Processing of chicken progastrin at post-Phe bonds by an aspartyl protease.
Prohormones mature to biologically active peptide hormones through posttranslational modifications, which include endoproteolytic cleavages. Cleavages at mono- and dibasic sites are well characterized, and several of the responsible prohormone convertases have been identified. There is, however, evidence that endoproteolytic maturation occurs also at other sites. Among these, post-Phe cleavage occurs in the maturation of chicken progastrin, where the processing to gastrin-30 has been examined in detail. In this study we have characterized an endoprotease of the aspartic acid protease family in chicken and human tissue capable of cleaving at the Phe site. Enzymatic activity was monitored by radioimmunoassays using antibodies specific for the N- and C-termini exposed after cleavage. Analysis showed that only pepstatin, a specific inhibitor of aspartic proteases, inhibited the enzyme. The pH optimum of the enzyme ranged from pH 2 to pH 5. Amino acid substitution from Phe to Ala in the substrate completely abolished enzyme activity. The endoproteolytic activity was identified in chicken antrum and pectoral muscle as well as human cardiac and prostate extracts, suggesting that the enzyme has widespread biological functions. Experiments using recombinant cathepsin D and E indicated that neither is responsible for the endoproteolytic cleavage of chicken progastrin at post-Phe bonds.
Jensen
Hanne
H
Department of Clinical Biochemistry, KB 3013, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen O, Denmark.
Yamamoto
Kenji
K
Bundgaard
Jens R
JR
Rehfeld
Jens F
JF
Johnsen
Anders H
AH
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism; D020059:Cathepsin E / Q000378:metabolism; D002645:Chickens / Q000378:metabolism*; D005755:Gastrins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D016415:Sequence Alignment
D005755:Gastrins; D011498:Protein Precursors; C013706:big gastrin; D010649:Phenylalanine; D016282:Aspartic Acid Endopeptidases; D020059:Cathepsin E; D002402:Cathepsin D