The Set of Serine Peptidases of the Tenebrio molitor Beetle: Transcriptomic Analysis on Different Developmental Stages.

Serine peptidases (SPs) of the chymotrypsin S1A subfamily are an extensive group of enzymes found in all animal organisms, including insects. Here, we provide analysis of SPs in the yellow mealworm Tenebrio molitor transcriptomes and genomes datasets and profile their expression patterns at various stages of ontogeny. A total of 269 SPs were identified, including 137 with conserved catalytic triad residues, while 125 others lacking conservation were proposed as non-active serine peptidase homologs (SPHs). Seven deduced sequences exhibit a complex domain organization with two or three peptidase units (domains), predicted both as active or non-active. The largest group of 84 SPs and 102 SPHs had no regulatory domains in the propeptide, and the majority of them were expressed only in the feeding life stages, larvae and adults, presumably playing an important role in digestion. The remaining 53 SPs and 23 SPHs had different regulatory domains, showed constitutive or upregulated expression at eggs or/and pupae stages, participating in regulation of various physiological processes. The majority of polypeptidases were mainly expressed at the pupal and adult stages. The data obtained expand our knowledge on SPs/SPHs and provide the basis for further studies of the functions of proteins from the S1A subfamily in T. molitor.

The dataset of predicted trypsin serine peptidases and their inactive homologs in Tenebrio molitor transcriptomes.

Tenebrio molitor is an important coleopteran model insect and agricultural pest from the Tenebrionidae family. We used RNA-Seq transcriptome data from T. molitor to annotate trypsin-like sequences from the chymotrypsin S1 family of serine peptidases, including sequences of active serine peptidases (SerP) and their inactive homologs (SerPH) in T. molitor transcriptomes. A total of 63 S1 family tryspin-like serine peptidase sequences were de novo assembled. Among the sequences, 58 were predicted to be active trypsins and five inactive SerPH. The length of preproenzyme and mature form of the predicted enzyme, position of signal peptide and proenzyme cleavage sites, molecular mass, active site and S1 substrate binding subsite residues, and transmembrane and regulatory domains were analyzed using bioinformatic tools. The data can be used for further physiological, biochemical, and phylogenetic study of tenebrionid pests and other animal systems.

New Glutamine-Containing Substrates for the Assay of Cysteine Peptidases From the C1 Papain Family.

New substrates with glutamine in the P1-position are introduced for the assay of peptidases from the C1 papain family, with a general formula of Glp-Phe-Gln-X, where Glp is pyroglutamyl and X is pNA (p-nitroanilide) or AMC (4-amino-7-methylcoumaride). The substrates have a simple structure, and C1 cysteine peptidases of various origins cleave them with high efficiency. The main advantage of the substrates is their selectivity for cysteine peptidases of the C1 family. Peptidases of other clans, including serine trypsin-like peptidases, do not cleave glutamine-containing substrates. We demonstrate that using Glp-Phe-Gln-pNA in combination with a commercially available substrate, Z-Arg-Arg-pNA, provided differential determination of cathepsins L and B. In terms of specific activity and kinetic parameters, the proposed substrates offer improvement over the previously described alanine-containing prototypes. The efficiency and selectivity of the substrates was demonstrated by the example of chromatographic and electrophoretic analysis of a multi-enzyme digestive complex of stored product pests from the Tenebrionidae family.