IDENTIFICATION OF A 175 KDA PROTEIN AS THE LIGAND-BINDING SUBUNIT OF THE RAT-LIVER SINUSOIDAL ENDOTHELIAL-CELL HYALURONAN RECEPTOR

The rat liver sinusoidal endothelial cell (LEG) hyaluronan (HA) recept or was previously identified using a photoaffinity HA derivative (J. B iol, Chem., 267, 20451-20456, 1992), Two polypeptides with M(r) = 175, 000 and 166,000, were consistently crosslinked, suggesting that the LE C HA receptor is an oligomer, Whether one or both subunits participate in HA binding, was not determined, Here we investigate the HA-subunit interactions and the potential oligomeric nature of the LEC HA recept or, When Sephacryl-400 gel filtration chromatography was used to enric h the HA receptor, the 175 kDa polypeptide was the major band seen by SDS-PAGE analysis, Little staining was seen at 166 kDa, suggesting tha t the 175 kDa protein could be separated from the 166 kDa protein and still retain HA-binding activity, A ligand blot assay was used to dete rmine if each individual subunit could bind HA, LEC proteins were sepa rated by nonreducing SDS-PAGE, and then immobilized onto nitrocellulos e. I-125-HA bound to a 175 kDa polypeptide but not to the 166 kDa prot ein, A high molecular weight band of similar to 300,000 also bound I-1 25-HA. I-125-HA binding to the 175 and 300 kDa proteins showed the sam e specificity of competition with a panel of carbohydrates as the bona fide LEC HA receptor, The 175 kDa HA-binding subunit may be nonglobul ar (asymmetric), since its apparent size by SDS-PAGE is dependent on t he polyacrylamide gel pore size; M(r) increases as porosity decreases, LECs were crosslinked to an I-125-labeled photoaffinity HA derivative and the HA saccharides were then released with hyaluronidase. After S DS-PAGE without reduction, radio-labeled bands were seen at 175 and 16 6 kDa (3:1 ratio), and a high MW (similar to 300,000) species was also detected, These data support an oligomeric model of the LEC HA recept or, and show that the 175 kDa protein possesses HA-binding activity in dependent from the 166 kDa polypeptide.