Purification and molecular identification of the human hyaluronan receptor for endocytosis.
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 approximately 175 kDa and approximately 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-purified 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 approximately 190 kDa and approximately 315 kDa; both proteins are approximately 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 approximately 315-kDa HARE after reduction. The reduced approximately 315-kDa HARE yielded two major proteins at approximately 250 kDa and approximately 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.
Zhou
Bin
B
Department of Biochemistry and Molecular Biology, and The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.
McGary
Carl T
CT
Weigel
Janet A
JA
Saxena
Amit
A
Weigel
Paul H
PH
D000595:Amino Acid Sequence; D015153:Blotting, Western; D015816:Cell Adhesion Molecules, Neuronal / Q000235:genetics* / Q000302:isolation & purification; D002881:Chromosomes, Human, Pair 12 / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D042783:Endothelial Cells / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics* / Q000302:isolation & purification*; D007150:Immunohistochemistry; D008198:Lymph Nodes / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D013154:Spleen / Q000378:metabolism
D015816:Cell Adhesion Molecules, Neuronal; D018960:Hyaluronan Receptors; C449927:STAB2 protein, human