A conserved sequence within the propeptide domain of membrane type 1 matrix metalloproteinase is critical for function as an intramolecular chaperone.
The propeptide domain of secreted matrix metalloproteinases (MMPs) is responsible for maintaining the latency of these proteinases. Recently, the propeptide domain of the prototype membrane type matrix metalloproteinase (MT1-MMP) was demonstrated to act as an intramolecular chaperone (Cao, J., Hymowitz, M., Conner, C., Bahou, W. F., and Zucker, S. (2000) J. Biol. Chem. 275, 29648-29653). In the current study, the role of an unique four-amino acid sequence in the propeptide domain of MT1-MMP was examined. The sequence (42)YGYL(45) is conserved in the propeptide domain of all six members of the MT-MMP subfamily, but not in secreted MMPs. Mutant MT1-MMP cDNAs coding for alanine substitutions (single and double amino acid sequences) in this conserved propeptide region were transfected into COS-1 cells deficient in endogenous MT1-MMP. As demonstrated by immunofluorescence, mutant MT1-MMP protein was synthesized and displayed on the plasma membrane of transfected cells. Alanine substitutions within the (42)YGYL(45) sequence proved to be detrimental for enzyme function in terms of activation of proMMP-2 and binding TIMP-2 to the cell surface (MT1-MMP serves as a cell surface receptor for TIMP-2). In contrast to wild-type MT1-MMP-transfected cells, mutant MT1-MMP-transfected cells were incapable of degrading and migrating on a fibronectin substrate. These data indicate that the conserved (42)YGYL(45) sequence within the propeptide domain of MT-MMPs is required for intramolecular chaperone function of these intrinsic membrane proteinases.
Pavlaki
Maria
M
Department of Veterans Affairs Medical Center, Northport, New York 11768, USA.
Cao
Jian
J
Hymowitz
Michelle
M
Chen
Wen-Tien
WT
Bahou
Wadie
W
Zucker
Stanley
S
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002465:Cell Movement; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D020781:Matrix Metalloproteinase 1 / Q000737:chemistry* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D005353:Fibronectins; D018832:Molecular Chaperones; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D020781:Matrix Metalloproteinase 1; D000409:Alanine