Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE)

Edward N. Harris, Janet A. Weigel, Paul H. Weigel

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Abstract

The human hyaluronan receptor for endocytosis (hHARE) mediates the endocytic clearance of hyaluronan (HA) and chondroitin sulfate from lymph fluid and blood. Two hHARE isoforms (190 and 315 kDa) are present in sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). Here we report the specificity and function of the 190-kDa HARE, expressed without the larger isoform, in Flp-In 293 cell lines (19OhHARE cells). Like the native protein, recombinant hHARE contains ∼25 kDa of N-linked oligosaccharides, binds HA in a ligand blot assay, cross-reacts with three anti-rat HARE monoclonal antibodies, and is inactivated by reduction. The 190hHARE cell lines mediated rapid, continuous 125I-HA endocytosis and degradation for >1 day. About 30-50% of the total cellular receptors were on the cell surface, and their recycling time for reutilization was ∼8.5 min. The average Kd for the binding of HA to the 190-kDa hHARE at 4 °C was 7 nM with 118,000 total HA binding sites per cell. Competition studies at 37 °C indicated that the 190-kDa hHARE binds HA and chondroitin better than dermatan sulfate and chondroitin sulfates A, C, D, and E, but it does not bind to heparin, heparan sulfate, or keratan sulfate. Although competition was observed at 37 °C, none of the glycosaminoglycans tested, except HA, competed for 125I-HA binding by 19OhHARE cells at 4 °C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit 125I-HA uptake mediated by the 175-kDa rat HARE, partially blocked HA endocytosis by the 190-kDa hHARE. We conclude that the 190-kDa hHARE can function independently of other hHARE isoforms to mediate the endocytosis of multiple glycosaminoglycans. Furthermore, the rat and human small HARE isoforms have different glycosaminoglycan specificities and sensitivities to inhibition by cross-reacting antibodies.

Original languageEnglish (US)
Pages (from-to)36201-36209
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number35
DOIs
StatePublished - Aug 27 2004

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CD44 Antigens
Antibody Specificity
Endocytosis
Glycosaminoglycans
Hyaluronic Acid
Sensitivity and Specificity
Antibodies
Protein Isoforms
Chondroitin Sulfates
Monoclonal Antibodies
Cells
Chondroitin
Keratan Sulfate
Glycomics
Heparitin Sulfate
Endothelial cells
Cell Line
Oligosaccharides
Recombinant Proteins
Liver

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE). / Harris, Edward N.; Weigel, Janet A.; Weigel, Paul H.

In: Journal of Biological Chemistry, Vol. 279, No. 35, 27.08.2004, p. 36201-36209.

Research output: Contribution to journalArticle

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abstract = "The human hyaluronan receptor for endocytosis (hHARE) mediates the endocytic clearance of hyaluronan (HA) and chondroitin sulfate from lymph fluid and blood. Two hHARE isoforms (190 and 315 kDa) are present in sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). Here we report the specificity and function of the 190-kDa HARE, expressed without the larger isoform, in Flp-In 293 cell lines (19OhHARE cells). Like the native protein, recombinant hHARE contains ∼25 kDa of N-linked oligosaccharides, binds HA in a ligand blot assay, cross-reacts with three anti-rat HARE monoclonal antibodies, and is inactivated by reduction. The 190hHARE cell lines mediated rapid, continuous 125I-HA endocytosis and degradation for >1 day. About 30-50{\%} of the total cellular receptors were on the cell surface, and their recycling time for reutilization was ∼8.5 min. The average Kd for the binding of HA to the 190-kDa hHARE at 4 °C was 7 nM with 118,000 total HA binding sites per cell. Competition studies at 37 °C indicated that the 190-kDa hHARE binds HA and chondroitin better than dermatan sulfate and chondroitin sulfates A, C, D, and E, but it does not bind to heparin, heparan sulfate, or keratan sulfate. Although competition was observed at 37 °C, none of the glycosaminoglycans tested, except HA, competed for 125I-HA binding by 19OhHARE cells at 4 °C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit 125I-HA uptake mediated by the 175-kDa rat HARE, partially blocked HA endocytosis by the 190-kDa hHARE. We conclude that the 190-kDa hHARE can function independently of other hHARE isoforms to mediate the endocytosis of multiple glycosaminoglycans. Furthermore, the rat and human small HARE isoforms have different glycosaminoglycan specificities and sensitivities to inhibition by cross-reacting antibodies.",
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N2 - The human hyaluronan receptor for endocytosis (hHARE) mediates the endocytic clearance of hyaluronan (HA) and chondroitin sulfate from lymph fluid and blood. Two hHARE isoforms (190 and 315 kDa) are present in sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). Here we report the specificity and function of the 190-kDa HARE, expressed without the larger isoform, in Flp-In 293 cell lines (19OhHARE cells). Like the native protein, recombinant hHARE contains ∼25 kDa of N-linked oligosaccharides, binds HA in a ligand blot assay, cross-reacts with three anti-rat HARE monoclonal antibodies, and is inactivated by reduction. The 190hHARE cell lines mediated rapid, continuous 125I-HA endocytosis and degradation for >1 day. About 30-50% of the total cellular receptors were on the cell surface, and their recycling time for reutilization was ∼8.5 min. The average Kd for the binding of HA to the 190-kDa hHARE at 4 °C was 7 nM with 118,000 total HA binding sites per cell. Competition studies at 37 °C indicated that the 190-kDa hHARE binds HA and chondroitin better than dermatan sulfate and chondroitin sulfates A, C, D, and E, but it does not bind to heparin, heparan sulfate, or keratan sulfate. Although competition was observed at 37 °C, none of the glycosaminoglycans tested, except HA, competed for 125I-HA binding by 19OhHARE cells at 4 °C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit 125I-HA uptake mediated by the 175-kDa rat HARE, partially blocked HA endocytosis by the 190-kDa hHARE. We conclude that the 190-kDa hHARE can function independently of other hHARE isoforms to mediate the endocytosis of multiple glycosaminoglycans. Furthermore, the rat and human small HARE isoforms have different glycosaminoglycan specificities and sensitivities to inhibition by cross-reacting antibodies.

AB - The human hyaluronan receptor for endocytosis (hHARE) mediates the endocytic clearance of hyaluronan (HA) and chondroitin sulfate from lymph fluid and blood. Two hHARE isoforms (190 and 315 kDa) are present in sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). Here we report the specificity and function of the 190-kDa HARE, expressed without the larger isoform, in Flp-In 293 cell lines (19OhHARE cells). Like the native protein, recombinant hHARE contains ∼25 kDa of N-linked oligosaccharides, binds HA in a ligand blot assay, cross-reacts with three anti-rat HARE monoclonal antibodies, and is inactivated by reduction. The 190hHARE cell lines mediated rapid, continuous 125I-HA endocytosis and degradation for >1 day. About 30-50% of the total cellular receptors were on the cell surface, and their recycling time for reutilization was ∼8.5 min. The average Kd for the binding of HA to the 190-kDa hHARE at 4 °C was 7 nM with 118,000 total HA binding sites per cell. Competition studies at 37 °C indicated that the 190-kDa hHARE binds HA and chondroitin better than dermatan sulfate and chondroitin sulfates A, C, D, and E, but it does not bind to heparin, heparan sulfate, or keratan sulfate. Although competition was observed at 37 °C, none of the glycosaminoglycans tested, except HA, competed for 125I-HA binding by 19OhHARE cells at 4 °C. Anti-HARE monoclonal antibodies #30 and #154, which do not inhibit 125I-HA uptake mediated by the 175-kDa rat HARE, partially blocked HA endocytosis by the 190-kDa hHARE. We conclude that the 190-kDa hHARE can function independently of other hHARE isoforms to mediate the endocytosis of multiple glycosaminoglycans. Furthermore, the rat and human small HARE isoforms have different glycosaminoglycan specificities and sensitivities to inhibition by cross-reacting antibodies.

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