The structural basis for the elastolytic activity of the 92-kDa and 72-kDa gelatinases. Role of the fibronectin type II-like repeats.
Several matrix metalloproteinases, including the 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME), and matrilysin degrade insoluble elastin. Because elastolytically active MME and matrilysin consist only of a catalytic domain (CD), we speculated that the homologous CDs of the 92-kDa and 72-kDa gelatinases would confer their elastolytic activities. In contrast to the MME CD, the 92 and 72 CDs expressed in Escherichia coli (lacking the internal fibronectin type II-like repeats) had no elastase activity, although both were gelatinolytic and cleaved a thiopeptolide substrate at rates comparable to the full-length gelatinases. To test the role of the fibronectin type II-like repeats in elastolytic activity, we expressed the 92-kDa gelatinase CD with its fibronectin type II-like repeats (92 CD/FN) in yeast. 92 CD/FN degraded insoluble elastin with activity comparable to full-length 92-kDa gelatinase. 92 and 72 CDs lacking the fibronectin type II-like repeats did not bind elastin, whereas the parent enzymes and 92 CD/FN did bind elastin. Furthermore, recombinant 92-kDa fibronectin type II-like repeats inhibited binding of the 92-kDa gelatinase to elastin. We conclude that the 92- and 72-kDa gelatinases require the fibronectin type II-like repeats for elastase activity.
Shipley
J M
JM
Respiratory and Critical Care Division, Washington University School of Medicine at Jewish Hospital, St. Louis, Missouri 63110, USA.
Doyle
G A
GA
Fliszar
C J
CJ
Ye
Q Z
QZ
Johnson
L L
LL
Shapiro
S D
SD
Welgus
H G
HG
Senior
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers; D004549:Elastin; D004926:Escherichia coli; D005353:Fibronectins / Q000737:chemistry*; D018093:Gelatinases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000737:chemistry* / Q000378:metabolism*; D010843:Pichia; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D005353:Fibronectins; D011994:Recombinant Proteins; D004549:Elastin; D010196:Pancreatic Elastase; D018093:Gelatinases; D008666:Metalloendopeptidases; D020783:Matrix Metalloproteinase 7