Abstract
The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of M(r) 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys(532) is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys(530) and Cys(534) form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of alpha-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.
Highlights
The MMP3 family of zinc-dependent endopeptidases includes both secreted and membrane-anchored proteases that can hydrolyze multiple substrates at distinct cellular and extracellular locations in physiological and pathological conditions [1]
MT6-matrix metalloproteinase (MMP) Is Induced in Differentiated HL-60 Cells—MT6MMP is known to be highly expressed in neutrophils [6, 18], where it is detected as a major ϳ120-kDa form, as we previously reported [9]
We showed that MT6-MMP is displayed at the cell surface as a ϳ120-kDa species [9]
Summary
The MMP3 family of zinc-dependent endopeptidases includes both secreted and membrane-anchored proteases that can hydrolyze multiple substrates at distinct cellular and extracellular locations in physiological and pathological conditions [1]. The presence of membrane-anchoring domains confers unique properties and regulatory mechanisms to this subset of MMPs, which serve to tightly control the localization and amount of protease at the cell surface. These regulatory mechanisms include targeting to cell-matrix contacts, insertion into membrane microdomains, internalization and recycling, processing, oligomerization, and ectodomain shedding [3, 4]. As opposed to the transmembrane MT-MMPs, the stem region of MT6and MT4-MMP contains three and two unique cysteine residues, respectively, which may play a role in formation of disulfide bridges [8]. These studies establish a unique role for the stem region of MT6MMP in mediating disulfide-linked homodimerization
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