A proteomic approach to identify substrates of matrix metalloproteinase-14 in human plasma

Biochim Biophys Acta. 2004 Oct 1;1702(1):79-87. doi: 10.1016/j.bbapap.2004.08.001.

Abstract

Matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases that proteolyze extracellular matrix components as well as a variety of functional proteins. Here we describe a "degradomics" method that efficiently identifies substrates of MMP-14 in a complex protein mixture, such as plasma. Plasma proteins were incubated in the presence or absence of the MMP-14 catalytic domain and displayed on two-dimensional (2-D) gels. After a comparison of the gels, we selected 40 protein spots that reproducibly showed disparities. Upon in-gel digestion, mass determination, and peptide mass fingerprinting, we identified 15 different proteins from 31 spots. These proteins included six known substrates and nine potential substrates of MMP-14. Among the latter, the purified forms of apolipoprotein A-I, apolipoprotein E, and plasma gelsolin were cleaved in vitro by MMP-14, confirming that each of them is a novel substrate of MMP-14. These results demonstrate that our method rapidly and selectively identifies MMP-14 substrates from human plasma proteins. This method would thus constitute a powerful tool for identifying the substrates of MMPs and other proteases in highly complex mixtures of proteins and would enhance our understanding of the biological roles of these enzymes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Apolipoprotein A-I / blood
  • Apolipoproteins E / blood
  • Blood Proteins / isolation & purification
  • Blood Proteins / metabolism
  • Catalytic Domain
  • Electrophoresis, Gel, Two-Dimensional
  • Gelsolin / blood
  • Humans
  • In Vitro Techniques
  • Matrix Metalloproteinases, Membrane-Associated
  • Metalloendopeptidases / blood
  • Metalloendopeptidases / chemistry
  • Metalloendopeptidases / genetics
  • Metalloendopeptidases / metabolism*
  • Peptide Mapping
  • Protein Conformation
  • Protein Structure, Tertiary
  • Proteomics
  • Substrate Specificity

Substances

  • Apolipoprotein A-I
  • Apolipoproteins E
  • Blood Proteins
  • Gelsolin
  • Matrix Metalloproteinases, Membrane-Associated
  • Metalloendopeptidases