Abstract
Heparin/heparan sulfate (HP/HS), HS proteoglycans, and their binding proteins play important roles in a variety of biological processes. Previously, we identified a novel cell surface HP/HS interacting protein (HIP) from human uterine epithelia and a variety of other human epithelial and endothelial cells and cell lines (Liu, S., Smith, S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D. (1996) J. Biol. Chem. 271, 11817-11823; Rohde, L. H., Julian, J., Babaknia, A., and Carson, D. D. (1996) J. Biol. Chem. 271, 11824-11830). In the current studies, we have purified and characterized HIP from HEC cells, a human uterine epithelial cell line, as well as recombinant HIP from a bacterial expression system. HIP supports attachment of the human trophoblastic cell line, JAR, in a HS-dependent fashion. Predigestion of JAR cells with a mixture of heparitinases, but not chondroitinase AC, abolished cell attachment to HIP. In addition, JAR cell attachment to HIP is highly sensitive to HP inhibition and much more selective for HP/HS than other glycosaminoglycans. Dermatan sulfate displays partial inhibitory activity as well, consistent with the observation that chondroitinase ABC digestion partially reduces JAR cell attachment to HIP. Solid-phase binding assays indicate HIP binds [3H]HP with high affinity (apparent KD = 8 nM). Furthermore, HIP bound cell surface/extracellular matrix-associated HS, expressed by RL95 cells, a human uterine epithelial cell line. Anti-HIP antibody generated against a synthetic peptide derived from a putative HP/HS-binding motif resident within HIP inhibited about half of [3H]HP binding to HIP, indicating that this domain is a functional HP-binding domain of HIP. Similarly, this same synthetic peptide motif of HIP could block about 50% of [3H]HP binding to HIP; however, this peptide almost completely inhibited cell attachment to HIP, suggesting a critical role, in this regard. Collectively, these results suggest that HIP can function as a HP/HS-binding cell-cell/cell-matrix adhesion molecule.
Highlights
Interactions between heparin/heparan sulfate (HP/HS),1 heparan sulfate proteoglycans, and their binding proteins occur in a variety of biological processes, including cell adhesion, growth and differentiation, modulation of growth factor activities, maintenance of extracellular matrix integrity, and cytokine functions [1,2,3,4]
Our results show that: 1) HS interacting protein (HIP) from either source displays similar activities; 2) HIP supports attachment of JAR cells, a human trophoblastic cell line, in a HS-dependent fashion; 3) a synthetic peptide generated to a putative HP/HS-binding motif greatly inhibits JAR cell attachment to HIP; 4) HIP selectively binds HP with high affinity and anti-HIP antibody, generated against the same synthetic peptide that inhibits cell attachment, partially blocks [3H]HP binding to HIP; 5) HIP binds cell surface and extracellular matrix forms of HS
These data demonstrate that HIP supports cell attachment and binds HP/HS with high affinity, indicating that it can function as a HP/HS-binding cell-cell/cell-extracellular matrix adhesion molecule
Summary
Materials—Heparin, low molecular weight heparin, bovine intestinal mucosa heparan sulfate, bovine kidney heparan sulfate, dermatan sulfate, chondroitin sulfate C, keratan sulfate, hyaluronic acid, heparinases I, II, and III, chondroitinases AC and ABC, BSA, and heparinagarose were purchased from Sigma. [3H]HP (0.44 mCi/mg) was purchased from NEN Life Science Products. [35S]Sulfate (43 Ci/mg) and 125I-protein A (30 Ci/g) were purchased from ICN Biochemical Inc. (Irvine, CA). Heparin-Agarose and Anti-HIP Immunoaffinity Chromatography for Purification of Natural HIP—Confluent HEC cell cultures were rinsed with Dulbecco’s PBS three times and cells released from dishes with 10 mM EDTA, 1 M NaCl, and 10 mM Tris-HCl, pH 7.2. The concentrated 1.0 M eluate was diluted with 10 mM Tris-HCl, pH 7.2, to reach a final NaCl concentration of 0.15 M, and incubated with anti-HIP immunoaffinity matrix, in the presence of PICS at 4 °C overnight in a rotary agitator. 10 or 100 g/ml HP, low molecular weight HP, bovine intestinal mucosa HS, bovine kidney HS, dermatan sulfate, chondroitin sulfate C, hyaluronic acid, or keratan sulfate were included in the incubation medium and used as competitors in the assays of JAR cell attachment to HIP. Five l of a 50% slurry of protein A-Sepharose was added to this solution and the mixture incubated with constant rotary
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