Purification and subunit characterization of the rat liver endocytic hyaluronan receptor

The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cell s (LECs) is responsible for the clearance of HA and other glycosaminoglycan s from the circulation in mammals. We report here for the first time the pu rification of this liver HA receptor. Using lectin and immune-affinity chro matography, two HA receptor species were purified from detergent-solubilize d membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylam ide gel electrophoresis (PAGE), these two proteins migrated at 175- and sim ilar to 300 kDa corresponding to the two species previously identified by p hotoaffinity 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 r enaturation, After reduction, the 175-kDa protein migrates as a similar to 185-kDa protein and is not able to bind HA The 300s-kDa HA receptor is a co mplex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at similar to 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 t hree subunits of the 300-kDa species contain N-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosi dase F, Both of the deglycosylated, nonreduced HA receptor proteins still b ind HA.