title: Expression, processing, and glycosaminoglycan binding activity of the recombinant human 315-kDa hyaluronic acid receptor for endocytosis (HARE).
abstract: The hyaluronic acid (HA) receptor for endocytosis (HARE; also designated stabilin-2 and FEEL-2) mediates systemic clearance of glycosaminoglycans from the circulatory and lymphatic systems via coated pit-mediated uptake. HARE is primarily found as two isoforms (315- and 190-kDa) in sinusoidal endothelial cells of the liver, lymph node, and spleen. Here we characterize the ligand specificity and function of the large stably expressed 315-HARE isoform in Flp-In 293 cell lines. Like human spleen sinusoidal endothelial cells, Flp-In 293 cell lines transfected with a single cDNA encoding the full-length 315-HARE express both the 315-kDa and the proteolytically truncated 190-kDa isoforms in a ratio of approximately 3-4:1. The 190-kDa HARE isoform generated from the 315-kDa HARE and the 315-kDa HARE specifically bound 125I-HA. Like the 190-kDa HARE expressed alone (Harris, E. N., Weigel, J. A., and Weigel, P. H. (2004) J. Biol. Chem. 279, 36201-36209), the 190- and 315-kDa HARE isoforms expressed in 315-HARE cell lines were recognized by anti-HARE monoclonal antibodies 30, 154, and 159. All 315-HARE cell lines could endocytose and degrade 125I-HA. Competition studies with live cells indicate that 190-HARE and 315-HARE bind HA with higher apparent affinity (Kd approximately 10-20 nM) than chondroitin sulfate (CS) types A, C, D, or E. Only slight competition of HA endocytosis was observed with CS-B (dermatan sulfate) and chondroitin. Direct binding assays with the 315-HARE ectodomain revealed high affinity HA binding, and lower binding affinities for CS-C, CS-D, and CS-E. A majority of each HARE isoform was intracellular, within the endocytic system, suggesting transient surface residency typical of an active endocytic recycling receptor.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
authors.forename: Edward N
authors.initials: EN
authors.lastname: Harris
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Kyosseva
authors.forename: Janet A
authors.initials: JA
authors.lastname: Weigel
authors.forename: Paul H
authors.initials: PH
authors.lastname: Weigel
mesh_terms: D001483:Base Sequence; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism*; D002460:Cell Line; D003124:Colorimetry; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004705:Endocytosis / Q000502:physiology*; D005822:Genetic Vectors; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D015816:Cell Adhesion Molecules, Neuronal; D017931:DNA Primers; D018076:DNA, Complementary; D006025:Glycosaminoglycans; D011994:Recombinant Proteins; C449927:STAB2 protein, human; D006820:Hyaluronic Acid