Functional analysis of C1 family cysteine peptidases in the larval gut of Тenebrio molitor and Tribolium castaneum

Abstract

BackgroundLarvae of the tenebrionids Tenebrio molitor and Tribolium castaneum have highly compartmentalized guts, with primarily cysteine peptidases in the acidic anterior midgut that contribute to the early stages of protein digestion.ResultsHigh throughput sequencing was used to quantify and characterize transcripts encoding cysteine peptidases from the C1 papain family in the gut of tenebrionid larvae. For T. castaneum, 25 genes and one questionable pseudogene encoding cysteine peptidases were identified, including 11 cathepsin L or L-like, 11 cathepsin B or B-like, and one each F, K, and O. The majority of transcript expression was from two cathepsin L genes on chromosome 10 (LOC659441 and LOC659502). For cathepsin B, the major expression was from genes on chromosome 3 (LOC663145 and LOC663117). Some transcripts were expressed at lower levels or not at all in the larval gut, including cathepsins F, K, and O. For T. molitor, there were 29 predicted cysteine peptidase genes, including 14 cathepsin L or L-like, 13 cathepsin B or B-like, and one each cathepsin O and F. One cathepsin L and one cathepsin B were also highly expressed, orthologous to those in T. castaneum. Peptidases lacking conservation in active site residues were identified in both insects, and sequence analysis of orthologs indicated that changes in these residues occurred prior to evolutionary divergence. 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 seed storage proteins and inhibitors. Predicted cathepsin B peptidases from both insects included some with a shortened occluding loop without active site residues in the middle, apparently lacking exopeptidase activity and unique to tenebrionid insects. Docking of specific substrates with models of T. molitor cysteine peptidases indicated that some insect cathepsins B and L bind substrates with affinities similar to human cathepsin L, while others do not and have presumably different substrate specificity.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1306-x) contains supplementary material, which is available to authorized users.

RNA interference and dietary inhibitors induce a similar compensation response in Tribolium castaneum larvae

“…The T. castaneum genome has 25 annotated cysteine cathepsin genes and one pseudogene (Tribolium Genome Sequencing Consortium, ; Martynov et al ., ). Some cysteine peptidase genes are expressed at relatively high levels in the T. castaneum larval gut (Morris et al ., ), but high level expression is found only in the feeding stages (larvae and adult), presumably for digestion of food (Perkin et al ., ).…”

“…In larvae fed cysteine peptidase inhibitors, the expression of some cysteine peptidase genes were decreased whereas others were increased, as part of an integrated compensation response (Oppert et al ., ). The majority of cysteine cathepsin activity in the larval gut is a result of the expression of TC011001 , found on chromosome 10 (Morris et al ., ; Martynov et al ., ). Cathepsin L genes on chromosome 10 form a multigene family ( TC010999 , TC011000 , TC011001 , TC011002 , TC011003 ) and are probably the result of gene duplication.…”

“…T. castaneum has a midgut with a pH gradient of an acidic anterior to a neutral to basic posterior. In the acidic anterior midgut, proteolytic activity is mostly by cathepsin L and B cysteine peptidases (Vinokurov et al, 2009;Martynov et al, 2015). Reliance on cysteine peptidase activity in the anterior midgut of tenebrionid larvae is probably an adaptation to degrade inhibitors and protect serine peptidases in the posterior midgut (Prabhakar et al, 2007;Oppert et al, 2010).…”

Biochemical characterization and structural analysis of trypsin from Plodia interpunctella midgut: implication of determinants in extremely alkaline pH activity profile

“…Although the predominant proteolytic activity of beetles is reported to occur at acidic pH, there are serine proteases (e.g. trypsin and chymotrypsin) that are optimally active at the basic pH of the posterior midgut of T. castaneum (Oppert et al ., , ; Martynov et al ., ; Perkin et al ., ); in the present study, to compare midgut trypsins in these insects, we perform multiple sequence alignment comparisons. Multiple alignment of the binding site amino acid sequence reveals six amino acid substitutions in the enzyme binding pockets: Ser24Ile and Thr28Ser in S 1 , Phe42Thr in S 2 , Gln172Ala in S ′ 1, Leu195Ala in S ′ 2 and Asn206Tyr in S ′ 3 .…”

Prolidase is a critical enzyme for complete gliadin digestion in Tenebrio molitor larvae

“…The expression levels of T. molitor DPP 4, prolidase, and PTRs mRNAs were estimated as in Martynov et al. ().…”