Journal
GLYCOBIOLOGY
Volume 20, Issue 8, Pages 991-1001Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/glycob/cwq057
Keywords
coated pit mediated; conformation; glycosaminoglycan turnover; HA binding affinity; Stabilin-2
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Funding
- National Institutes of Health/National Institute of General Medical Sciences [GM69961]
- Biotechnology and Biological Sciences Research Council [BBF0083091, B19088]
- Biotechnology and Biological Sciences Research Council [B19088] Funding Source: researchfish
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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 similar to 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.
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