Abstract
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.
Highlights
Fibrillar procollagen precursors contain N- and C-terminal propeptide extensions at both ends of their pro ␣ chains
Kinetic data were fitted by the Langmuir binding model
We have previously shown that kinetic data of the binding of endostatin, the C-terminal domain of collagen XVIII [30] and of HepV, a fragment of collagen V [31], to heparin/heparan sulfate were well fitted by a heterogeneous ligand model
Summary
Construction of an Expression Vector for Recombinant Human PCPE-1-Flag Production—The full-length cDNA for human PCPE-1 (1474 bp) was excised from clone KT11 in pBlueSkriptII KS [6] and was ligated into the EcoRI site of pCDNA3.1(ϩ) (Invitrogen). For inhibition studies, increasing amounts of heparin (low mol wt; Sigma H3400) or a monoclonal antibody (IgG fraction) recognizing the NTR domain (7A11/5; Santa Cruz) or murine immunoglobulins (Sigma) as a negative control, were allowed to bind to the PCPE-1 coated wells for 2 h before addition of the cell suspension. In another set of inhibition experiments, the cells were preincubated with or without various concentrations of the NTR fragment before attachment to biotinylated PCPE-1. Chondroitin sulfate from bovine trachea (C8529), heparin (H3393), and dermatan sulfate (C3788) from porcine intestinal
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