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W2017704712 abstract "We previously reported that versican, a large chondroitin/dermatan sulfate (CS/DS) proteoglycan, interacts through its CS/DS chains with adhesion molecules L- and P-selectin and CD44, as well as chemokines. Here, we have characterized these interactions further. Using a metabolic inhibitor of sulfation, sodium chlorate, we show that the interactions of the CS/DS chains of versican with L- and P-selectin and chemokines are sulfation-dependent but the interaction with CD44 is sulfation-independent. Consistently, versican's binding to L- and P-selectin and chemokines is specifically inhibited by oversulfated CS/DS chains containing GlcAβ1–3GalNAc(4,6-O-disulfate) or IdoAα1–3GalNAc(4,6-O-disulfate), but its binding to CD44 is inhibited by all the CS/DS chains, including low-sulfated and unsulfated ones. Affinity and kinetic analyses using surface plasmon resonance revealed that the oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) bind directly to selectins and chemokines with high affinity (Kd21.1 to 293 nm). In addition, a tetrasaccharide fragment of repeating GlcAβ1–3GalNAc(4,6-O-disulfate) units directly interacts with L- and P-selectin and chemokines and oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) inhibit chemokine-induced Ca2+ mobilization. Taken together, our results show that oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) are recognized by L- and P-selectin and chemokines, and imply that these chains are important in selectin- and/or chemokine-mediated cellular responses. We previously reported that versican, a large chondroitin/dermatan sulfate (CS/DS) proteoglycan, interacts through its CS/DS chains with adhesion molecules L- and P-selectin and CD44, as well as chemokines. Here, we have characterized these interactions further. Using a metabolic inhibitor of sulfation, sodium chlorate, we show that the interactions of the CS/DS chains of versican with L- and P-selectin and chemokines are sulfation-dependent but the interaction with CD44 is sulfation-independent. Consistently, versican's binding to L- and P-selectin and chemokines is specifically inhibited by oversulfated CS/DS chains containing GlcAβ1–3GalNAc(4,6-O-disulfate) or IdoAα1–3GalNAc(4,6-O-disulfate), but its binding to CD44 is inhibited by all the CS/DS chains, including low-sulfated and unsulfated ones. Affinity and kinetic analyses using surface plasmon resonance revealed that the oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) bind directly to selectins and chemokines with high affinity (Kd21.1 to 293 nm). In addition, a tetrasaccharide fragment of repeating GlcAβ1–3GalNAc(4,6-O-disulfate) units directly interacts with L- and P-selectin and chemokines and oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) inhibit chemokine-induced Ca2+ mobilization. Taken together, our results show that oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) are recognized by L- and P-selectin and chemokines, and imply that these chains are important in selectin- and/or chemokine-mediated cellular responses. glycosaminoglycan chondroitin sulfate dermatan sulfate heparan sulfate keratan sulfate hyaluronic acid chondroitin polysulfate dermatan polysulfate chondroitin Δ4,5HexAα1–3GalNAc Δ4,5HexAα1–3GalNAc(4-O-sulfate) Δ4,5HexAα1–3GalNAc(6-O-sulfate) 6)S, Δ4,5HexA(2-O-sulfate)α1–3GalNAc(6-O-sulfate) 4)S, Δ4,5HexA(2-O-sulfate)α1–3GalNAc(4-sulfate) 6)S, Δ4,5HexAα1–3GalNAc(4,6-O-disulfate) 4,6)S, Δ4,5HexA(2-O-sulfate)α1–3GalNAc(4,6-O-disulfate) Δ4,5HexA(2-O-sulfate)α1–3GalNAc GlcAβ1–3GalNAc GlcAβ1–3GalNAc(4-O-sulfate) GlcAβ1–3GalNAc(6-O-sulfate) 6)S, GlcAβ1–3GalNAc(4,6-O-disulfate) secondary lymphoid tissue chemokine γ-interferon inducible protein-10 platelet factor 4 stromal cell-derived factor truncated SLC high performance liquid chromatography bovine serum albumin 2-aminobenzamide Proteoglycans are ubiquitous components of cell surface membranes, basement membranes, and extracellular matrices in various tissues. They belong to a family of macromolecules that consist of core proteins to which glycosaminoglycans (GAGs),1 sulfated polysaccharides, are attached. GAGs are linear polysaccharides made up of disaccharide units composed of hexosamine and hexuronic acid (or hexose). They are classified into chondroitin sulfate (CS), dermatan sulfate (DS), heparin, heparan sulfate (HS), keratan sulfate (KS), and hyaluronic acid (HA). Because of the high sulfate and carboxyl group content of their GAG moieties, proteoglycans have strong negative charges. This property allows them to interact with a wide range of proteins, including growth factors, enzymes, cytokines, chemokines, lipoproteins, and adhesion molecules (1.Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 11: 1270-1279Crossref Scopus (393) Google Scholar, 2.Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2295) Google Scholar).We previously showed that a large CS/DS proteoglycan, versican (also called PG-M), that was derived from a renal adenocarcinoma cell line, ACHN, interacts through its CS/DS chains with adhesion molecules such as L- and P-selectin and CD44 (3.Kawashima H. Li Y.-F. Watanabe N. Hirose J. Hirose M. Miyasaka M. Int. Immunol. 1999; 11: 393-405Crossref PubMed Scopus (58) Google Scholar, 4.Kawashima H. Hirose M. Hirose J. Nagakubo D. Plaas A.H.K. Miyasaka M. J. Biol. Chem. 2000; 275: 35448-35456Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), and various chemokines (5.Hirose J. Kawashima H. Yoshie O. Tashiro K. Miyasaka M. J. Biol. Chem. 2001; 276: 5228-5234Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), all of which have been implicated in leukocyte trafficking. Versican possesses a hyaluronic acid-binding domain at its N terminus, a GAG attachment domain in the middle, and a set of epidermal growth factor-like, C-type lectin-like, and complement regulatory protein-like domains at its C terminus (6.Zimmermann D.R. Ruoslahti E. EMBO J. 1989; 8: 2975-2981Crossref PubMed Scopus (499) Google Scholar). Alternative splicing of the versican gene generates four versican isoforms: V0, V1, and V2, which bear GAG attachment domains of different lengths, and V3, which is without a GAG attachment domain (7.Zako M. Shinomura T. Ujita M. Ito K. Kimata K. J. Biol. Chem. 1995; 270: 3914-3918Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Versican is widely expressed in many tissues, including the kidney, skin, brain, and aorta (8.Bode-Lesniewska B. Dours-Zimmermann M.T. Odermatt B.F. Briner J. Heitz P.U. Zimmermann D.R. J. Histochem. Cytochem. 1996; 44: 303-312Crossref PubMed Scopus (162) Google Scholar). Our previous studies indicated that versican from certain cell types, such as ACHN and Vero cells, but not that from 293T cells or human skin fibroblasts, interacts with L-selectin, suggesting that at least one glycoform of versican species is reactive with L-selectin (3.Kawashima H. Li Y.-F. Watanabe N. Hirose J. Hirose M. Miyasaka M. Int. Immunol. 1999; 11: 393-405Crossref PubMed Scopus (58) Google Scholar). We have also shown that a subset of GAGs, including CS B from pig skin, CS E from squid cartilage, and HS from bovine kidney, interacts with L- and P-selectin and chemokines and inhibits the interaction with versican (4.Kawashima H. Hirose M. Hirose J. Nagakubo D. Plaas A.H.K. Miyasaka M. J. Biol. Chem. 2000; 275: 35448-35456Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 5.Hirose J. Kawashima H. Yoshie O. Tashiro K. Miyasaka M. J. Biol. Chem. 2001; 276: 5228-5234Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Thus, it seems likely that a nonspecific electrostatic interaction is not the sole factor determining the interaction, and that a specific carbohydrate structure is recognized by L- and P-selectin and chemokines, although the precise structural features remain undefined.In the present study, we have extended our previous work to examine the structural requirement for the interaction of CS/DS chains with adhesion molecules and chemokines. We report here affinity and kinetic parameters for the interaction of various GAGs with L- and P-selectin and chemokines. We also provide evidence that a tetrasaccharide fragment composed of two GlcAβ1–3GalNAc(4,6-O-disulfate) units directly interacts with L- and P-selectin and chemokines.DISCUSSIONIn this study, having demonstrated that sulfation is essential for the interaction of CS/DS chains with L- and P-selectin and chemokines, we analyzed the carbohydrate structures that bind to these molecules and have shown that oversulfated CS/DS chains containing GlcAβ1–3GalNAc(4,6-O-disulfate) or IdoAα1–3GalNAc(4,6-O-disulfate) bind L- and P-selectin and chemokines with high affinity. We have also provided evidence that a tetrasaccharide fragment composed of repeating GlcAβ1–3GalNAc(4,6-O-disulfate) units directly interacts with L- and P-selectin and chemokines, and that the oversulfated CS/DS chains inhibit chemokine activity in vitro.Sulfation plays an important role in the interactions of L- and P-selectin with the majority of ligands hitherto reported. For example, the tyrosine sulfation of P-selectin glycoprotein ligand-1 is required for its interaction with L- and P-selectin (17.Sako D. Comess K.M. Barone K.M. Camphausen R.T. Cumming D.A. Shaw G.D. Cell. 1995; 83: 323-331Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 18.Pouyani T. Seed B. Cell. 1995; 83: 333-343Abstract Full Text PDF PubMed Scopus (356) Google Scholar, 19.Spertini O. Cordey A.-S. Monai N. Giuffrè L. Schapira M. J. Cell Biol. 1996; 135: 523-531Crossref PubMed Scopus (183) Google Scholar). The ligands for L-selectin on the high endothelial venules bind L-selectin in a sulfation-dependent manner (20.Imai Y. Lasky L.A. Rosen S.D. Nature. 1993; 361: 555-557Crossref PubMed Scopus (330) Google Scholar, 21.Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.-C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 22.Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.-R. Huang C.-C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar). HNK-1-reactive sulfoglucuronyl glycolipids (23.Needham L.K. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1359-1363Crossref PubMed Scopus (180) Google Scholar), heparin oligosaccharides (24.Nelson R.M. Cecconi O. Roberts W.G. Aruffo A. Linhardt R.J. Bevilacqua M.P. Blood. 1993; 11: 3253-3258Crossref Google Scholar), and HS GAGs (25.Koenig A. Norgard-Sumnicht K. Linhardt R. Varki A. J. Clin. Invest. 1998; 101: 877-889Crossref PubMed Scopus (358) Google Scholar) bind L- and P-selectin. Our results showing that sulfation is required for versican's binding to L- and P-selectin (Figs. 1 and 4) are thus consistent with these previous findings. Extending these observations, we also showed that the sulfation of versican is required for the interaction with chemokines but not CD44 (Figs. 1 and 4). The absence of a sulfation requirement for versican's binding to CD44 is reminiscent of the binding of unsulfated GAG and HA to CD44 (26.Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2135) Google Scholar).Surface plasmon resonance analysis showed that the binding affinities of soluble monomeric L- and P-selectin to oversulfated CS/DS chains are higher than those for known ligands (Fig. 6 and Table II). It has been reported that monomeric L-selectin binds to immobilized glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) with low affinity (Kd = 108 μm) and with very fast association (≥105m−1 s−1) and dissociation (≥10 s−1) rates (27.Nicholson M.W. Barclay A.N. Singer M.S. Rosen S.D. van der Merwe P.A. J. Biol. Chem. 1998; 273: 763-770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). P-selectin has been reported to bind to P-selectin glycoprotein ligand-1 with relatively high affinity (Kd ∼300 nm) and with fast association (4.4 × 106m−1s−1) and dissociation (1.4 s−1) rates (28.Mehta P. Cummings R.D. McEver R.P. J. Biol. Chem. 1998; 273: 32506-32513Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). These properties have been proposed to be critical for the dynamic selectin-mediated rolling adhesion that is mediated by rapid adhesion and de-adhesion (27.Nicholson M.W. Barclay A.N. Singer M.S. Rosen S.D. van der Merwe P.A. J. Biol. Chem. 1998; 273: 763-770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 28.Mehta P. Cummings R.D. McEver R.P. J. Biol. Chem. 1998; 273: 32506-32513Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). We speculate that the high-affinity binding of L- and P-selectin to oversulfated CS/DS chains with a slow dissociation rate (Table II) may enable rolling interactions with different rolling velocity and/or static adhesive interactions of leukocytes, if appropriate CS/DS chains are expressed locally.The oversulfated CS/DS chains interact with certain chemokines with high affinity as observed with the selectins (Table II). The high affinity binding of chemokines SLC, IP-10, and SDF-1β suggest that these chemokines might be readily trapped by oversulfated CS/DS chains in vivo. This hypothesis is supported by the surface plasmon resonance kinetic analysis, which demonstrated that the oversulfated CS/DS-chemokine complex formation is characterized by fast association rates (0.864 to 4.15 × 104m−1 s−1). It is conceivable that the oversulfated CS/DS-bound form of chemokines may not function as agonists for chemokine receptors, because those GAGs inhibit chemokine activity (Fig. 8). Rather, the oversulfated CS/DS-chemokine complex may function as a reservoir for chemokines in vivo. The slow dissociation rate (2.78 × 10−4 to 5.30 × 10−3 s−1) that is observed in the interaction of chemokines with oversulfated CS/DS chains supports this idea.Hints about the mechanism of CS/DS binding to chemokines are provided by the observation that versican interacts with SDF-1β but not SDF-1α (Figs. 4 and 5). Because SDF-1α and SDF-1β are produced by an alternative splicing of a single gene with the latter lacking only 4 amino acid residues in the C terminus (29.Shirozu M. Nakano T. Inazawa J. Tashiro K. Tada H. Shinohara T. Honjo T. Genomics. 1995; 28: 495-500Crossref PubMed Scopus (533) Google Scholar), versican, or its CS/DS chains, appears to interact with the chemokine's C terminus. Consistent with this idea, versican failed to bind to a mutant SLC that lacked the C-terminal basic amino acid clusters (Fig. 4), and CS B and CS E also failed to bind the C terminus truncated SLC. 2J. Hirose, H. Kawashima, and M. Miyasaka, submitted for publication. Therefore, these data strongly suggest that CS/DS interacts with the C terminus of these chemokines.Structural analysis indicated that GlcAβ1–3GalNAc(4,6-O-disulfate) units are present in the GAG moiety of versican (Fig. 3). Given that L- and P-selectin and chemokines bind preferentially to a tetrasaccharide composed of repeating GlcAβ1–3GalNAc(4,6-O-disulfate) units (Fig. 7), and that CD44 preferentially interacts with unsulfated or low-sulfated CS chains (Table II), it seems likely that GlcAβ1–3GalNAc(4,6-O-disulfate) units interact with selectins and chemokines if these units are present as a cluster in the GAG chain, whereas a different structure containing GlcAβ1–3GalNAc(4-O-sulfate) or GlcAβ1–3GalNAc(6-O-sulfate) appears to interact with CD44.Although CS B (DS) from pig skin inhibited the interaction of versican with L- and P-selectin and chemokines in our previous studies (4.Kawashima H. Hirose M. Hirose J. Nagakubo D. Plaas A.H.K. Miyasaka M. J. Biol. Chem. 2000; 275: 35448-35456Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 5.Hirose J. Kawashima H. Yoshie O. Tashiro K. Miyasaka M. J. Biol. Chem. 2001; 276: 5228-5234Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), the DS from rooster comb that was used in this study failed to inhibit the interaction (Fig. 2). Disaccharide composition analysis of these DS chains, however, showed no striking difference between them;i.e. DS from pig skin is composed of ΔDi-6S (1.3%), ΔDi-4S (91.5%), and ΔDi-di(2,4)S (7.2%), whereas DS from rooster comb is composed of ΔDi-0S (5.0%), ΔDi-6S (3.4%), ΔDi-4S (85.2%), and ΔDi-di(2, 4)S (6.3%) (data not shown). Because a highly sulfated oligosaccharide composed of repeating disulfated disaccharides interacted well with selectins and chemokines (Fig. 7), we think it possible that a difference in the amount of highly sulfated clusters containing IdoA(2-O-disulfate)α1–3GalNAc(4-O-disulfate) in these DS chains may in part explain the differential reactivity, although further structural characterization is required.Oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) have been found in squid cartilage (30.Suzuki S. Saito H. Yamagata T. Anno K. Seno N. Kawai Y. Furuhashi T. J. Biol. Chem. 1968; 243: 1543-1550Abstract Full Text PDF PubMed Google Scholar), mast cells (31.Katz H.R. Austen K.F. Caterson B. Stevens R.L. J. Biol. Chem. 1986; 261: 13393-13396Abstract Full Text PDF PubMed Google Scholar, 32.Stevens R.L. Fox C.C. Lichtenstein L.M. Austen K.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2284-2287Crossref PubMed Scopus (99) Google Scholar), neutrophils (33.Ohhashi Y. Hasumi F. Mori Y. Biochem. J. 1984; 217: 199-207Crossref PubMed Scopus (18) Google Scholar, 34.Petersen F. Brandt E. Lindahl U. Spillmann D. J. Biol. Chem. 1999; 274: 12376-12382Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), monocytes (35.Uhlin-Hansen L. Eskeland T. Kolset S.O. J. Biol. Chem. 1989; 264: 14916-14922Abstract Full Text PDF PubMed Google Scholar, 36.McGee M.P. Teuschler H. Parthasarathy N. Wagner W.D. J. Biol. Chem. 1995; 270: 26109-26115Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), glomeruli (37.Kobayashi S. Oguri K. Yaoita E. Kobayashi K. Okayama M. Biochim. Biophys. Acta. 1985; 841: 71-80Crossref PubMed Scopus (25) Google Scholar), and mesangial cells (38.Yaoita E. Oguri K. Okayama E. Kawasaki K. Kobayashi S. Kihara I. Okayama M. J. Biol. Chem. 1990; 265: 522-531Abstract Full Text PDF PubMed Google Scholar), and have been reported to interact with various biologically active molecules and regulate their functions in vitro. For example, CS E inhibits the adhesion of cortical neuronal cells to the neurotrophic factor midkine through direct interaction with midkine (39.Ueoka C. Kaneda N. Okazaki I. Nadanaka S. Muramatsu T. Sugahara K. J. Biol. Chem. 2000; 275: 37407-37413Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), promotes neurite outgrowth in embryonic day 18 rat hippocampal neurons (40.Nadanaka S. Clement A. Masayama K. Faissner A. Sugahara K. J. Biol. Chem. 1998; 273: 3296-3307Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), serves as a receptor for PF4 on the surface of neutrophils (34.Petersen F. Brandt E. Lindahl U. Spillmann D. J. Biol. Chem. 1999; 274: 12376-12382Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and inhibits procoagulant activity (36.McGee M.P. Teuschler H. Parthasarathy N. Wagner W.D. J. Biol. Chem. 1995; 270: 26109-26115Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Our results showing that oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) bind to proinflammatory molecules, such as selectins and chemokines, and regulate chemokine activity, further indicate the versatility of these oversulfated chains in regulating biological responses. It is now extremely important to understand the exact tissue distribution of the oversulfated CS/DS chains bearing the ability to interact with these proinflammatory molecules and the regulatory mechanisms whereby these GAGs are generated. Proteoglycans are ubiquitous components of cell surface membranes, basement membranes, and extracellular matrices in various tissues. They belong to a family of macromolecules that consist of core proteins to which glycosaminoglycans (GAGs),1 sulfated polysaccharides, are attached. GAGs are linear polysaccharides made up of disaccharide units composed of hexosamine and hexuronic acid (or hexose). They are classified into chondroitin sulfate (CS), dermatan sulfate (DS), heparin, heparan sulfate (HS), keratan sulfate (KS), and hyaluronic acid (HA). Because of the high sulfate and carboxyl group content of their GAG moieties, proteoglycans have strong negative charges. This property allows them to interact with a wide range of proteins, including growth factors, enzymes, cytokines, chemokines, lipoproteins, and adhesion molecules (1.Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 11: 1270-1279Crossref Scopus (393) Google Scholar, 2.Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2295) Google Scholar). We previously showed that a large CS/DS proteoglycan, versican (also called PG-M), that was derived from a renal adenocarcinoma cell line, ACHN, interacts through its CS/DS chains with adhesion molecules such as L- and P-selectin and CD44 (3.Kawashima H. Li Y.-F. Watanabe N. Hirose J. Hirose M. Miyasaka M. Int. Immunol. 1999; 11: 393-405Crossref PubMed Scopus (58) Google Scholar, 4.Kawashima H. Hirose M. Hirose J. Nagakubo D. Plaas A.H.K. Miyasaka M. J. Biol. Chem. 2000; 275: 35448-35456Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), and various chemokines (5.Hirose J. Kawashima H. Yoshie O. Tashiro K. Miyasaka M. J. Biol. Chem. 2001; 276: 5228-5234Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), all of which have been implicated in leukocyte trafficking. Versican possesses a hyaluronic acid-binding domain at its N terminus, a GAG attachment domain in the middle, and a set of epidermal growth factor-like, C-type lectin-like, and complement regulatory protein-like domains at its C terminus (6.Zimmermann D.R. Ruoslahti E. EMBO J. 1989; 8: 2975-2981Crossref PubMed Scopus (499) Google Scholar). Alternative splicing of the versican gene generates four versican isoforms: V0, V1, and V2, which bear GAG attachment domains of different lengths, and V3, which is without a GAG attachment domain (7.Zako M. Shinomura T. Ujita M. Ito K. Kimata K. J. Biol. Chem. 1995; 270: 3914-3918Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Versican is widely expressed in many tissues, including the kidney, skin, brain, and aorta (8.Bode-Lesniewska B. Dours-Zimmermann M.T. Odermatt B.F. Briner J. Heitz P.U. Zimmermann D.R. J. Histochem. Cytochem. 1996; 44: 303-312Crossref PubMed Scopus (162) Google Scholar). Our previous studies indicated that versican from certain cell types, such as ACHN and Vero cells, but not that from 293T cells or human skin fibroblasts, interacts with L-selectin, suggesting that at least one glycoform of versican species is reactive with L-selectin (3.Kawashima H. Li Y.-F. Watanabe N. Hirose J. Hirose M. Miyasaka M. Int. Immunol. 1999; 11: 393-405Crossref PubMed Scopus (58) Google Scholar). We have also shown that a subset of GAGs, including CS B from pig skin, CS E from squid cartilage, and HS from bovine kidney, interacts with L- and P-selectin and chemokines and inhibits the interaction with versican (4.Kawashima H. Hirose M. Hirose J. Nagakubo D. Plaas A.H.K. Miyasaka M. J. Biol. Chem. 2000; 275: 35448-35456Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 5.Hirose J. Kawashima H. Yoshie O. Tashiro K. Miyasaka M. J. Biol. Chem. 2001; 276: 5228-5234Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Thus, it seems likely that a nonspecific electrostatic interaction is not the sole factor determining the interaction, and that a specific carbohydrate structure is recognized by L- and P-selectin and chemokines, although the precise structural features remain undefined. In the present study, we have extended our previous work to examine the structural requirement for the interaction of CS/DS chains with adhesion molecules and chemokines. We report here affinity and kinetic parameters for the interaction of various GAGs with L- and P-selectin and chemokines. We also provide evidence that a tetrasaccharide fragment composed of two GlcAβ1–3GalNAc(4,6-O-disulfate) units directly interacts with L- and P-selectin and chemokines. DISCUSSIONIn this study, having demonstrated that sulfation is essential for the interaction of CS/DS chains with L- and P-selectin and chemokines, we analyzed the carbohydrate structures that bind to these molecules and have shown that oversulfated CS/DS chains containing GlcAβ1–3GalNAc(4,6-O-disulfate) or IdoAα1–3GalNAc(4,6-O-disulfate) bind L- and P-selectin and chemokines with high affinity. We have also provided evidence that a tetrasaccharide fragment composed of repeating GlcAβ1–3GalNAc(4,6-O-disulfate) units directly interacts with L- and P-selectin and chemokines, and that the oversulfated CS/DS chains inhibit chemokine activity in vitro.Sulfation plays an important role in the interactions of L- and P-selectin with the majority of ligands hitherto reported. For example, the tyrosine sulfation of P-selectin glycoprotein ligand-1 is required for its interaction with L- and P-selectin (17.Sako D. Comess K.M. Barone K.M. Camphausen R.T. Cumming D.A. Shaw G.D. Cell. 1995; 83: 323-331Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 18.Pouyani T. Seed B. Cell. 1995; 83: 333-343Abstract Full Text PDF PubMed Scopus (356) Google Scholar, 19.Spertini O. Cordey A.-S. Monai N. Giuffrè L. Schapira M. J. Cell Biol. 1996; 135: 523-531Crossref PubMed Scopus (183) Google Scholar). The ligands for L-selectin on the high endothelial venules bind L-selectin in a sulfation-dependent manner (20.Imai Y. Lasky L.A. Rosen S.D. Nature. 1993; 361: 555-557Crossref PubMed Scopus (330) Google Scholar, 21.Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.-C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 22.Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.-R. Huang C.-C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar). HNK-1-reactive sulfoglucuronyl glycolipids (23.Needham L.K. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1359-1363Crossref PubMed Scopus (180) Google Scholar), heparin oligosaccharides (24.Nelson R.M. Cecconi O. Roberts W.G. Aruffo A. Linhardt R.J. Bevilacqua M.P. Blood. 1993; 11: 3253-3258Crossref Google Scholar), and HS GAGs (25.Koenig A. Norgard-Sumnicht K. Linhardt R. Varki A. J. Clin. Invest. 1998; 101: 877-889Crossref PubMed Scopus (358) Google Scholar) bind L- and P-selectin. Our results showing that sulfation is required for versican's binding to L- and P-selectin (Figs. 1 and 4) are thus consistent with these previous findings. Extending these observations, we also showed that the sulfation of versican is required for the interaction with chemokines but not CD44 (Figs. 1 and 4). The absence of a sulfation requirement for versican's binding to CD44 is reminiscent of the binding of unsulfated GAG and HA to CD44 (26.Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2135) Google Scholar).Surface plasmon resonance analysis showed that the binding affinities of soluble monomeric L- and P-selectin to oversulfated CS/DS chains are higher than those for known ligands (Fig. 6 and Table II). It has been reported that monomeric L-selectin binds to immobilized glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) with low affinity (Kd = 108 μm) and with very fast association (≥105m−1 s−1) and dissociation (≥10 s−1) rates (27.Nicholson M.W. Barclay A.N. Singer M.S. Rosen S.D. van der Merwe P.A. J. Biol. Chem. 1998; 273: 763-770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). P-selectin has been reported to bind to P-selectin glycoprotein ligand-1 with relatively high affinity (Kd ∼300 nm) and with fast association (4.4 × 106m−1s−1) and dissociation (1.4 s−1) rates (28.Mehta P. Cummings R.D. McEver R.P. J. Biol. Chem. 1998; 273: 32506-32513Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). These properties have been proposed to be critical for the dynamic selectin-mediated rolling adhesion that is mediated by rapid adhesion and de-adhesion (27.Nicholson M.W. Barclay A.N. Singer M.S. Rosen S.D. van der Merwe P.A. J. Biol. Chem. 1998; 273: 763-770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 28.Mehta P. Cummings R.D. McEver R.P. J. Biol. Chem. 1998; 273: 32506-32513Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). We speculate that the high-affinity binding of L- and P-selectin to oversulfated CS/DS chains with a slow dissociation rate (Table II) may enable rolling interactions with different rolling velocity and/or static adhesive interactions of leukocytes, if appropriate CS/DS chains are expressed locally.The oversulfated CS/DS chains interact with certain chemokines with high affinity as observed with the selectins (Table II). The high affinity binding of chemokines SLC, IP-10, and SDF-1β suggest that these chemokines might be readily trapped by oversulfated CS/DS chains in vivo. This hypothesis is supported by the surface plasmon resonance kinetic analysis, which demonstrated that the oversulfated CS/DS-chemokine complex formation is characterized by fast association rates (0.864 to 4.15 × 104m−1 s−1). It is conceivable that the oversulfated CS/DS-bound form of chemokines may not function as agonists for chemokine receptors, because those GAGs inhibit chemokine activity (Fig. 8). Rather, the oversulfated CS/DS-chemokine complex may function as a reservoir for chemokines in vivo. The slow dissociation rate (2.78 × 10−4 to 5.30 × 10−3 s−1) that is observed in the interaction of chemokines with oversulfated CS/DS chains supports this idea.Hints about the mechanism of CS/DS binding to chemokines are provided by the observation that versican interacts with SDF-1β but not SDF-1α (Figs. 4 and 5). Because SDF-1α and SDF-1β are produced by an alternative splicing of a single gene with the latter lacking only 4 amino acid residues in the C terminus (29.Shirozu M. Nakano T. Inazawa J. Tashiro K. Tada H. Shinohara T. Honjo T. Genomics. 1995; 28: 495-500Crossref PubMed Scopus (533) Google Scholar), versican, or its CS/DS chains, appears to interact with the chemokine's C terminus. Consistent with this idea, versican failed to bind to a mutant SLC that lacked the C-terminal basic amino acid clusters (Fig. 4), and CS B and CS E also failed to bind the C terminus truncated SLC. 2J. Hirose, H. Kawashima, and M. Miyasaka, submitted for publication. Therefore, these data strongly suggest that CS/DS interacts with the C terminus of these chemokines.Structural analysis indicated that GlcAβ1–3GalNAc(4,6-O-disulfate) units are present in the GAG moiety of versican (Fig. 3). Given that L- and P-selectin and chemokines bind preferentially to a tetrasaccharide composed of repeating GlcAβ1–3GalNAc(4,6-O-disulfate) units (Fig. 7), and that CD44 preferentially interacts with unsulfated or low-sulfated CS chains (Table II), it seems likely that GlcAβ1–3GalNAc(4,6-O-disulfate) units interact with selectins and chemokines if these units are present as a cluster in the GAG chain, whereas a different structure containing GlcAβ1–3GalNAc(4-O-sulfate) or GlcAβ1–3GalNAc(6-O-sulfate) appears to interact with CD44.Although CS B (DS) from pig skin inhibited the interaction of versican with L- and P-selectin and chemokines in our previous studies (4.Kawashima H. Hirose M. Hirose J. Nagakubo D. Plaas A.H.K. Miyasaka M. J. Biol. Chem. 2000; 275: 35448-35456Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 5.Hirose J. Kawashima H. Yoshie O. Tashiro K. Miyasaka M. J. Biol. Chem. 2001; 276: 5228-5234Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), the DS from rooster comb that was used in this study failed to inhibit the interaction (Fig. 2). Disaccharide composition analysis of these DS chains, however, showed no striking difference between them;i.e. DS from pig skin is composed of ΔDi-6S (1.3%), ΔDi-4S (91.5%), and ΔDi-di(2,4)S (7.2%), whereas DS from rooster comb is composed of ΔDi-0S (5.0%), ΔDi-6S (3.4%), ΔDi-4S (85.2%), and ΔDi-di(2, 4)S (6.3%) (data not shown). Because a highly sulfated oligosaccharide composed of repeating disulfated disaccharides interacted well with selectins and chemokines (Fig. 7), we think it possible that a difference in the amount of highly sulfated clusters containing IdoA(2-O-disulfate)α1–3GalNAc(4-O-disulfate) in these DS chains may in part explain the differential reactivity, although further structural characterization is required.Oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) have been found in squid cartilage (30.Suzuki S. Saito H. Yamagata T. Anno K. Seno N. Kawai Y. Furuhashi T. J. Biol. Chem. 1968; 243: 1543-1550Abstract Full Text PDF PubMed Google Scholar), mast cells (31.Katz H.R. Austen K.F. Caterson B. Stevens R.L. J. Biol. Chem. 1986; 261: 13393-13396Abstract Full Text PDF PubMed Google Scholar, 32.Stevens R.L. Fox C.C. Lichtenstein L.M. Austen K.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2284-2287Crossref PubMed Scopus (99) Google Scholar), neutrophils (33.Ohhashi Y. Hasumi F. Mori Y. Biochem. J. 1984; 217: 199-207Crossref PubMed Scopus (18) Google Scholar, 34.Petersen F. Brandt E. Lindahl U. Spillmann D. J. Biol. Chem. 1999; 274: 12376-12382Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), monocytes (35.Uhlin-Hansen L. Eskeland T. Kolset S.O. J. Biol. Chem. 1989; 264: 14916-14922Abstract Full Text PDF PubMed Google Scholar, 36.McGee M.P. Teuschler H. Parthasarathy N. Wagner W.D. J. Biol. Chem. 1995; 270: 26109-26115Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), glomeruli (37.Kobayashi S. Oguri K. Yaoita E. Kobayashi K. Okayama M. Biochim. Biophys. Acta. 1985; 841: 71-80Crossref PubMed Scopus (25) Google Scholar), and mesangial cells (38.Yaoita E. Oguri K. Okayama E. Kawasaki K. Kobayashi S. Kihara I. Okayama M. J. Biol. Chem. 1990; 265: 522-531Abstract Full Text PDF PubMed Google Scholar), and have been reported to interact with various biologically active molecules and regulate their functions in vitro. For example, CS E inhibits the adhesion of cortical neuronal cells to the neurotrophic factor midkine through direct interaction with midkine (39.Ueoka C. Kaneda N. Okazaki I. Nadanaka S. Muramatsu T. Sugahara K. J. Biol. Chem. 2000; 275: 37407-37413Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), promotes neurite outgrowth in embryonic day 18 rat hippocampal neurons (40.Nadanaka S. Clement A. Masayama K. Faissner A. Sugahara K. J. Biol. Chem. 1998; 273: 3296-3307Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), serves as a receptor for PF4 on the surface of neutrophils (34.Petersen F. Brandt E. Lindahl U. Spillmann D. J. Biol. Chem. 1999; 274: 12376-12382Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and inhibits procoagulant activity (36.McGee M.P. Teuschler H. Parthasarathy N. Wagner W.D. J. Biol. Chem. 1995; 270: 26109-26115Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Our results showing that oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) bind to proinflammatory molecules, such as selectins and chemokines, and regulate chemokine activity, further indicate the versatility of these oversulfated chains in regulating biological responses. It is now extremely important to understand the exact tissue distribution of the oversulfated CS/DS chains bearing the ability to interact with these proinflammatory molecules and the regulatory mechanisms whereby these GAGs are generated. In this study, having demonstrated that sulfation is essential for the interaction of CS/DS chains with L- and P-selectin and chemokines, we analyzed the carbohydrate structures that bind to these molecules and have shown that oversulfated CS/DS chains containing GlcAβ1–3GalNAc(4,6-O-disulfate) or IdoAα1–3GalNAc(4,6-O-disulfate) bind L- and P-selectin and chemokines with high affinity. We have also provided evidence that a tetrasaccharide fragment composed of repeating GlcAβ1–3GalNAc(4,6-O-disulfate) units directly interacts with L- and P-selectin and chemokines, and that the oversulfated CS/DS chains inhibit chemokine activity in vitro. Sulfation plays an important role in the interactions of L- and P-selectin with the majority of ligands hitherto reported. For example, the tyrosine sulfation of P-selectin glycoprotein ligand-1 is required for its interaction with L- and P-selectin (17.Sako D. Comess K.M. Barone K.M. Camphausen R.T. Cumming D.A. Shaw G.D. Cell. 1995; 83: 323-331Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 18.Pouyani T. Seed B. Cell. 1995; 83: 333-343Abstract Full Text PDF PubMed Scopus (356) Google Scholar, 19.Spertini O. Cordey A.-S. Monai N. Giuffrè L. Schapira M. J. Cell Biol. 1996; 135: 523-531Crossref PubMed Scopus (183) Google Scholar). The ligands for L-selectin on the high endothelial venules bind L-selectin in a sulfation-dependent manner (20.Imai Y. Lasky L.A. Rosen S.D. Nature. 1993; 361: 555-557Crossref PubMed Scopus (330) Google Scholar, 21.Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.-C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 22.Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.-R. Huang C.-C. Kannagi R. Rosen S.D. Hemmerich S. J. Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar). HNK-1-reactive sulfoglucuronyl glycolipids (23.Needham L.K. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1359-1363Crossref PubMed Scopus (180) Google Scholar), heparin oligosaccharides (24.Nelson R.M. Cecconi O. Roberts W.G. Aruffo A. Linhardt R.J. Bevilacqua M.P. Blood. 1993; 11: 3253-3258Crossref Google Scholar), and HS GAGs (25.Koenig A. Norgard-Sumnicht K. Linhardt R. Varki A. J. Clin. Invest. 1998; 101: 877-889Crossref PubMed Scopus (358) Google Scholar) bind L- and P-selectin. Our results showing that sulfation is required for versican's binding to L- and P-selectin (Figs. 1 and 4) are thus consistent with these previous findings. Extending these observations, we also showed that the sulfation of versican is required for the interaction with chemokines but not CD44 (Figs. 1 and 4). The absence of a sulfation requirement for versican's binding to CD44 is reminiscent of the binding of unsulfated GAG and HA to CD44 (26.Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2135) Google Scholar). Surface plasmon resonance analysis showed that the binding affinities of soluble monomeric L- and P-selectin to oversulfated CS/DS chains are higher than those for known ligands (Fig. 6 and Table II). It has been reported that monomeric L-selectin binds to immobilized glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) with low affinity (Kd = 108 μm) and with very fast association (≥105m−1 s−1) and dissociation (≥10 s−1) rates (27.Nicholson M.W. Barclay A.N. Singer M.S. Rosen S.D. van der Merwe P.A. J. Biol. Chem. 1998; 273: 763-770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). P-selectin has been reported to bind to P-selectin glycoprotein ligand-1 with relatively high affinity (Kd ∼300 nm) and with fast association (4.4 × 106m−1s−1) and dissociation (1.4 s−1) rates (28.Mehta P. Cummings R.D. McEver R.P. J. Biol. Chem. 1998; 273: 32506-32513Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). These properties have been proposed to be critical for the dynamic selectin-mediated rolling adhesion that is mediated by rapid adhesion and de-adhesion (27.Nicholson M.W. Barclay A.N. Singer M.S. Rosen S.D. van der Merwe P.A. J. Biol. Chem. 1998; 273: 763-770Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 28.Mehta P. Cummings R.D. McEver R.P. J. Biol. Chem. 1998; 273: 32506-32513Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). We speculate that the high-affinity binding of L- and P-selectin to oversulfated CS/DS chains with a slow dissociation rate (Table II) may enable rolling interactions with different rolling velocity and/or static adhesive interactions of leukocytes, if appropriate CS/DS chains are expressed locally. The oversulfated CS/DS chains interact with certain chemokines with high affinity as observed with the selectins (Table II). The high affinity binding of chemokines SLC, IP-10, and SDF-1β suggest that these chemokines might be readily trapped by oversulfated CS/DS chains in vivo. This hypothesis is supported by the surface plasmon resonance kinetic analysis, which demonstrated that the oversulfated CS/DS-chemokine complex formation is characterized by fast association rates (0.864 to 4.15 × 104m−1 s−1). It is conceivable that the oversulfated CS/DS-bound form of chemokines may not function as agonists for chemokine receptors, because those GAGs inhibit chemokine activity (Fig. 8). Rather, the oversulfated CS/DS-chemokine complex may function as a reservoir for chemokines in vivo. The slow dissociation rate (2.78 × 10−4 to 5.30 × 10−3 s−1) that is observed in the interaction of chemokines with oversulfated CS/DS chains supports this idea. Hints about the mechanism of CS/DS binding to chemokines are provided by the observation that versican interacts with SDF-1β but not SDF-1α (Figs. 4 and 5). Because SDF-1α and SDF-1β are produced by an alternative splicing of a single gene with the latter lacking only 4 amino acid residues in the C terminus (29.Shirozu M. Nakano T. Inazawa J. Tashiro K. Tada H. Shinohara T. Honjo T. Genomics. 1995; 28: 495-500Crossref PubMed Scopus (533) Google Scholar), versican, or its CS/DS chains, appears to interact with the chemokine's C terminus. Consistent with this idea, versican failed to bind to a mutant SLC that lacked the C-terminal basic amino acid clusters (Fig. 4), and CS B and CS E also failed to bind the C terminus truncated SLC. 2J. Hirose, H. Kawashima, and M. Miyasaka, submitted for publication. Therefore, these data strongly suggest that CS/DS interacts with the C terminus of these chemokines. Structural analysis indicated that GlcAβ1–3GalNAc(4,6-O-disulfate) units are present in the GAG moiety of versican (Fig. 3). Given that L- and P-selectin and chemokines bind preferentially to a tetrasaccharide composed of repeating GlcAβ1–3GalNAc(4,6-O-disulfate) units (Fig. 7), and that CD44 preferentially interacts with unsulfated or low-sulfated CS chains (Table II), it seems likely that GlcAβ1–3GalNAc(4,6-O-disulfate) units interact with selectins and chemokines if these units are present as a cluster in the GAG chain, whereas a different structure containing GlcAβ1–3GalNAc(4-O-sulfate) or GlcAβ1–3GalNAc(6-O-sulfate) appears to interact with CD44. Although CS B (DS) from pig skin inhibited the interaction of versican with L- and P-selectin and chemokines in our previous studies (4.Kawashima H. Hirose M. Hirose J. Nagakubo D. Plaas A.H.K. Miyasaka M. J. Biol. Chem. 2000; 275: 35448-35456Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 5.Hirose J. Kawashima H. Yoshie O. Tashiro K. Miyasaka M. J. Biol. Chem. 2001; 276: 5228-5234Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), the DS from rooster comb that was used in this study failed to inhibit the interaction (Fig. 2). Disaccharide composition analysis of these DS chains, however, showed no striking difference between them;i.e. DS from pig skin is composed of ΔDi-6S (1.3%), ΔDi-4S (91.5%), and ΔDi-di(2,4)S (7.2%), whereas DS from rooster comb is composed of ΔDi-0S (5.0%), ΔDi-6S (3.4%), ΔDi-4S (85.2%), and ΔDi-di(2, 4)S (6.3%) (data not shown). Because a highly sulfated oligosaccharide composed of repeating disulfated disaccharides interacted well with selectins and chemokines (Fig. 7), we think it possible that a difference in the amount of highly sulfated clusters containing IdoA(2-O-disulfate)α1–3GalNAc(4-O-disulfate) in these DS chains may in part explain the differential reactivity, although further structural characterization is required. Oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) have been found in squid cartilage (30.Suzuki S. Saito H. Yamagata T. Anno K. Seno N. Kawai Y. Furuhashi T. J. Biol. Chem. 1968; 243: 1543-1550Abstract Full Text PDF PubMed Google Scholar), mast cells (31.Katz H.R. Austen K.F. Caterson B. Stevens R.L. J. Biol. Chem. 1986; 261: 13393-13396Abstract Full Text PDF PubMed Google Scholar, 32.Stevens R.L. Fox C.C. Lichtenstein L.M. Austen K.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2284-2287Crossref PubMed Scopus (99) Google Scholar), neutrophils (33.Ohhashi Y. Hasumi F. Mori Y. Biochem. J. 1984; 217: 199-207Crossref PubMed Scopus (18) Google Scholar, 34.Petersen F. Brandt E. Lindahl U. Spillmann D. J. Biol. Chem. 1999; 274: 12376-12382Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), monocytes (35.Uhlin-Hansen L. Eskeland T. Kolset S.O. J. Biol. Chem. 1989; 264: 14916-14922Abstract Full Text PDF PubMed Google Scholar, 36.McGee M.P. Teuschler H. Parthasarathy N. Wagner W.D. J. Biol. Chem. 1995; 270: 26109-26115Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), glomeruli (37.Kobayashi S. Oguri K. Yaoita E. Kobayashi K. Okayama M. Biochim. Biophys. Acta. 1985; 841: 71-80Crossref PubMed Scopus (25) Google Scholar), and mesangial cells (38.Yaoita E. Oguri K. Okayama E. Kawasaki K. Kobayashi S. Kihara I. Okayama M. J. Biol. Chem. 1990; 265: 522-531Abstract Full Text PDF PubMed Google Scholar), and have been reported to interact with various biologically active molecules and regulate their functions in vitro. For example, CS E inhibits the adhesion of cortical neuronal cells to the neurotrophic factor midkine through direct interaction with midkine (39.Ueoka C. Kaneda N. Okazaki I. Nadanaka S. Muramatsu T. Sugahara K. J. Biol. Chem. 2000; 275: 37407-37413Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), promotes neurite outgrowth in embryonic day 18 rat hippocampal neurons (40.Nadanaka S. Clement A. Masayama K. Faissner A. Sugahara K. J. Biol. Chem. 1998; 273: 3296-3307Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), serves as a receptor for PF4 on the surface of neutrophils (34.Petersen F. Brandt E. Lindahl U. Spillmann D. J. Biol. Chem. 1999; 274: 12376-12382Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and inhibits procoagulant activity (36.McGee M.P. Teuschler H. Parthasarathy N. Wagner W.D. J. Biol. Chem. 1995; 270: 26109-26115Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Our results showing that oversulfated CS/DS chains containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) bind to proinflammatory molecules, such as selectins and chemokines, and regulate chemokine activity, further indicate the versatility of these oversulfated chains in regulating biological responses. It is now extremely important to understand the exact tissue distribution of the oversulfated CS/DS chains bearing the ability to interact with these proinflammatory molecules and the regulatory mechanisms whereby these GAGs are generated. We thank those who provided the antibodies and recombinant proteins listed under “Experimental Procedures.” We also thank Dr. Keiichi Yoshida (Tokyo Research Institute of Seikagaku Corporation) and Dr. Yoshiki Yamaguchi (Nagoya City University) for helpful discussions, and Momoyo Ueno (Kobe Pharmaceutical University), Bo-Gie Yang, and Yumi Takara for technical assistance." @default.
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W2017704712 title "Oversulfated Chondroitin/Dermatan Sulfates Containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) Interact with L- and P-selectin and Chemokines" @default.
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W2017704712 doi "https://doi.org/10.1074/jbc.m200396200" @default.
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