Purification and molecular identification of the human hyaluronan receptor for endocytosis

Bin Zhou, Carl T. McGary, Janet A. Weigel A., Amit Saxena, Paul H. Weigel

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44 Scopus citations

Abstract

The clearance of hyaluronan (HA) and chondroitin sulfates from the circulating blood and lymph in the body is mediated by the membrane-bound HA receptor for endocytosis (HARE). Previously, we found that two HARE species of ∼175 kDa and ∼300 kDa are abundant in the sinusoidal endothelial cells in rat liver, spleen, and lymph nodes (Zhou et al. [2000], J. Biol. Chem., 275, 37733-37741). In the present study, immunocytochemical analysis of human tissues showed a similar pattern with abundant expression of HARE in the sinusoidal endothelial cells of human liver, spleen, and lymph nodes. The two human HARE proteins were immunoaffinity-purifed from human spleen. Each protein was recognized in western blots using several anti-rat HARE monoclonal antibodies and was able to bind 125I-HA specifically. In nonreducing SDS-PAGE, these two human HARE species migrated at ∼190 kDa and ∼315 kDa; both proteins are ∼15 kDa larger than the corresponding rat HAREs, although the de-N-glycosylated core proteins are essentially the same mass. After reduction, the human 190-kDa HARE gave a single 196-kDa species, which was not seen in the ∼315-kDa HARE after reduction. The reduced ∼315-kDa HARE yielded two major proteins at ∼250 kDa and ∼220 kDa. We determined the sequence of the human 190-kDa HARE cDNA based on analysis of internal tryptic peptides, as well as RT-PCR and 5′ RACE analyses using human spleen and lymph node cDNA libraries. The human gene that encodes HARE is on chromosome 12.

Original languageEnglish (US)
Pages (from-to)339-349
Number of pages11
JournalGlycobiology
Volume13
Issue number5
DOIs
StatePublished - May 1 2003

Bibliographical note

Funding Information:
We thank Steve Smith and Dr. Alex Kurosky at the University of Texas Medical Branch at Galveston for internal peptide analyses; Robert Raymond and Dr. Ed Harris for performing the NH2-terminal sequence analysis; Anil Singh for technical assistance; and Sheryl Christofferson of the Oklahoma Medical Research Foundation DNA Sequencing Facility. This research was supported by NIH grant GM35978 from the National Institute of General Medical Sciences. The nucleic acid and protein sequences of human HARE reported here are in the GenBank database under accession number AY22744.

Keywords

  • Hyaluronan binding protein
  • Hyaluronic acid
  • Iso-receptors
  • Receptor-mediated endocytosis
  • Recycling receptor

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