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 ∼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.