Binding of procollagen C-proteinase enhancer-1 (PCPE-1) to heparin/heparan sulfate: properties and role in PCPE-1 interaction with cells

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.

FIGURE 1.

Binding of the NTR domain of PCPE-1 (645 nm) to immobilized heparin (154 RU) and heparan sulfate (175 RU). Flow rate: 30 μl/min, contact time: 4 min, sample buffer: HBS-P + 5 mm CaCl₂.

FIGURE 2.

Heparin inhibits attachment of 3T3 mouse fibroblasts and HEK-293 cells to PCPE-1. A, cell attachment to PCPE-1 in the absence or presence of increasing concentrations of heparin (0–400 μg/ml); B, cell binding to PCPE-1 expressed as percentage of binding of 3T3 mouse fibroblasts in the absence of heparin (100%). Each value represents mean ± S.D. (n = 4).

FIGURE 3.

Attachment of 3T3 mouse fibroblasts to PCPE-1 is inhibited by a monoclonal antibody recognizing the NTR domain of PCPE-1 (A) and the free NTR fragment (B). A, biotin-labeled PCPE-1 was incubated with the anti-NTR mAb (1, 2, or 4 μg/ml) for 2 h prior to addition to extravidin-coated wells for adsorption. Normal murine immunoglobulin (mIgG; 4 μg/ml) served as a control. B, cells (60,000/100 μl) were incubated for 20 min with increasing concentrations (0.5–10 μg/ml) of the NTR domain before addition to the biotin-PCPE-1-coated wells. The cell attachment assay in both A and B, was then performed as described under “Experimental Procedures.” Each value represents mean ± S.D. (n = 4).

FIGURE 4.

Heparin inhibits binding of PCPE-Flag to 3T3 mouse fibroblasts. Mouse fibroblasts at 70–80% confluency were incubated for 1.5 h with PCPE-Flag (200 ng/ml). Unbound PCPE-Flag was removed by washing with phosphate-buffered saline, and the cells were then placed on fresh medium supplemented with heparin (0, 1, 5, or 20 μg/ml as indicated) and incubated further for 16 h (37 °C, 7% CO₂). The cells were then washed and fixed with 3.7% paraformaldehyde. Cell-bound PCPE-Flag was detected by immunofluorescence using an anti-Flag antibody and a Cy2-coupled secondary antibody. Nuclei were stained with DAPI. C, control: cells were incubated with PCPE-Flag without subsequent addition of heparin. Immunodetection was without addition of the first (anti-Flag) antibody.

FIGURE 5.

Cell-bound PCPE-Flag is maintained on the cell surface and stimulates procollagen processing by BMP-1 at this location. PCPE-Flag was allowed to bind to 3T3 mouse fibroblasts for 90 min. Radioactively labeled procollagen type I was then added and allowed to settle on the cells for additional 90 min. BMP-1 was then added, and the cells were incubated further for 2 h for procollagen processing to occur (total incubation time of 5 h). A, immunofluorescence analysis with an anti-Flag antibody (performed after 5 h of incubation with 200 ng/ml of PCPE-Flag) showing that PCPE-Flag remains cell-bound throughout the experiment. B, immunoblot of cell layer extracts prepared after 5 h of incubation and probed with an anti-Flag antibody, showing that the amount of cell-bound PCPE-Flag is proportional to the original input of PCPE-Flag. C, autoradiogram showing cell association of the procollagen substrate. Cells incubated without BMP-1 were solubilized, radioactivity was determined, and approximately half of the total cell-associated radioactivity (6,000 cpm) was loaded per lane. SDS-PAGE was performed on a 6% gel with reduction. Bands corresponding to the proα1(I) and proα2(I) chains are indicated by arrows. D, autoradiogram showing a comparable degree of enhancement of PCP activity (2–2.6-fold) at a constant input of PCPE-Flag (200 ng/ml), essentially independent of BMP-1 concentration. E, autoradiogram showing that the degree of enhancement of PCP activity, assessed by the C-propeptide release, increases as a function of PCPE-Flag concentration. Fold enhancement, ratio between the amount of radioactivity released into the supernatants in the presence and absence of PCPE-Flag, respectively.