Purification and subunit characterization of the rat liver endocytic hyaluronan receptor.
The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cells (LECs) is responsible for the clearance of HA and other glycosaminoglycans from the circulation in mammals. We report here for the first time the purification of this liver HA receptor. Using lectin and immuno-affinity chromatography, two HA receptor species were purified from detergent-solubilized membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), these two proteins migrated at 175- and approximately 300 kDa corresponding to the two species previously identified by photoaffinity labeling of live cells as the HA receptor (Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) J. Biol. Chem. 267, 20451-20456). These two proteins co-purify in a molar ratio of 2:1 (175:300), and both proteins are active, able to bind HA after SDS-PAGE, electrotransfer, and renaturation. After reduction, the 175-kDa protein migrates as a approximately 185-kDa protein and is not able to bind HA. The 300-kDa HA receptor is a complex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at approximately 260, 230, and 97 kDa. These proteins designated, respectively, the alpha, beta, and gamma subunits are present in a molar ratio of 1:1:1 and are also unable to bind HA when reduced. The 175-kDa protein and all three subunits of the 300-kDa species contain N-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosidase F. Both of the deglycosylated, nonreduced HA receptor proteins still bind HA.
Zhou
B
B
Department of Biochemistry, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Oka
J A
JA
Singh
A
A
Weigel
P H
PH
D000818:Animals; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D006820:Hyaluronic Acid / Q000378:metabolism; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008958:Models, Molecular; D008970:Molecular Weight; D020033:Protein Isoforms / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D018960:Hyaluronan Receptors; D020033:Protein Isoforms; D006820:Hyaluronic Acid