Insect midgut carboxypeptidases with emphasis on S10 hemipteran and M14 lepidopteran carboxypeptidases.

We compared the whole complement of midgut carboxypeptidases from 10 insects pertaining to five orders based on transcriptomes obtained by deep sequencing and biochemical data. Most of the carboxypeptidases were metallocarboxypeptidases from family M14, with carboxypeptidase A (CPA) predominating over carboxypeptidase B (CPB). They were found in all of the insects studied except for the hemipterans and a bruchid beetle. M14 carboxypeptidases were expressed only in the midgut of Spodoptera frugiperda (Lepidoptera). The most expressed CPA from this insect (SfCPA) was cloned, sequenced and expressed as a recombinant enzyme. This enzyme was used to generate antibodies used to demonstrate that SfCPA is secreted by an exocytic route. Serine carboxypeptidases from family S10 were found in all of the insects studied here. In S. frugiperda, they are expressed in all tissues besides the midgut, in accordance with their presumed lysosomal role. In the hemipteran Dysdercus peruvianus, S10 carboxypeptidases are expressed only in midgut, suggesting that they are digestive enzymes. This was confirmed by enzyme assays of midgut contents. Furthermore, the substrate specificity of D. peruvianus S10 carboxypeptidases are predicted to be one CPC (preferring hydrophobic residues) and one CPD (preferring basic residues), thus able to hydrolyse the peptides formed by their digestive cathepsin D and cathepsin L, respectively. The role of S10 carboxypeptidases in bruchid beetles are suggested to be the same as in hemipterans.