|Martynov, Alexander - Moscow State University|
|Elpidina, Elena - Moscow State University|
Submitted to: Biomed Central (BMC) Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/30/2015
Publication Date: 2/14/2015
Publication URL: http://handle.nal.usda.gov/10113/60904
Citation: Martynov, A.G., Elpidina, E.N., Perkin, L.C., Oppert, B.S. 2015. Functional analysis of C1 family cysteine peptidases in the larval gut of Tenebrio molitor and Tribolium castaneum. Biomed Central (BMC) Genomics. 16(75):1-15. DOI: http://dx.doi.org/10.1186/s12864-015-1306-x.
Interpretive Summary: Insects digest proteins through a complex set of digestive enzymes. In some beetles, protein digestion relies on a set of enzymes called cysteine cathepsins. In tenebrionids, these cysteine cathepsins are in the first part of the digestive tract and are critical to digestion of their main food, cereal proteins. We have compared cysteine cathepsins in two tenebrionids, the red flour beetle and yellow mealworm, using high throughput sequencing. We find that each insect has approximately several dozen cysteine cathepsins, and it is apparent that many of these are the result of gene duplication through evolutionary time, probably because of inhibitors and toxins that they encounter in their food. The sequencing method allows us to quantitate expression of each cysteine cathepsin, and overall expression is similar in both insects. Changes in conserved sequences may indicate difference in substrate or enzyme specificity. In the course of this study, we discovered a new group of enzymes with atypical structures, thus far not found in other organisms. These data will help in the design of new biological control products for stored product beetles.
Technical Abstract: We studied protein digestion the tenebrionids Tenebrio molitor and Tribolium castaneum, pests of stored grains and grain products, to identify potential targets for biopesticide development. Tenebrionid larvae have highly compartmentalized guts, with primarily cysteine peptidases in the acidic anterior midgut that contribute to the early stages of protein digestion. In this study, we have used high throughput sequencing to quantify and characterize the more highly expressed transcripts encoding digestive cysteine peptidases in these tenebrionid larvae. For T. castaneum, transcript and genomic data identified 24 genes and three pseudogenes encoding cysteine peptidases, including 12 cathepsin L or L-like, nine cathepsin B or B-like, and one each F, K, and O. Many of these genes are clustered on four different chromosomes. The majority of transcript expression is from two cathepsin L genes on chromosome 10 (LOC659441 and LOC659502), and two on chromosome 8 (LOC660368, 26-29-p); for cathepsin B, the major genes in the T. castaneum larval gut are on chromosome 3 (LOC663145 and LOC663117). Some cysteine peptidases were expressed at lower levels or not at all in the larval gut, including cathepsin F, K, and O. Without a sequenced genome for T. molitor our data is still speculative, but suggests that there are 25 cysteine peptidase genes, which include 13 cathepsin L, 10 cathepsin B, and one each cathepsin O and F in sequences from the larval gut. Of these, there were three each of relatively highly expressed cathepsin L and B transcripts. Orthologs were found for all except seven genes from T. castaneum and six transcripts from T. molitor; additional sequencing, including a sequenced genome for T. molitor, may resolve some of the differences. In general, the more highly expressed transcripts encoded peptidases that were predicted to be extracellular, whereas the remainder were likely lysosomal; all were phylogenetically related, and clusters of genes found in tandem on chromosomes (and presumably due to duplication events) were grouped in a cladogram. Sequence analysis indicated that nonfunctional (lacking conserved residues in the active site) orthologs were found in both insects, suggesting that changes in these residues occurred prior to evolutionary divergence. Cathepsin L sequences from both insects have a high degree of variability in the substrate binding regions, consistent with the ability of these enzymes to degrade a variety of cereal proteins and inhibitors. Cathepsin B sequences included those with a typical occluding loop, as well as atypical cathepsin B-like peptidases with a shortened occluded loop lacking the active site in the middle; these atypical cathepsin B-like peptidases are apparently unique to tenebrionid insects. Docking studies with characteristic substrates for human cysteine cathepsin L indicated that, while some tenebrionid cathepsin B and L peptidases have similar binding affinities, others do not and have presumably different substrate specificity, including the atypical cathepsin B-like peptidases. These studies have refined our model of protein digestion in the larval gut of tenebrionid insects, and suggest genes that may be targeted by inhibitors or RNA interference for the control of cereal pests in storage areas.