Characterization and purification of the hyaluronan-receptor on liver endothelial cells.
In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.
Forsberg
N
N
Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
Gustafson
S
S
D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004586:Electrophoresis / Q000379:methods; D004727:Endothelium / Q000378:metabolism; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid / Q000378:metabolism*; D007120:Immunochemistry; D008099:Liver / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000302:isolation & purification
D018960:Hyaluronan Receptors; D011956:Receptors, Cell Surface; D006820:Hyaluronic Acid