Biochemical Journal

Research article

Family-wide characterization of matrix metalloproteinases from Arabidopsis thaliana reveals their distinct proteolytic activity and cleavage site specificity

Giada Marino, Pitter F. Huesgen, Ulrich Eckhard, Christopher M. Overall, Wolfgang P. Schröder, Christiane Funk

Abstract

MMPs (matrix metalloproteases) are a family of zinc-dependent endopeptidases widely distributed throughout all kingdoms of life. In mammals, MMPs play key roles in many physiological and pathological processes, including remodelling of the extracellular matrix. In the genome of the annual plant Arabidopsis thaliana, five MMP-like proteins (At-MMPs) are encoded, but their function is unknown. Previous work on these enzymes was limited to gene expression analysis, and so far proteolytic activity has been shown only for At1-MMP. We expressed and purified the catalytic domains of all five At-MMPs as His-tagged proteins in Escherichia coli cells to delineate the biochemical differences and similarities among the Arabidopsis MMP family members. We demonstrate that all five recombinant At-MMPs are active proteases with distinct preferences for different protease substrates. Furthermore, we performed a family-wide characterization of their biochemical properties and highlight similarities and differences in their cleavage site specificities as well as pH- and temperature-dependent activities. Detailed analysis of their sequence specificity using PICS (proteomic identification of protease cleavage sites) revealed profiles similar to human MMPs with the exception of At5-MMP; homology models of the At-MMP catalytic domains supported these results. Our results suggest that each At-MMP may be involved in different proteolytic processes during plant growth and development.

  • Arabidopsis thaliana matrix metalloproteinase (At-MMP)
  • extracellular matrix
  • plant protease
  • proteomic identification of protease cleavage sites (PICS)

Abbreviations: AHA, acetohydroxamic acid; At-MMP, Arabidopsis thaliana matrix metalloproteinase; ECM, extracellular matrix; ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol; HPX, haemopexin; MBP, myelin basic protein; MMP, matrix metalloprotease; MS/MS, tandem MS; Nt1-MMP, Nicotiana tabacum MMP1; PICS, proteomic identification of protease cleavage sites; QF24, Mca-PLGLDpaAR-NH2; QF75, Mca-KESAbuNLFVLKDpaR-NH2; TFA, trifluoroacetic acid

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