Characterization and cloning of tripeptidyl peptidase II from the fruit fly, Drosophila melanogaster.
We describe the characterization, cloning, and genetic analysis of tripeptidyl peptidase II (TPP II) from Drosophila melanogaster. Mammalian TPP II removes N-terminal tripeptides, has wide distribution, and has been identified as the cholecystokinin-degrading peptidase in rat brain. Size exclusion and ion exchange chromatography produced a 70-fold purification of dTPP II activity from Drosophila tissue extracts. The substrate specificity and the inhibitor sensitivity of dTPP II is comparable to that of the human enzyme. In particular, dTPP II is sensitive to butabindide, a specific inhibitor of the rat cholecystokinin-inactivating activity. We isolated a 4309-base pair dTPP II cDNA which predicts a 1354-amino acid protein. The deduced human and Drosophila TPP II proteins display 38% overall identity. The catalytic triad, its spacing, and the sequences that surround it are highly conserved; the C-terminal end of dTPP II contains a 100-amino acid insert not found in the mammalian proteins. Recombinant dTPP II displays the predicted activity following expression in HEK cells. TPP II maps to cytological position 49F4-7; animals deficient for this interval show reduced TPP II activity.
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
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Renn
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Tomkinson
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Taghert
D000595:Amino Acid Sequence; D000626:Aminopeptidases; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism
D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C048001:tripeptidyl-peptidase 2; D012697:Serine Endopeptidases