Oversulfated Chondroitin/Dermatan Sulfates Containing GlcAβ1/IdoAα1–3GalNAc(4,6-O-disulfate) Interact with L- and P-selectin and Chemokines

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 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...

The interaction of versican with its binding partners

“…As CD44 binds to both hyaluronan and versican, it is possible that the three may form complexes together. However, as versican and CD44 bind following hyaluronidase treatment, the complex association does not depend on the presence of hyaluronan [73].…”

“…Thus, versican appears to interact with selectins via its GAGs (Fig. 2), and may only do so when they are oversulfated [73]. Specific tetrasaccharides involved in the interaction were identified.…”

“…Interestingly, the binding of either L-or P-selectin to versican was inhibited with the addition of the solubilized other, but CD44 binding was not affected in the presence of selectins. This suggests that different GAG sites are responsible for these interactions [73].…”

Immunological Activity of Chondroitin Sulfate

“…Chondroitin sulfate-B [dermatan sulfate (DS)], while nearly structurally identical to CS-A (it contains IdoA instead of GlcA), shows lower activity. This is surprising as the greater flexibility of the IdoA residue in CS-B is commonly used to explain the propensity of IdoA-containing GAGs to interact with proteins and display a large number of different biological activities (Kawashima et al, 2002). Examination of the disulfated CS samples shows that the effects of CS-E on the Th2-inhibitory activity were higher than those of CS-D or -A (Fig.…”

Expression of potential lymphocyte trafficking mediator molecules in the mammary gland

“…Sgp90 was characterized as sialomucin CD34 and podocalyxin-like protein (PCLP) [5,43], however Sgp200 is not yet characterized. L-selectin recognizes a specific sulfated form of these molecules and basic structure was reported [13,26].…”