Identification and characterization of a divalent cation-dependent glycosaminoglycan-binding protein from rat liver endothelium.
Rat liver endothelial cells (LECs) express a membrane-associated Ca2+-dependent hyaluronan-binding activity (CaHA-BP) which is distinct from the Ca2+-independent, endocytic LEC HA receptor (Yannariello-Brown et al., J. Cell Biochem., 48, 73-80, 1992). The CaHA-BP is specific for a subset of glycosaminoglycans, since Ca2+-dependent binding of 125I-HA (approximately 80 kDa) to LECs was competed with a 100-fold excess (w/w) of HA, chondroitin sulfate, and heparin, but not with chondroitin. The CaHA-BP activity on intact LECs was pH-dependent. Optimal binding occurred at pH 6.0; no binding was detected at pH values <or= 5 or >or= 9. 125I-HA, pre-bound in the presence of Ca2+, could also be dissociated with an acidic buffer (pH 5.0), as well as the divalent cation chelators EDTA and EGTA. 125I-HA binding was stimulated by divalent cations other than Ca2+, such as Mg2+, Mn2+, and Ba2+; with the exception of Zn2+. A photoaffinity crosslinking reagent (125I-ASD-HA) was used to identify specifically crosslinked polypeptides on LECs. In the absence of Ca2+, and in the presence of EGTA, only bands at 175/166 kDa were consistently crosslinked. These bands have been previously identified as the LEC Ca2+-independent endocytic HA receptor (Yannariello-Brown et al., J. Biol. Chem., 267, 20451-20455, 1992). In the presence of Ca2+, crosslinking was consistently seen to a 68 kDa polypeptide. Crosslinking was competed with a 100-fold excess (w/w) of HA. These and other data suggest that a 68 kDa protein is the most likely candidate for the CaHA-BP in LECs.
Yannariello-Brown
J
J
Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston 77555-0787, USA.
D000818:Animals; D001386:Azides; D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002413:Cations, Divalent; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D003470:Culture Media; D004727:Endothelium / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D010777:Photochemistry; D051381:Rats; D013388:Succinimides
D001386:Azides; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D003470:Culture Media; C082315:GAG-BP protein, Streptococcus pyogenes; D007457:Iodine Radioisotopes; D013388:Succinimides; C063086:sulfosuccinimidyl 3-((2-(4-azidosalicylamido)ethyl)dithio)propionate; D006820:Hyaluronic Acid; D002118:Calcium