Role of the netrin-like domain of procollagen C-proteinase enhancer-1 in the control of metalloproteinase activity

Abstract

The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments.

FIGURE 1.

Amino acid sequence of human PCPE-1 in the region of the CUB2-NTR linker, showing the start positions of the NTR containing fragments previously identified by Mott et al. (17) and the N-terminal sequence of the construct used by Liepinsh et al. (10), as well as the MMP-2 cleavage site determined here and the start positions of the different forms of NTR used in this study.

FIGURE 2.

Circular dichroism spectra of the PCPE-1 NTR domain. The spectra for NTRs and NTRt were measured at 25 °C, and that for NTRt was also measured at 65 °C.

FIGURE 3.

Effects of NTRs and NTRt on the activities of MMP-2, BMP-1, and ADAMTS-2. MMP-2 activity was assayed on a quenched fluorogenic peptide (NTR concentration, 1 μm), BMP-1 activity was assayed on fluorescently labeled mini-procollagen III (NTR concentration, 1 μm), and ADAMTS-2 activity was assayed on radioactively labeled pN-collagen I (NTR concentration, 2.5 μm). The activities are expressed relative to activity in the absence of NTR, for each enzyme/substrate combination. The error bars show standard deviations.

FIGURE 4.

Binding of PCPE-1 to BMP-1 studied by surface plasmon resonance (Biacore). A, concentration dependence of binding of full-length PCPE-1 (12.5, 25, 50, 100, 200, 400, 800, 1600, 3200, and 6400 nm) to immobilized BMP-1 (500 RU). Inset, fit of the response at infinite time (RU eq) as a function of PCPE-1 concentration using the equation given under “Experimental Procedures.” B, interactions of PCPE-1 fragments CUB1CUB2, NTRt, and CUB2-NTR (all at 500 nm) with immobilized BMP-1 (1463 RU).

FIGURE 5.

Superstimulation of PCPE-1 enhancing activity by heparin. Effect of heparin (50 μg/ml; Sigma H-3393) on the activity of BMP-1 (10 nm) on mini-procollagen III (mini III, 400 nm) in the presence and absence of PCPE-1His, its CUB1CUB2 or NTRt fragments, or a mixture of CUB1CUB2 and NTRt (all at 400 nm). Incubations were carried out at 37 °C for 15 min. The migration positions of the released C-propeptide trimer (CPIII) and the N-terminal fragment (Nter) are indicated. Discontinuous SDS-PAGE (4–20% acrylamide in the separating gel) was carried out in nonreducing conditions, followed by staining with Coomassie Blue.

FIGURE 6.

A, concentration dependence of the effect of heparin on PCPE-1 enhancing activity, determined by SDS-PAGE followed by SYPRO Ruby staining, showing the percentage of conversion of the mini-procollagen III substrate (200 nm) in the presence of BMP-1 alone (5 nm), BMP-1 + PCPE-1His (200 nm), and BMP-1 + PCPE-1His + increasing concentrations of heparin. The incubations were carried out at 37 °C for 10 min. B, concentration dependence of the effect of heparan sulfate on PCPE-1 enhancing activity. The experimental conditions were as in A.

FIGURE 7.

Interaction of PCPE-1, mini-procollagen III (miniIII), the procollagen III C-propeptide trimer (CPIII), and BMP-1 with heparin. Biotinylated heparin was first bound to immobilized streptavidin to reach 44 RU and then solutions containing 50 nm of each protein were injected in 10 mm HEPES, 0.15 m NaCl, 0.05% P20 for 150 s at 30 μl/min.

FIGURE 8.

Possible mechanism by which heparin-like glycosaminoglycans might superstimulate the enhancing activity of PCPE-1 in the C-terminal processing of procollagen III. PCPE-1 binds to procollagen III in the region of the C-propeptides with nanomolar affinity. This complex then binds to cell surface or extracellular matrix associated heparin-like glycosaminoglycans via interactions with PCPE-1 NTR and with procollagen III. In addition, BMP-1 interacts with PCPE-1 NTR, the CUB2-NTR linker, and heparin. These interactions might increase the local concentrations of the reactants and/or create a new recognition site that further facilitates the action of BMP-1, resulting in superstimulation. Approximate molecular dimensions are based on known low resolution structures (27, 64, 65). C1C2, CUB1CUB2.