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.
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.
Harris
Edward N
EN
Department of Biochemistry & Molecular Biology and The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.
Parry
Simon
S
Sutton-Smith
Mark
M
Pandey
Madhu S
MS
Panico
Maria
M
Morris
Howard R
HR
Haslam
Stuart M
SM
Dell
Anne
A
Weigel
Paul H
PH
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
D015816:Cell Adhesion Molecules, Neuronal; D011134:Polysaccharides; C449927:STAB2 protein, human; D006820:Hyaluronic Acid
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