Abstract
The extracellular matrix (ECM) is a highly dynamic network constantly remodeled by a fine-tuned protein formation and degradation balance. Matrix metalloproteinases (MMPs) constitute key orchestrators of ECM degradation. Their activity is controlled by tissue inhibitors of metalloproteinases (TIMPs) and glycosaminoglycans (GAG). Here, we investigated the molecular interplay of MMP2 with different GAG (chondroitin sulfate, hyaluronan (HA), sulfated hyaluronan (SH) and heparin (HE)) and the impact of GAG on MMP2/TIMP3 complex formation using in vitro-experiments with human bone marrow stromal cells, in silico docking and molecular dynamics simulations. SH and HE influenced MMP2 and TIMP3 protein levels and MMP2 activity. Only SH supported the alignment of both proteins in fibrillar-like structures, which, based on our molecular models, would be due to a stabilization of the interactions between MMP2-hemopexin domain and TIMP3-C-terminal tail. Dependent on the temporal sequential order in which the final ternary complex was formed, our models indicated that SH and HA can affect TIMP3-induced MMP2 inhibition through precluding or supporting their interactions, respectively. Our combined experimental and theoretical approach provides valuable new insights on how GAG interfere with MMP2 activity and MMP2/TIMP3 complex formation. The results obtained evidence GAG as promising molecules for fine-balanced intervention of ECM remodeling.
Highlights
Www.nature.com/scientificreports and are inserted into the catalytic domain
TIMP2–4 participate in the activation of proMMP2 due to a latent activation mechanism that involves the interaction of the tissue inhibitors of metalloproteinases (TIMPs) C-terminal tail and the third and fourth blade propellers of the zymogen PEX domain[7]
To evaluate whether the decreased MMP2 activity resulted from a decreased amount of enzyme, MMP2 activity was normalized to MMP2 protein
Summary
Www.nature.com/scientificreports and are inserted into the catalytic domain. A flexible proline-rich linker connects the C-terminus of the catalytic domain with the PEX domain, which is involved in mediating protein-protein interactions (e.g. to TIMP3 and the membrane type-1 matrix metalloproteinase (MT1-MMP), known as MMP14) and appropriate substrate recognition, among others[5]. The N-terminal tail of TIMPs binds to the active site of MMPs and, precludes substrate recognition. TIMP1, 2 and 4 have been reported to diffuse in the extracellular environment[6], whereas TIMP3 is the only member of the TIMP-family that tightly sticks to the ECM10–12 This is due to its interaction with sulfated glycosaminoglycans (GAG), e.g. with certain heparan sulfate proteoglycans. There are many indications of GAG containing a “code” defined by their chemical structure (e.g. sugar backbone, degree and position of sulfation), which affects various binding partners (extracellular mediators) in a different way[14]. Modified GAG derivatives providing a defined sulfation pattern and chain length constitute attractive molecules for structure-function relationships studies on GAG recognition by MMPs and TIMPs as they minimize batch-to-batch variabilities. As a consequence of decreased proteolytic activity, the protein level of several ECM components deposited by hBMSC such as fibronectin was increased[22,27]
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