Role of the NTR domain of procollagen C-proteinase enhancer-1 in the control of metalloproteinase activity*

The NTR (netrin-like) 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 (MMPs) 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 astacinlike tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of PCPE-1 NTR, the latter beginning at the Nterminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including MMP-2, bone morphogenetic protein-1 (a tolloid proteinase) and different ADAMTS proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of BMP-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. Introduction It is now well established that different associations of modules in multi-domain proteins can provide increased diversity and specificity of function in many areas of cell biology, including receptor signaling, enzyme (particularly proteinase) activity and extracellular matrix function. The NTR domain (1), found solely in extracellular proteins, occurs in TIMPs (2), agrin (3;4), netrins (5), complement proteins C3/C4/C5 (6), secreted frizzled-related proteins (7), growth and differentiation factor binding proteins (8), ADAMTSL-5 (9) and procollagen Cproteinase enhancers (PCPE-1 and -2) (10;11). In the TIMPs, the NTR module corresponds to the N-terminal domain, which forms a tight 1:1 complex with the active site of MMPs, thereby inhibiting their activitiy (12;13). Within such complexes, the TIMP N-terminal cysteine residue plays a key role in expelling the catalytic water molecule bound to the zinc atom in the MMP active site (12;13), such that addition of an alanine residue at the Nterminus abolishes TIMP inhibition (14). The identification of NTR domains in proteins other than TIMPs prompted the suggestion that these might also be involved in the regulation of metalloproteinase activity (1). In agrin (4), the NTR domain (also called NtA) also occurs at the N-terminus of the molecule, and its 3D structure (an OB-fold) is strikingly similar to that of TIMP-1 (3). In contrast however, the first residue in agrin, just before the TIMP-like http://www.jbc.org/cgi/doi/10.1074/jbc.M109.086447 The latest version is at JBC Papers in Press. Published on March 5, 2010 as Manuscript M109.086447 Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 7, 2017 hp://w w w .jb.org/ D ow nladed from