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

Edward N Harris, Simon Parry, Mark Sutton-Smith, Madhu S. Pandey, Maria Panico, Howard R. Morris, Stuart M. Haslam, Anne Dell, Paul H. Weigel

Research output: Contribution to journalArticle

11 Citations (Scopus)

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

Original languageEnglish (US)
Article numbercwq057
Pages (from-to)991-1001
Number of pages11
JournalGlycobiology
Volume20
Issue number8
DOIs
StatePublished - Apr 14 2010

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Hyaluronic Acid
Endocytosis
Polysaccharides
Ligands
Membranes
Scavenger Receptors
Surface Plasmon Resonance
Chondroitin Sulfates
Surface plasmon resonance

Keywords

  • Coated pit mediated
  • Conformation
  • Glycosaminoglycan turnover
  • HA binding affinity
  • Stabilin-2

ASJC Scopus subject areas

  • Biochemistry

Cite this

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. / Harris, Edward N; Parry, Simon; Sutton-Smith, Mark; Pandey, Madhu S.; Panico, Maria; Morris, Howard R.; Haslam, Stuart M.; Dell, Anne; Weigel, Paul H.

In: Glycobiology, Vol. 20, No. 8, cwq057, 14.04.2010, p. 991-1001.

Research output: Contribution to journalArticle

Harris, EN, Parry, S, Sutton-Smith, M, Pandey, MS, Panico, M, Morris, HR, Haslam, SM, Dell, A & Weigel, PH 2010, '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', Glycobiology, vol. 20, no. 8, cwq057, pp. 991-1001. https://doi.org/10.1093/glycob/cwq057
Harris, Edward N ; Parry, Simon ; Sutton-Smith, Mark ; Pandey, Madhu S. ; Panico, Maria ; Morris, Howard R. ; Haslam, Stuart M. ; Dell, Anne ; Weigel, Paul H. / 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. In: Glycobiology. 2010 ; Vol. 20, No. 8. pp. 991-1001.
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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; Kd = 5.2 nM). The results indicate that Link domain Nglycans stabilize interactions that facilitate HA binding to HARE.",
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