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

Authors

Ed Harris, University of Nebraska - LincolnFollow
Simon Parry, Imperial College, London
Mark Sutton-Smith, Imperial College, London
Madhu S. Pandey, University of Oklahoma Health Sciences Center
Maria Panico, Imperial College, London
Howard R. Morris, Imperial College, London
Stuart M. Haslam, Imperial College, London
Anne Dell, Imperial College, London
Paul H. Weigel, University of Oklahoma Health Sciences CenterFollow

Document Type

Article

Date of this Version

2010

Comments

Published in Glycobiology 20:8 (2010), pp. 991–1001; doi: 10.1093/glycob/cwq057 Copyright © 2010 Edward N. Harris, Simon Parry, Mark Sutton-Smith, Madhu S. Pandey, Maria Panico, Howard R. Morris, Stuart M. Haslam, Anne Dell, and Paul H. Weigel. Published by Oxford University Press. Used by permission.

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 ~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 N²²⁸⁰, 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.