Expression, processing, and glycosaminoglycan binding activity of the recombinant human 315-kDa Hyaluronic Acid Receptor for Endocytosis (HARE)

Edward N. Harris, Svetlana V. Kyosseva, Janet A. Weigel, Paul H. Weigel

Research output: Contribution to journalArticle

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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 ∼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∼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.

Original languageEnglish (US)
Pages (from-to)2785-2797
Number of pages13
JournalJournal of Biological Chemistry
Volume282
Issue number5
DOIs
StatePublished - Jan 2 2007

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Hyaluronic Acid
Endocytosis
Glycosaminoglycans
Human Activities
Processing
Protein Isoforms
Chondroitin Sulfates
Cells
Cell Line
Dermatan Sulfate
Endothelial cells
Spleen
Endothelial Cells
Chondroitin
Lymphatic System

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Expression, processing, and glycosaminoglycan binding activity of the recombinant human 315-kDa Hyaluronic Acid Receptor for Endocytosis (HARE). / Harris, Edward N.; Kyosseva, Svetlana V.; Weigel, Janet A.; Weigel, Paul H.

In: Journal of Biological Chemistry, Vol. 282, No. 5, 02.01.2007, p. 2785-2797.

Research output: Contribution to journalArticle

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