Characterization of Drosophila carboxypeptidase D

Galyna Sidyelyeva, Lloyd D. Fricker

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Metallocarboxypeptidase D (CPD), is a 180-kDa protein that contains three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail and which functions in the processing of proteins that transit the secretory pathway. An initial report on the Drosophila melanogaster silver gene indicated a CPD-like protein with only two and a half carboxypeptidase-like domains with no transmembrane region (Settle, S. H., Jr., Green, M. M., and Burtis, K. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9470-9474). A variety of bioinformatics and experimental approaches were used to determine that the Drosophila silver gene corresponds to a CPD-like protein with three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail. In addition, two alternative exons were found, which result in proteins with different carboxypeptidase-like domains, termed domains 1A and 1B. Northern blot, reverse transcriptase PCR, and sequence analysis were used to confirm the presence of the various mRNA forms. Individual domains of Drosophila CPD were expressed in insect Sf9 cells using the baculovirus expression system. Media from domain 1B- and domain 2-expressing cells showed substantial enzymatic activity, whereas medium from domain 1A-expressing cells was no different from cells infected with wild-type virus. Domains 1B and 2 were purified, and the enzymatic properties were examined. Both enzymes cleaved substrates with C-terminal Arg or Lys, but not Leu, and were inhibited by conventional metallopeptidase inhibitors and some divalent cations. Drosophila domain 1B is more active at neutral pH and greatly prefers C-terminal Arg over Lys, whereas domain 2 is more active at pH 5-6 and slightly prefers C-terminal Lys over Arg. The differences in pH optima and substrate specificity between Drosophila domains 1B and 2 are similar to the differences between duck CPD domains 1 and 2, suggesting that these properties are essential to CPD function.

Original languageEnglish (US)
Pages (from-to)49613-49620
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number51
DOIs
StatePublished - Dec 20 2002

Fingerprint

Carboxypeptidases
Drosophila
Proteins
Silver
Tail
Genes
Sf9 Cells
Ducks
Baculoviridae
Secretory Pathway
RNA-Directed DNA Polymerase
Divalent Cations
Metalloproteases
Substrates
Bioinformatics
Substrate Specificity
Computational Biology
Drosophila melanogaster
Reverse Transcriptase Polymerase Chain Reaction
Viruses

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of Drosophila carboxypeptidase D. / Sidyelyeva, Galyna; Fricker, Lloyd D.

In: Journal of Biological Chemistry, Vol. 277, No. 51, 20.12.2002, p. 49613-49620.

Research output: Contribution to journalArticle

Sidyelyeva, Galyna ; Fricker, Lloyd D. / Characterization of Drosophila carboxypeptidase D. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 51. pp. 49613-49620.
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