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J. Biol. Chem., Vol. 279, Issue 35, 36201-36209, August 27, 2004

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Endocytic Function, Glycosaminoglycan Specificity, and Antibody Sensitivity of the Recombinant Human 190-kDa Hyaluronan Receptor for Endocytosis (HARE)^*

Edward N. Harris, Janet A. Weigel, and Paul H. Weigel

From the Department of Biochemistry & Molecular Biology, The Oklahoma Center for Medical Glycobiology and University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190

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 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 ¹²⁵I-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 8.5 min. The average K_d for the binding of HA to the 190-kDa hHARE at 4 °C was 7 nM with 118,000 total HA binding sites per cell. Competition studies at 37 °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 °C, none of the glycosaminoglycans tested, except HA, competed for ¹²⁵I-HA binding by 190hHARE cells at 4 °C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit ¹²⁵I-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.

Received for publication, May 12, 2004

^* This work was supported by NIGMS Grants GM35978 and GM69961 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry & Molecular Biology, The Oklahoma Center for Medical Glycobiology and University of Oklahoma Health Sciences Center, BMSB, Rm. 860, Box 26901, Oklahoma City, OK 73190. Tel.: 405-271-1288; Fax: 405-271-3092; E-mail: paul-weigel{at}ouhsc.edu.

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