Biochemistry, Department of


Date of this Version



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.


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; Kd = 5.2 nM). The results indicate that Link domain N-glycans stabilize interactions that facilitate HA binding to HARE.