Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin-binding domains in insects

Abstract

In insects, chitin is a major structural component of the cuticle and the peritrophic membrane (PM). In nature, chitin is always associated with proteins among which chitin-binding proteins (CBPs) are the most important for forming, maintaining and regulating the functions of these extracellular structures. In this study, a genome-wide search for genes encoding proteins with ChtBD2-type (peritrophin A-type) chitin-binding domains (CBDs) was conducted. A total of 53 genes encoding 56 CBPs were identified, including 15 CPAP1s (cuticular proteins analogous to peritrophins with 1 CBD), 11 CPAP3s (CPAPs with 3 CBDs) and 17 PMPs (PM proteins) with a variable number of CBDs, which are structural components of cuticle or of the PM. CBDs were also identified in enzymes of chitin metabolism including 6 chitinases and 7 chitin deacetylases encoded by 6 and 5 genes, respectively. RNA-seq analysis confirmed that PMP and CPAP genes have differential spatial expression patterns. The expression of PMP genes is midgut-specific, while CPAP genes are widely expressed in different cuticle forming tissues. Phylogenetic analysis of CBDs of proteins in insects belonging to different orders revealed that CPAP1s from different species constitute a separate family with 16 different groups, including 6 new groups identified in this study. The CPAP3s are clustered into a separate family of 7 groups present in all insect orders. Altogether, they reveal that duplication events of CBDs in CPAP1s and CPAP3s occurred prior to the evolutionary radiation of insect species. In contrast to the CPAPs, all CBDs from individual PMPs are generally clustered and distinct from other PMPs in the same species in phylogenetic analyses, indicating that the duplication of CBDs in each of these PMPs occurred after divergence of insect species. Phylogenetic analysis of these three CBP families showed that the CBDs in CPAP1s form a clearly separate family, while those found in PMPs and CPAP3s were clustered together in the phylogenetic tree. For chitinases and chitin deacetylases, most of phylogenetic analysis performed with the CBD sequences resulted in similar clustering to the one obtained by using catalytic domain sequences alone, suggesting that CBDs were incorporated into these enzymes and evolved in tandem with the catalytic domains before the diversification of different insect orders. Based on these results, the evolution of CBDs in insect CBPs is discussed to provide a new insight into the CBD sequence structure and diversity, and their evolution and expression in insects.

Annotation and expression analysis of cuticular proteins from the tobacco hornworm, Manduca sexta

“…A few genes from A. pisum , A. mellifera , and P. humanus could not be clearly grouped within any of the 16 CPAP1 branches and were left unclassified. It should be noted that although both our analysis (this report and Tetreau et al ., 2014, this issue) and that of Ioannidou et al . (2014) independently verified the close relationship of the new members of the CPAP1 group to the original ten genes described previously for T. castaneum , the presence of the encoded proteins in the cuticle must still be established (and hence are they truly cuticle proteins).…”

“…Since similar work had not yet been performed for B. mori , we named the M. sexta genes based on orthology to T. castaneum and homologous genes in other insects. This analysis is described in a companion paper detailing the different groups of proteins containing peritrophin A-type chitin-binding domains in M. sexta (Tetreau et al ., 2014, this issue) but will be summarized here. M. sexta has orthologs to 9 of the 10 CPAP1 genes in T. castaneum with 1-E being the exception.…”

“…Details of the CPAP analysis can be found in a companion paper (Tetreau et al ., 2014, this issue). Analysis was performed using MEGA software (v5.2.1; Tamura et al ., 2011).…”

VP24 Is a Chitin-Binding Protein Involved in White Spot Syndrome Virus Infection

“…In nature, CBPs are the major group of proteins that associate with chitin and participate in the formation, maintenance, and regulation of the functions of these extracellular structures (42). Other classes of CBPs function as lectins and chitinases (43,44).…”

The Evolution and Metamorphosis of Arthropod Proteomics and Genomics

“…But every other protein known to bind chitin had cysteine residues, and our earlier amino acid studies and work by others had found almost no cysteine in CPs (1,57). Exceptions to this are now known (24,25,40). During that dinner, we arranged that Rebers should spend a sabbatical in my lab testing whether the Consensus binds to chitin.…”