Characterization of the Mechanisms by which Gelatinase A, Neutrophil Collagenase, and Membrane-Type Metalloproteinase MMP-14 Recognize Collagen I and Enzymatically Process the Two α-Chains

Abstract

The turnover of native collagen has been ascribed to different members of the matrix metalloproteinase (MMP) family. Here, the mechanisms by which neutrophil collagenase (MMP-8), gelatinase A (MMP-2), and the ectodomain of MT1-MMP (ectMMP-14) degrade fibrillar collagen were examined. In particular, the hydrolysis of type I collagen at 37°C was investigated to identify functional differences in the processing of the two α-chain types of fibrillar collagen. Thermodynamic and kinetic parameters were used for a quantitative comparison of the binding, unwinding, and hydrolysis of triple helical collagen. We demonstrate that the MMP family has developed at least two distinct mechanisms for collagen unwinding and cleavage. MMP-8 and ectMMP-14 display a similar mechanism (although with different catalytic parameters), which is characterized by binding (likely through the hemopexin-like domain) and cleavage of α-1 and/or α-2 chains without distinguishing between them and keeping the gross conformation of the triple helix (at least during the first cleavage step). On the other hand, MMP-2 binds preferentially the α-1 chains (likely through the fibronectin-like domain, which is not present in MMP-8 and ectMMP-14), grossly altering the whole triple helical arrangement of the collagen molecule and cleaving preferentially the α-2 chain. These distinctive mechanisms underly a drastically different mode of interaction with triple helical fibrillar collagen I, according to which the MMP domain is involved in binding. These findings can be related to the different role exerted by these MMPs on collagen homeostasis in the extracellular matrix.

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

“…The enzymatic activity of several MMPs on different natural substrates, such as collagen I, II and IV has been investigated by several groups (Gioia et al, 2002(Gioia et al, , 2007Patterson et al, 2001;Tam et al, 2002Tam et al, , 2004Xu et al, 2004a), unraveling various functional features, which imply an important role of different domains in the macromolecular substrate proteolytic processing. Notably, MMP cleavage of bioactive substrates is temperature-dependent Gioia et al, 2002;Salsas-Escat et al, 2010), as also supported by the experiments on triple-helical synthetic substrates (Minond et al, 2006).…”

The Collagen Binding Domain of Gelatinase A Modulates Degradation of Collagen IV by Gelatinase B

“…30,40 The binding of the MMP2 CBD unwinds native collagen I, facilitating its cleavage. 30,34 Here, we investigated the effect of MMP2 CBD on collagen IV degradation by MMP9. Figure 3 shows the processing of type IV collagen by MMP9 in the absence and in the presence of MMP2 CBD, which is known to bind collagen IV in the micromolar range.…”

“…In this regard, it is worth stressing that the reverse investigation (i.e., the synergy of MMP9 CBD and MMP2 in degrading native collagen IV) cannot be studied as the CBD of MMP9 has been reported to bind collagen IV only at high concentrations. [31][32][33][34][35][36] The biological relevance of the reported modulation is validated by the accompanying observations on the effect of this interaction on neutrophil chemokinesis, raising important questions on the strategy for the design of efficient and selective inhibitors against macromolecular substrates.…”

“…27,28 Also, the role of the CBD exosite in modulating type I collagen degradation has been extensively documented. [29][30][31][32][33][34] Nevertheless, despite the importance of type IV collagen degradation, it has been little studied. 35 We have recently shown that MMP2 is able to cleave the α1 and α2 chains of type IV collagen at 37°C and that its CBD is involved in the recognition of both chains by MMP2.…”

Enzymatic processing by MMP‐2 and MMP‐9 of wild‐type and mutated mouse β‐dystroglycan

“…Recombinant human full-length MMP-2 (R&D Systems, Minneapolis, MN, USA) was obtained as a proteolytically active enzyme, thus not requiring any chemical or enzymatic activation. Both these enzymes have been used in previous investigations on different macromolecular substrates (22,23), clearly demonstrating that no contamination by other proteolytic activities was present.…”