Binding of procollagen C-proteinase enhancer-1 (PCPE-1) to heparin/heparan sulfate: properties and role in PCPE-1 interaction with cells.
Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of collagen deposition. PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptides and are required for PCP enhancing activity, and one NTR domain that binds heparin. To understand the biological role of the NTR domain, we performed surface plasmon resonance (SPR) binding assays, cell attachment assays as well as immunofluorescence and activity assays, all indicating that the NTR domain can mediate PCPE-1 binding to cell surface heparan sulfate proteoglycans (HSPGs). The SPR data revealed binding affinities to heparin/HSPGs in the high nanomolar range and dependence on calcium. Both 3T3 mouse fibroblasts and human embryonic kidney cells (HEK-293) attached to PCPE-1, an interaction that was inhibited by heparin. Cell attachment was also inhibited by an NTR-specific antibody and the NTR fragment. Immunofluorescence analysis revealed that PCPE-Flag binds to mouse fibroblasts and heparin competes for this binding. Cell-associated PCPE-Flag stimulated procollagen processing by BMP-1 several fold. Our data suggest that through interaction with cell surface HSPGs, the NTR domain can anchor PCPE-1 to the cell membrane, permitting pericellular enhancement of PCP activity. This points to the cell surface as a physiological site of PCPE-1 action.
Weiss
Tali
T
Maurice and Gabriela Goldschleger Eye Research Institute, Tel-Aviv University Sackler Faculty of Medicine, Sheba Medical Center, Tel Hashomer 52621, Israel.
Ricard-Blum
Sylvie
S
Moschcovich
Laura
L
Wineman
Eitan
E
Mesilaty
Shlomit
S
Kessler
Efrat
E
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D003094:Collagen / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D011347:Procollagen / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D003094:Collagen; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D006493:Heparin; D006497:Heparan Sulfate; D011347:Procollagen; C491555:PCOLCE protein, human; C108243:Pcolce protein, mouse