Carboxypeptidase D: A multitasking enzyme involved in Drosophila viability, development, behavior, and memory

Abstract

Carboxypeptidase D is an evolutionary conserved metallocarboxypeptidase, which is suggested to be involved in the production of biologically active molecules. In all species CPD has two active domains, a catalytically inactive third domain, a transmembrane domain and a cytosolic tail and is active towards C-terminal Lys and Arg. CPD displays broad pH optima and differential substrate preference. In contrast to mammalian CPD, Drosophila CPD undergoes alternative splicing and is represented by two soluble (inactive 1A and active 1B) and four full length forms.;Drosophila CPD is encoded by the silver gene. Two naturally occurring mutants, svr1 and svr poi demonstrate altered cuticle coloration and a pointed wing shape. Another silver mutant svrPG33 is lethal at the early larval stage. svr1 has a missing Leu in the transthyretin-like subdomain in the second CP domain, which affects folding of the full length protein and renders the affected domain inactive. svrpoi has a ∼1 kb duplication of the gene, inserted in the middle of the second CP domain, thus introducing a stop codon and creating a truncated protein. Soluble CPD forms are not affected in silver mutants. The lethal svrPG33 mutant has a P-element insertion just upstream of the initiating ATG codon and no CPD forms are made.;To further understand the importance of various CPD forms and its individual domains, we created several transgenic CPD lines in the svr PG33 background. All CPD forms tested except the inactive IA soluble form could rescue the lethality to various degrees. Forms with altered third CP domain showed reduced viability, suggesting that the inactive third CP domain is important for survival. svr1, svrpoi, and transgenic CPD mutants with overexpressed active domains displayed altered phenotypes in behaviors involving neuropeptides, which are similar to each other. All mutants showed affected cold and ethanol sensitivity; svr mutants showed memory deficit. Overexpression of both active CP domains reduced the levels of adipokinetic hormone Lys/Arg-extended intermediates. To summarize, our findings suggest that both enzymatically active CP domains have redundant functions in the processing of neuropeptides, hormones and growth factors and the inactive third CP domain is important for Drosophila viability.