CELL-SURFACE EXPRESSION OF HIP, A NOVEL HEPARIN HEPARAN SULFATE-BINDING PROTEIN, OF HUMAN UTERINE EPITHELIAL-CELLS AND CELL-LINES/

Previous studies established that uterine epithelial cells and cell li nes express cell surface heparin/heparan sulfate (HP/HS)-binding prote ins (Wilson, O., Jacobs, A. L., Stewart, S., and Carson, D. D. (1990) J. Cell. Physiol. 143, 60-67; Raboudi, N., Julian, J., Rohde, L. H., a nd Carson, D. D. (1992) J. Biol. Chem. 267, 11930-11939). The accompan ying paper (Liu, S., Smith, S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D. (1996) J. Biol. Chem. 271, 11817-11823) describ es the cloning of a full-length cDNA corresponding to a candidate cell surface HP/HS interacting protein, HIP, expressed by a variety of hum an epithelia. A synthetic peptide was synthesized corresponding to an amino acid sequence predicted from the cDNA sequence and used to prepa re a rabbit polyclonal antibody. This antibody reacted with a protein with an apparent M(r) of 24,000 by SDS-polyacrylamide gel electrophore sis that was highly enriched in the 100,000 x g particulate fraction o f RL95 cells. This molecular weight is similar to that of the protein expressed by 3T3 cells transfected with HIP cDNA. HIP was solubilized from this particulate fraction with NaCl concentrations greater than o r equal to 0.8 M demonstrating a peripheral association consistent wit h the lack of a membrane spanning domain in the predicted cDNA sequenc e. HIP was not released by heparinase digestion suggesting that the as sociation is not via membrane-bound HS proteoglycans. NaCl solubilized HIP bound to heparin-agarose in physiological saline and eluted with NaCl concentrations of 0.75 M and above. Furthermore, incubation of I- 125-HP With transblots of the NaCl solubilized HIP preparations separa ted by two-dimensional gel electrophoresis demonstrated direct binding of HP to HIP. Indirect immunofluorescence studies demonstrated that H IP is expressed on the surfaces of intact RL95 cells. Binding of HIP a ntibodies to RL95 cell surfaces at 4 degrees C was saturable and block ed by preincubation with the peptide antigen. Single cell suspensions of RL95 cells formed large aggregates when incubated with antibodies d irected against HIP but not irrelevant antibodies. Finally, indirect i mmunofluorescence studies demonstrate that HIP is expressed in both lu menal and glandular epithelium of normal human endometrium throughout the menstrual cycle. In addition, HIP expression increases in the pred ecidual cells of post ovulatory day 13-15 stroma. Collectively, these data indicate that HIP is a membrane-associated HP-binding protein exp ressed on the surface of normal human uterine epithelia and uterine ep ithelial cell lines.