Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE).
The human hyaluronan receptor for endocytosis (hHARE) mediates the endocytic clearance of hyaluronan (HA) and chondroitin sulfate from lymph fluid and blood. Two hHARE isoforms (190 and 315 kDa) are present in sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). Here we report the specificity and function of the 190-kDa HARE, expressed without the larger isoform, in Flp-In 293 cell lines (190hHARE cells). Like the native protein, recombinant hHARE contains approximately 25 kDa of N-linked oligosaccharides, binds HA in a ligand blot assay, cross-reacts with three anti-rat HARE monoclonal antibodies, and is inactivated by reduction. The 190hHARE cell lines mediated rapid, continuous (125)I-HA endocytosis and degradation for >1 day. About 30-50% of the total cellular receptors were on the cell surface, and their recycling time for reutilization was approximately 8.5 min. The average K(d) for the binding of HA to the 190-kDa hHARE at 4 degrees C was 7 nm with 118,000 total HA binding sites per cell. Competition studies at 37 degrees C indicated that the 190-kDa hHARE binds HA and chondroitin better than dermatan sulfate and chondroitin sulfates A, C, D, and E, but it does not bind to heparin, heparan sulfate, or keratan sulfate. Although competition was observed at 37 degrees C, none of the glycosaminoglycans tested, except HA, competed for (125)I-HA binding by 190hHARE cells at 4 degrees C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit (125)I-HA uptake mediated by the 175-kDa rat HARE, partially blocked HA endocytosis by the 190-kDa hHARE. We conclude that the 190-kDa hHARE can function independently of other hHARE isoforms to mediate the endocytosis of multiple glycosaminoglycans. Furthermore, the rat and human small HARE isoforms have different glycosaminoglycan specificities and sensitivities to inhibition by cross-reacting antibodies.
Harris
Edward N
EN
Department of Biochemistry & Molecular Biology, The Oklahoma Center for Medical Glycobiology and University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.
Weigel
Janet A
JA
Weigel
Paul H
PH
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015153:Blotting, Western; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000737:chemistry; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004306:Dose-Response Relationship, Immunologic; D004705:Endocytosis; D042783:Endothelial Cells / Q000378:metabolism; D005822:Genetic Vectors; D006025:Glycosaminoglycans / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors; D014162:Transfection
D000906:Antibodies; D000911:Antibodies, Monoclonal; D015816:Cell Adhesion Molecules, Neuronal; D002809:Chondroitin Sulfates; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D008024:Ligands; D009844:Oligosaccharides; D020033:Protein Isoforms; D011994:Recombinant Proteins; C449927:STAB2 protein, human