title: N-Glycans on the link domain of human HARE/Stabilin-2 are needed for hyaluronan binding to purified ecto-domain, but not for cellular endocytosis of hyaluronan.
abstract: The hyaluronic acid receptor for endocytosis (HARE)/Stabilin-2 is the primary systemic scavenger receptor for 13 ligands including hyaluronan (HA), heparin and chondroitin sulfates. Most ligand-binding sites are within the 190 kDa isoform, which contains approximately 25 kDa of N-glycans and is the C-terminal half of the full-length 315 kDa HARE. Glycoproteomic analyses of purified recombinant human 190-HARE ecto-domain identified a diverse population of glycans at 10 of 17 consensus sites. The most diversity (and the only sialylated structures) occurred at N(2280), within the HA-binding Link domain. To determine if these N-glycans are required for HA binding, we created human Flp-In 293 cell lines expressing membrane-bound or soluble ecto-domain variants of 190-HARE(N2280A). Membrane-bound HARE lacking Link domain N-glycans mediated rapid HA endocytosis, but purified 190-HARE(N2280A) ecto-domain showed little or no HA binding in ELISA-like, HA-HARE pull-down assays or by surface plasmon resonance analysis (which detected very high apparent affinity for 190-HARE ecto-domain binding to HA; K(d) = 5.2 nM). The results indicate that Link domain N-glycans stabilize interactions that facilitate HA binding to HARE.
authors.affiliation: Department of Biochemistry & Molecular Biology and The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.
authors.forename: Edward N
authors.initials: EN
authors.lastname: Harris
authors.forename: Simon
authors.initials: S
authors.lastname: Parry
authors.forename: Mark
authors.initials: M
authors.lastname: Sutton-Smith
authors.forename: Madhu S
authors.initials: MS
authors.lastname: Pandey
authors.forename: Maria
authors.initials: M
authors.lastname: Panico
authors.forename: Howard R
authors.initials: HR
authors.lastname: Morris
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Haslam
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.forename: Paul H
authors.initials: PH
authors.lastname: Weigel
mesh_terms: D001665:Binding Sites; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004705:Endocytosis; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D015816:Cell Adhesion Molecules, Neuronal; D011134:Polysaccharides; C449927:STAB2 protein, human; D006820:Hyaluronic Acid
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