Abstract
Matrix metalloproteinases (MMPs) and the related families of disintegrin metalloproteinases (ADAMs) and ADAMs with thrombospondin repeats (ADAMTSs) play a crucial role in extracellular matrix (ECM) turnover and shedding of cell-surface molecules. The proteolytic activity of metalloproteinases is post-translationally regulated by their endogenous inhibitors, known as tissue inhibitors of metalloproteinases (TIMPs). Several MMPs, ADAMTSs and TIMPs have been reported to be endocytosed by the low-density lipoprotein receptor-related protein-1 (LRP-1). Different binding affinities of these proteins for the endocytic receptor correlate with different turnover rates which, together with differences in their mRNA expression, determines their nett extracellular levels. In this study, we used surface plasmon resonance to evaluate the affinity between LRP-1 and a number of MMPs, ADAMs, ADAMTSs, TIMPs and metalloproteinase/TIMP complexes. This identified MMP-1 as a new LRP-1 ligand. Among the proteins analyzed, TIMP-3 bound to LRP-1 with highest affinity (KD = 1.68 nM). Additionally, we found that TIMP-3 can facilitate the clearance of its target metalloproteinases by bridging their binding to LRP-1. For example, the free form of MMP-1 was found to have a KD of 34.6 nM for LRP-1, while the MMP-1/TIMP-3 complex had a sevenfold higher affinity (KD = 4.96 nM) for the receptor. TIMP-3 similarly bridged binding of MMP-13 and MMP-14 to LRP-1. TIMP-1 and TIMP-2 were also found to increase the affinity of target metalloproteinases for LRP-1, albeit to a lesser extent. This suggests that LRP-1 scavenging of TIMP/metalloproteinase complexes may be a general mechanism by which inhibited metalloproteinases are removed from the extracellular environment.
Highlights
Matrix metalloproteinases (MMPs) and the related families of disintegrin metalloproteinases (ADAMs) and ADAMs with thrombospondin repeats (ADAMTSs) play a crucial role in extracellular matrix (ECM) turnover and shedding of cell-surface molecules
In order to analyze the binding of tissue inhibitors of metalloproteinases (TIMPs)-3/MMP complexes to lipoprotein receptor-related protein-1 (LRP-1), we first investigated the inhibition of selected metalloproteinases by TIMP-3 in the low nanomolar range
We determined a Ki(app) for TIMP-3 inhibition of MMP-1ΔC of 1.86 nM (Table 1), in agreement with previous reports[21,33], meaning that around 50% of enzyme activity was blocked by equimolar amounts of enzyme
Summary
TIMP‐3 forms stable complexes with selected metalloproteinases in the low nanomolar range. When enzyme concentrations were increased to 5 nM, 90% of MMP-1 activity was blocked by an equimolar amount of TIMP-3, indicating that the large majority of the enzyme is in a stable complex with the inhibitor (Fig. 1A). TIMP-3 inhibited ADAM17 with a Ki(app) of 3.61 nM (Table 1), with 5 nM TIMP-3 being able to largely block the activity of 1 nM ADAM17 (Table 1, Fig. 1F) These enzymatic assays demonstrated that TIMP-3 and a number of its target metalloproteinases form stable complexes in vitro at concentrations in the very low nanomolar range. When 10 nM [35S]MMP-1 was pre-incubated with an equimolar concentration of TIMP-3 to form a [35S]MMP-1/TIMP-3 complex, its internalization increased, showing endocytosis kinetics similar to that of [35S]TIMP-3 alone (Fig. 4B and E). TIMP-3, but not TIMP-1 or TIMP-2, increased MMP-1 internalization rate when in complex with the proteinase
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