The ligand-binding profile of HARE: hyaluronan and chondroitin sulfates A, C, and D bind to overlapping sites distinct from the sites for heparin, acetylated low-density lipoprotein, dermatan sulfate, and CS-E

Abstract

The hyaluronic acid receptor for endocytosis (HARE)/ Stabilin-2 is the primary systemic scavenger receptor for hyaluronan (HA), the chondroitin sulfates (CS), dermatan sulfate (DS), and nonglycosaminoglycan (GAG) ligands such as acetylated low-density lipoprotein (AcLDL), pro-collagen propeptides, and advanced glycation end products. We recently discovered that HARE is also a systemic scavenger receptor for heparin (Hep) (Harris EN, Weigel JA, Weigel PH. 2008. The human hyaluronan receptor for endocytosis [HARE/Stabilin-2] is a systemic clearance receptor for heparin. J Biol Chem. 283:17341-17350). Our goal was to map the binding sites of eight different ligands within HARE. We used biotinylated GAGs and radio-iodinated streptavidin or AcLDL to assess the binding activities of ligands directly or indirectly (by competition with unlabeled ligands) in endocytosis assays using stable cell lines expressing the 315 or 190 kDa HA receptor for endocytosis (315- or 190-HARE) isoforms, and ELISA-like assays, with purified recombinant soluble 190-HARE ecto-domain. For example, Hep binding to HARE was competed by DS, CS-E, AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. (125)I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both Hep and HA to some degree. Hep and HA binding or endocytosis is mutually inclusive; binding of these two GAGs occurs with functionally separate, noncompetitive, and apparently noninteracting domains. Thus, HARE binds to HA and Hep simultaneously. Although the domain(s) responsible for Hep binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding activity map for multiple ligands of HARE.

Genome-wide association studies identify genetic loci for low von Willebrand factor levels

“…STAB2 can bind various ligands, such heparin, LDL, bacteria and advanced glycosylation products, and subjects them to endocytosis. 40 STAB2 variation might be important in the regulation of VWF levels via the clearance of VWF molecules.…”

Inhibition of Stabilin-2 elevates circulating hyaluronic acid levels and prevents tumor metastasis

“…Stab2 also recognizes membrane phosphatidylserine of apoptotic cells (11). Previous studies found that unlabeled chondroitin sulfate inhibited the uptake of 125 -I-HA (12), and that ac-LDL binding to Stab2 was partially competed by heparin and dextran sulfate, but not competed by HA (13). These findings suggest that the HA binding site overlaps with the binding site of chondroitin sulfate but differs from the binding sites of ac-LDL, heparin, and dextran sulfate.…”

Probing Structural Selectivity of Synthetic Heparin Binding to Stabilin Protein Receptors

“…Anti-V5 antibodies and resins were from Bethyl Laboratories (Montgomery, TX). Other materials, reagents, and kits were obtained as described recently (9). Tris-buffered saline with Tween 20 (TBST) contains 20 mM Tris-HCl, pH 7.0, 150 mM NaCl, and 0.1% Tween 20.…”

“…Stab2 receptors are also responsible for clearing other glycosaminoglycans, including hyaluronic acid and chondroitin sulfates. It is known that the Stab2 binding domain(s) for heparin are distinct from the Link domain for binding hyaluronic acid and chondroitin sulfates (9).…”