Here we show that a large chondroitin sulfate proteoglycan, versican, derived from a renal adenocarcinoma cell line ACHN, binds L-selectin, P-selectin, and CD44. The binding was mediated by the interaction of the chondroitin sulfate (CS) chain of versican with the carbohydrate-binding domain of L-and P-selectin and CD44. The binding of versican to L-and P-selectin was inhibited by CS B, CS E, and heparan sulfate (HS) but not by any other glycosaminoglycans tested. On the other hand, the binding to CD44 was inhibited by hyaluronic acid, chondroitin (CH), CS A, CS B, CS C, CS D, and CS E but not by HS or keratan sulfate. A crossblocking study indicated that L-and P-selectin recognize close or overlapping sites on versican, whereas CD44 recognizes separate sites. We also show that soluble L-and P-selectin directly bind to immobilized CS B, CS E, and HS and that soluble CD44 directly binds to immobilized hyaluronic acid, CH, and all the CS chains examined. Consistent with these results, structural analysis showed that versican is modified with at least CS B and CS C. Thus, proteoglycans sufficiently modified with the appropriate glycosaminoglycans should be able to bind L-selectin, P-selectin, and/or CD44.

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 sulfationdependent but the interaction with CD44 is sulfationindependent. Consistently, versican's binding to L-and P-selectin and chemokines is specifically inhibited by oversulfated CS/DS chains containing GlcA␤1-3Gal-NAc(4,6-O-disulfate) or IdoA␣1-3GalNAc(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 GlcA␤1/IdoA␣1-3GalNAc(4,6-O-disulfate) bind directly to selectins and chemokines with high affinity (K d 21.1 to 293 nM). In addition, a tetrasaccharide fragment of repeating GlcA␤1-3GalNAc(4,6-Odisulfate) units directly interacts with L-and P-selectin and chemokines and oversulfated CS/DS chains containing GlcA␤1/IdoA␣1-3GalNAc(4,6-O-disulfate) inhibit chemokine-induced Ca 2؉ mobilization. Taken together, our results show that oversulfated CS/DS chains containing GlcA␤1/IdoA␣1-3GalNAc(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.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, 2). 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, 4), and various chemokines (5), 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). Alternative splicing of the versican gene generates four ver...

We previously reported that versican, a large chondroitin sulfate proteoglycan, isolated from a renal adenocarcinoma cell line, ACHN, binds L-selectin. Here we report that versican also binds certain chemokines and regulates chemokine function. This binding was strongly inhibited by the chondroitinase digestion of versican or by the addition of soluble chondroitin sulfate (CS) B, CS E, or heparan sulfate. Furthermore, these glycosaminoglycans (GAGs) could bind directly to the chemokines that bind versican. Thus, versican appears to interact with chemokines via its GAGs. We next examined if versican or GAGs affect secondary lymphoid tissue chemokine (SLC)-induced integrin activation and Ca 2؉ mobilization in lymphoid cells expressing a receptor for SLC, CC chemokine receptor 7. Interestingly, whereas heparan sulfate supported both ␣ 4 ␤ 7 integrindependent binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-IgG and Ca 2؉ mobilization induced by SLC, versican or CS B inhibited these cellular responses, and the extent of inhibition was dependent on the dose of versican or CS B added. These findings suggest that different proteoglycans have different functions in the regulation of chemokine activities and that versican may negatively regulate the function of SLC via its GAG chains.

Clostridium butyricum could produce hydrogen from a sweet potato starch residue upon supplementation of nitrogen sources. A repeated batch culture using a mixed culture of C. butyricum and Enterobacter aerogenes produced hydrogen with a yield of 2.4 mol H2/mol glucose under a controlled culture pH of 5.25 in a medium consisting of the sweet potato starch residue and 0.1% Polypepton without addition of any reducing agents. Rhodobacter sp. M-19 produced hydrogen from the supernatant of the culture broth obtained in the repeated batch culture containing C. butyricum and E. aerogenes when 50 microg/l Na2MoO4.2H2O and 20 mg/l EDTA were added to the supernatant and it was cultured under a controlled culture pH of 7.5. A high yield of hydrogen of 7.0 mol H2/mol glucose from the starch remaining in the starch residue was attained in two-step repeated batch cultures containing C. butyricum and E. aerogenes, and by Rhodobacter sp. M-19.

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