Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/10/2006
Publication Date: 7/2/2006
Citation: Liu, X., Fellers, J.P., Zhu, Y., Mutti, N.S., El-Bouhssini, M., Chen, M. 2006. Cloning and characterization of cdnas encoding carboxypeptidase-like proteins from the gut of hessian fly [mayetiola destructor (say)] larvae. Insect Biochemistry and Molecular Biology. 36:665-673 Interpretive Summary: One important method for controlling pest insects that attack plants is to inhibit their digestive enzymes. Previous work has focused on one type of digestive enzyme called an endopeptidase because it breaks down proteins by cutting them apart in the middle. This work describes our efforts to characterize a different type of digestive enzyme called an exopeptidase because it breaks down proteins by cutting pieces off each end rather than in the middle. We analyzed the digestive enzymes in the larval stage of the Hessian fly, one of the most destructive pests of wheat plants. We found five genes that code for three different exopeptidase enzymes called carboxypeptidase A, carboxypeptidase B, and carboxypeptidase D. Our results show that carboxypeptidase A and B are the major digestive enzymes in Hessian fly larvae. Therefore, future research will focus on finding methods to inhibit these enzymes as means of controlling this significant insect pest.
Technical Abstract: Transcriptomic analysis of the gut from Hessian fly [Mayetiola destructor (Say)] larvae identified nine unique cDNA clones that encode carboxypeptidase-like proteins. Sequence comparison and phylogenetic analysis sorted these cDNAs into five groups named MDCP-A1, MDCP-A2, MDCP-B1, MDCP-B2, and MDCP-D, according to their putative functional specificity. Among the five groups, MDCP-A1 and MDCP-A2 encoded carboxypeptidase A; MDCP-B1 and MDCP-B2 encoded carboxypeptidase B; and MDCD-D encoded carboxypeptidase D based on sequence similarities to those of well characterized proteins in public databases. All residues characteristic of metallocarboxypeptidases including the HXXE motif were conserved among all members. Northern blot analysis revealed various expression patterns for different gene groups in different developmental stages of M. destructor, suggesting that different carboxypeptidases perform different functions or have different specificities. Enzymatic activity assays demonstrated that both carboxypeptidase A and B are predominant in the larval stage, indicating a role of carboxypeptidase A and B in food digestion since larva is the only feeding stage of M. destructor. The digestive role is further supported by the fact that over 80% of the enzymatic activitiy in larvae occurred in the gut. Among these two types of enzymes, the enzymatic activity of carboxypeptidase A was at least four times higher than that of carboxypeptidase B under the same conditions, suggesting that carboxypeptidase A is the major digestive enzymes in the gut of M. destructor larvae.