Cloning, expression, and characterization of human metalloprotease 1: a novel member of the pitrilysin family of metalloendoproteases.
A novel cDNA, designated human metalloendoprotease 1 (hMP1), was identified on the basis of homology to known metalloendoproteases of the pitrilysin family. The full-length MP1 codes for a protein with an open reading frame of 1038 amino acids. The N-terminal region contains the HXXEH(X)76E catalytic domain that is conserved in the members of pitrilysin family, namely insulin-degrading enzyme and NRD convertase. The hMP1 mRNA is expressed in a number of cell lines and tissues as a single species of about 3.4 kb. The expression of hMP1 mRNA is higher in muscle and heart than in brain, pancreas, liver, lung, and placenta. The full-length hMP1 was expressed in the baculovirus system and purified to homogeneity using isoelectrofocusing and ion-exchange chromatography. The enzyme exhibited a neutral pH optimum and high sensitivity to thiol reagents. HMP1 was inactivated by 1,10-phenanthroline, a specific inhibitor of Zn(+2)-dependent metalloproteases. The enzyme was not inhibited by agents that inhibit neutral metalloendoproteases of the thermolysin family such as thimet endo-oligopeptidase, enkephalinase, or angiotensin-converting enzyme. HMP1 cleaved a prodynorphin-derived peptide, leumorphin, N-terminal to Arg in the monobasic processing site, as evidenced by MALDI-TOF mass spectrometry. However, the enzyme did not exhibit strict monobasic cleavage specificity, as peptide substrates with amino acid substitutions around the monobasic site was cleaved efficiently by hMP1. Taken together, these results suggest that hMP1 is a novel member of the metalloendoprotease superfamily with ubiquitous distribution that could play a broad role in general cellular regulation.
Mzhavia
N
N
Department of Pharmacology, New York University School of Medicine, NY 10016, USA.
Berman
Y L
YL
Qian
Y
Y
Yan
L
L
Devi
L A
LA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D006801:Humans; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D008666:Metalloendopeptidases; C119694:PITRM1 protein, human; C046583:pitrilysin