Identification of the Ca(2+)-independent endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells using a photoaffinity cross-linking reagent.
The Ca(2+)-independent endocytic hyaluronan (HA) receptor in rat liver sinusoidal endothelial cells (LECs) was identified using a novel cross-linking derivative of HA. The heterobifunctional, photoactivatable, reducible reagent sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) was coupled to the terminal amino group of uniquely modified HA-amine oligosaccharides (M(r) approximately 60,000) and subsequently iodinated. 125I-ASD-HA bound to cultured LECs with similar specificity and affinity as a previously characterized 125I-HA-amine/Bolton-Hunter adduct. Permeabilized LECs were incubated with 125I-ASD-HA with 10 mM EGTA and photolysed with UV light. Detergent extracts were reduced to release the HA oligosaccharides and radiolabeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Two polypeptides were consistently and equally labeled at M(r) = 175,000 and 166,000. Photoaffinity labeling of these two proteins was virtually identical in cultured LECs or membranes and was competed greater than 90% with a 100-fold excess of HA. As with the previously characterized bona fide LEC HA receptor, cross-linking was also competed by chondroitin sulfate and heparin, but less efficiently by chondroitin and not with galacturonan. We conclude that the Ca(2+)-independent LEC HA receptor is composed of at least two polypeptides of M(r) approximately 175,000 and 166,000 and may exist as a heterodimer of M(r) approximately 340,000. We also conclude that the LEC HA receptor is distinct from the CD44 family of HA-binding proteins.
Yannariello-Brown
J
J
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0647.
Frost
S J
SJ
Weigel
P H
PH
D000818:Animals; D001345:Autoradiography; D001386:Azides; D002118:Calcium / Q000502:physiology; D002413:Cations, Divalent; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D004727:Endothelium / Q000737:chemistry; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid; D008099:Liver / Q000737:chemistry* / Q000166:cytology; D008297:Male; D010777:Photochemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000032:analysis*; D013388:Succinimides
D001386:Azides; D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D018960:Hyaluronan Receptors; D011956:Receptors, Cell Surface; D013388:Succinimides; D006820:Hyaluronic Acid; C047257:sulfosuccinimidyl 2-(4-azidosalicylamido)ethyl-1,3'-dithiopropionate; D002118:Calcium