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KMID : 0368420220650060487
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Journal of Plant Biology
2022 Volume.65 No. 6 p.487 ~ p.503
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Characterization of the Arabidopsis At5-MMP Matrix Metalloproteinase
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Tang Guozhu
Wu Shuchi
Zhao Bingyu
Flinn Barry S.
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Abstract
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The plant extracellular matrix (ECM) represents the continuum between the plasma membrane and the apoplast, and undergoes significant modifications during plant growth, development and stress responses. While matrix metalloproteinases (MMPs) are significant proteolytic modulators of the vertebrate ECM, they remain poorly understood in plant biology. Of the five Arabidopsis thaliana MMPs, At5-MMP is the most constitutively expressed and was chosen for further characterization. Autoactivation of recombinant At5-MMP generated a functional metalloproteinase which exhibited maximum proteolytic activity at pH 7.5, and a broad level of activity into the basic range. Furthermore, an E to A mutation within the HEIGH portion of the At5-MMP catalytic domain abolished proteolytic activity. Using GFP-tagged At5-MMP variants expressed in Nicotiana benthamiana, the predicted signal peptide was required for targeting to the cell periphery, and deletion of this peptide resulted in intracellular, localized At5-MMP accumulation. Using a generic MMP cleavage PXG(L/I) peptide sequence, the predicted Arabidopsis extracellular proteome was screened for potential candidate MMP substrates. The extracellular domain of one candidate, WAK1, was expressed as a recombinant protein and shown to be cleaved internally at specific sites by At5-MMP in vitro. These same cleavage sites were identified using mammalian MMP cleavage prediction software.
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KEYWORD
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Extracellular matrix, Matrix metalloproteinase, At5-MMP, Cysteine switch, Autocatalytic activation
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