Glycobiology vol 7 no 1 pp. 15-21, 1997
© 1997
research-article |
Identification of a 175 kDa protein as the ligand-binding subunit of the rat liver sinusoidal endothelial cell hyaluronan receptor
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center Oklahoma City, OK 73190, USA
1Departments of Ophthalmology & Visual Sciences, and Human Biological Chemistiy & Genetics, University of Texas Medical Branch, Galveston, TX 77555-0787, USA
2To whom correspondence should be addressed
Received on March 11, 1996; revised on August 13, 1996; accepted on August 23, 1996
The rat liver sinusoidal endothelial cell (LEC) hyaluronan (HA) receptor was previously identified using a photoaffinity HA derivative (J. BioL Chem., 267, 20451–20456, 1992). Two polypeptides with Mr = 175,000 and 166,000, were consistently crosslinked, suggesting that the LEC 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 receptor. When Sephacryl-400 gel filtration chromatography was used to enrich the HA receptor, the 175 kDa polypeptide was the major band seen by SDS-PAGE analysis. Little staining was seen at 166 kDa, suggesting that 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 determine if each individual subunit could bind HA. LEC proteins were separated by nonreducing SDS-PAGE, and then immobilized onto nitrocellulose. 125I-HA bound to a 175 kDa polypeptide but not to the 166 kDa protein. A high molecular weight band of 
300,000 also bound 125I-HA. 125I-HA binding to the 175 and 300 kDa proteins showed the same specificity of competition with a panel of carbohydrates as the bona fide EEC HA receptor. The 175 kDa HA-binding subunit may be nonglobular (asymmetric), since its apparent size by SDS-PAGE is dependent on the polyacrylamide gel pore size; Mr increases as porosity decreases. LECs were crosslinked to an 125I-labeled photoaffinity HA derivative and the HA saccharides were then released with hyaluronidase. After SDS-PAGE without reduction, radio-labeled bands were seen at 175 and 166 kDa (3:1 ratio), and a high MW (300,000) species was also detected. These data support an oligomeric model of the EEC HA receptor, and show that the 175 kDa protein possesses HA-binding activity independent from the 166 kDa polypeptide.
hyaluronan receptor endocytosis ligand blot assay liver sinusoidal endothelial cells
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