved in 10 ml of water, dialyzed, and lyophilized. Digestion of C4S and Its N-Acylated Derivatives with Chondroitinase ABC—The C4S and its derivatives (100 μg each) were digested with chondroitinase ABC (50 milliunits) in 50 μl of 100 mm Tris-HCl, pH 8.0, containing 30 mm NaOAc and 0.01% BSA at 37 °C for 5 h as described previously (13Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The samples were analyzed by gel filtration on Bio-Gel P-10 columns (1.5 × 70 cm). Analysis of N-Deacetylated C4S and Re-N-acylated C4S by Nitrous Acid Degradation—The N-deacetylated, re-N-acylated, or re-N-benzoylated C4Ss (100 μg) were dissolved in 100 μl of 200 mm sodium acetate buffer, pH 3.8 (35Cifonelli J.A. Methods Carbohydr. Chem. 1976; 8: 139-141Google Scholar). An equal volume of 1 m sodium nitrite was added, and the reaction mixture was allowed to stand at room temperature for 24 h and then chromatographed on Bio-Gel P-10 columns. Gel Permeation Chromatography—The analysis of modified C4S and its nitrous acid or chondroitinase ABC degradation products was performed on columns of Sepharose CL-6B (1 × 50 cm), Bio Gel P-6 (1.5 × 70 cm), or Bio-Gel P-10 (1.5 × 70 cm) using either 0.2 m sodium chloride or 0.1 m pyridine, 0.1 m acetic acid, pH 5.5. Fractions (0.67 or 2 ml) were collected and assayed for the uronic acid content. Disaccharide Compositional Analysis—The C4S (5–10 μg) in 50 μl of 100 mm Tris-HCl, pH 8.0, containing 30 mm NaOAc and 0.01% BSA was digested with chondroitinase ABC (2.5–5 milliunits) at 37 °C for 6 h (13Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The unsaturated disaccharides that are formed were analyzed on an amino-bonded silica PA03 column (YMC Inc., Milford, MA) using a Waters 600E HPLC system with a linear gradient of 16–530 mm NaH2PO4 over a period of 70 min at room temperature at a flow rate of 1 ml/min as described by Sugahara et al. (38Sugahara K. Shigeno K. Masuda M. Fujii N. Kurosaka A. Takeda K. Carbohydr. Res. 1994; 255: 145-163Crossref PubMed Scopus (82) Google Scholar). The elution of disaccharides was monitored on line by measuring absorption at 232 nm with a Waters 484 variable wavelength UV detector. The data were processed with the Millennium 2010 chromatography manager using an NEC PowerMate 433 data processing system. Polyacrylamide Gel Electrophoresis of C4S

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