Characterization of the Molecular Basis of the Drosophila Mutations in Carboxypeptidase D

Abstract

Carboxypeptidase D (CPD) functions in the processing of proteins and peptides in the secretory pathway. Drosophila CPD is encoded by the silver gene (svr), which is differentially spliced to produce long transmembrane protein forms with three metallocarboxypeptidase (CP)-like domains and short soluble forms with a single CP domain. Many svr mutants have been reported, but the precise molecular defects have not been previously determined. In the present study, three mutant lines were characterized. svr Metallocarboxypeptidases (CPs)3 perform many physiological functions, ranging from the digestion of food to the biosynthesis of neuropeptides (1-10). CPs are divided into three subfamilies based on amino acid analysis: the A/B subfamily (1-3, 9), the N/E subfamily (4 -8, 10), and a recently discovered subfamily that includes Nna1 and related proteins but has not yet been demonstrated to have CP activity (11). Within each subfamily, the members show 35-60% amino acid sequence identity, but between subfamilies, there is only 15-25% amino acid sequence conservation. The A/B subfamily is primarily involved in protein digestion, either in the digestive track or elsewhere in the body. In contrast, the N/E subfamily plays more of a biosynthetic role by selectively removing specific residues from peptide processing intermediates, and this step often affects the biological properties of the substrate. In humans and mice, there are eight members of this N/E family, of which five show enzymatic activity (CPE, CPN, CPM, CPZ, and CPD); the remaining members of this subfamily (CPX1, CPX2, and AEBP-1/ACLP) are not active toward standard substrates (4 -8, 10, 12-15). In contrast, Drosophila contains only two members of this subfamily, one that has high homology to CPM and another that is a CPD homolog (16).CPD is unique among CPs in that it contains multiple CP-like domains, a transmembrane domain, and a short cytosolic tail (5). Humans, rats, mice, duck, and Drosophila CPD all contain three CP-like domains, of which the first two are enzymatically active and the third is missing key catalytic residues but still appears to retain the basic CP-like structure (16 -27). The functional significance of the three CP-like domains in CPD is not clear. One proposal is that the distinct pH optima of the first two domains enable CPD to be active throughout the secretory pathway, which ranges from neutral to acidic pH values (17, 28). In both duck and Drosophila, the first domain is more active at neutral pH, whereas the second domain is optimally active at pH 5-6 (16, 17, 28). Protein containing both domains together showed a broader pH optimum (17, 28).There are several known mutations for CPD in Drosophila that are collectively known as the silver, or svr, mutants based on the silvery body color of the viable mutants (27,29). In addition to the body color, the viable svr mutants have been reported to show altered wing shape and mating behavior in light (30, 31), although these results have not been adequately described in the literature....

Identification of Novel Genes That Modify Phenotypes Induced by Alzheimer's β-Amyloid Overexpression in Drosophila

“…taining multiple carboxypeptidase domains and it has been proposed that this special structure increases the range of pH at which it can be active, ensuring activity throughout the secretory pathway (Novikova et al 1999). The Drosophila silver protein contains three carboxypeptidase domains, a transmembrane domain, and a cytosolic tail (Sidyelyeva and Fricker 2002). We found that three independent loss-of-function mutations in the silver gene act as enhancers of Ab-induced phenotypes.…”

Metallocarboxypeptidases: Emerging Drug Targets in Biomedicine

“…Unlike all other MCPs, CPD contains multiple CP-like domains. Human, rat, mouse, bovine, duck, chicken, and Drosophila CPD all contain three CP-like domains [37][38][39][40]. It is remarkable that only the first two CPlike domains contain all of the requisite active site and substrate-binding residues; the third domain is more similar to the CPX1/2 family members in that it lacks residues considered critical for enzymatic activity and is devoid of activity toward standard CP substrates [41].…”

Cloning and characterization of cDNAS encoding carboxypeptidase-like proteins from the gut of Hessian fly larvae [Mayetiola destructor (Say)]☆

“…Of the conserved residues, His69, Glu72, and His196 (Gln in the MDCP-A1 group) are involved in zinc binding, whereas Arg71, Arg127, and Arg145 along with Tyr248 (His in the MDCP-A1 group) and Glu270 (either Leu or Ile in the MDCP-A1 group) are involved in substrate binding and cleavage (Auld, 1998;Estebanez-Perpina et al, 2001). carboxpeptidase-like domain with similar size (Sidyelyeva and Fricker, 2002). The sequence identity between MDCP-D and the Drosophila carboxypeptidase D is 56%.…”