Abstract
Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and β-dystroglycan (β -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of β -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483–628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621–628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613–651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613–651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor.
Highlights
Dystroglycan (DG) is a membrane receptor belonging to the dystrophin-glycoprotein complex (DGC)
The α-DG (613–651) peptide inhibits the α-DG (483–628) domain proteolysis catalyzed by catalytic domain of members of the metalloproteinase (MMP)-2 (cdMMP-2), which can occur independently on the presence or not of the 621–628 a.a. region, disrupting both the entire protein construct and its fragmentation product
The hemopexin-like domain seems to play a role in substrate recognition, since, when it does not assist the catalytic domain, induces a loss of the interaction specificity, making the enzymatic fragmentation of α-DG(483–621) possible
Summary
Dystroglycan (DG) is a membrane receptor belonging to the dystrophin-glycoprotein complex (DGC). The two subunits are held together by non-covalent interactions occurring between the C-terminal domain of α-DG and the extracellular N-terminal domain of β -DG [7] These interactions play a crucial role for the integrity of the entire DGC [8], that is in turn responsible for the stability of the plasma membrane, especially in the skeletal muscle and in the central nervous system, where DG is mainly expressed [9]. The peculiarity of the CBD domain concerns its involvement in the recognition and binding of protein substrates, a role exerted prevalently by the hemopexin-like domain in other MMPs, providing an ancillary binding surface which governs the cleavage specificity of very complex macromolecular substrates, such as 3D triple helical collagens [21,22,23,24,25,26]
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