Abstract
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.
Keywords: Serine peptidases, Pseudopeptidases, Inactive peptidase homologs, Trypsin, Tenebrio molitor, Tenebrionidae
Specifications Table
| Subject | Bioinformatics/Omics: Transcriptomics |
| Specific subject area | Assembly, annotation and structural analysis of trypsin-like SerP and SerPH sequences from the Tenebrio molitor transcriptome using bioinformatics tools. |
| Type of data | Tables and Supplementary text file, containing assembled sequences in fasta format. |
| How data were acquired | RNA sequencing by Illumina HiSeq 2000, structural analysis with appropriate software |
| Data format | Raw |
| Parameters for data collection | Laboratory colonies of T. molitor were maintained on a diet consisting of either 50–100% oat flakes or 85% stabilized wheat germ. RNA was isolated and sequenced from the larval midgut and from eggs, I and IV instar larvae, pupae, young males and females with non-chitinized integuments and two week old adult males and females of T. molitor. Parameters for sequencing, contigs assembly and analysis programs were default. |
| Description of data collection | mRNA-isolation, high throughput RNA-sequencing using Illumina HiSeq 2000, automated and manual assembly of contigs to full-length sequences, annotation and structural analysis using bioinformatic tools. |
| Data source location | Institution: USDA Agricultural Research Service, Center for Grain and Animal Health Research City/Town/Region: Manhattan, KS 66502 Country: USA Latitude and longitude: 27.2046° N, 77.4977° E Institution: Lomonosov Moscow State University, A.N. Belozersky Institute of Physico-Chemical Biology City/Town/Region: Moscow, 119992 Country: Russia Latitude and longitude: 55.705836° N, 37.521456° E |
| Data accessibility | Repository name: NCBI Data identification number: Accession numbers unique to each sequence are listed in Tables 1 and 2. Direct URL to data: Direct URLs to data are presented as hyperlinks in Tables 1 and 2, which can be opened by clicking on the corresponding accession number. Data are also available with the article in the Supplementary file. |
Value of the Data
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Serine peptidases of the S1 chymotrypsin family and their inactive homologs were identified in transcriptome data from Tenebrio molitor, a serious pest of stored products and а biochemical and coleopteran model. This information is important for clarification of the organization of the digestive process in Tenebrionidae insects as well as the role of serine peptidases in life functions of the insect.
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The data obtained can help agricultural industries and researchers in developing measures to control these pests, as well as food industries incorporating insects as supplemental protein.
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The dataset revealed the presence of inactive serine peptidases homologs (pseudoenzymes) as have been observed in several other insects. The study can contribute to understanding their functional significance and, in particular, their role in the regulation of the action of active peptidases.
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These data form the basis for further bioinformatic and biochemical analyzes to clarify the relationship between the structure and function of serine peptidases and their inactive homologs.
1. Data Description
The sequences of trypsins and their inactive homologs from the S1 chymotrypsin family, according to MEROPS classification [1], are attached to this article in the Supplementary file with the reference accession numbers unique to each sequence received during submission to the open public repository NCBI database (https://www.ncbi.nlm.nih.gov) [2] and listed in Tables 1 and 2. Direct URLs to data are presented as hyperlinks in Tables 1 and 2, which can be opened by clicking on the corresponding accession number. The data represent assembled transcriptome sequences of serine peptidases and their inactive homologs for which structural analysis was carried out using bioinformatic tools. The data include 58 sequences of trypsins and five trypsin-like SerPH that were found in the T. molitor transcriptome obtained via a combination of data from the larval midgut, eggs, I and IV instar whole larvae, pupae, adult males and females. Of these, the sequences of 34 active trypsins and five SerPH were found in the larval midgut transcriptome (Table 1) and 24 active trypsins were absent in the larval midgut transcriptome and are considered non-gut peptidases (Table 2). The custom ID and corresponding NCBI accession numbers are listed in Tables 1 and 2. Sequences SerP1 [3], SerP183 [4], SerPH223 [5] and SerPH415 [6] were previously submitted by other research groups and coincided with those assembled by us, so we used existing NCBI accession numbers with relevant references.
Table 1.
Sequences of trypsins (SerP) and tripsin-like homologs (SerPH) found in the larval gut transcriptome of Tenebrio molitor. All active sites of active serine peptidase sequences consist of H D S, except for SerPH70 (H D D) and other SerPH (H D G), and all S1 subsites are D G G.
| Identification | NCBI Nucleotide Accession | NCBI Protein Accession | Preproenzyme/Mature Enzyme (aa) | Mature Enzyme (Da) | Signal Peptide (aa) | Proenzyme Cleavage Site | Regulatory Domains(aa position) |
|---|---|---|---|---|---|---|---|
| SerP11 | DQ356014 | ABC88729 | 258/227 | 22,742 | 16 | R|IVGG | - |
| SerP2 | MW603455 | QWS65012 | 252/227 | 23,618 | 16 | R|IVGG | - |
| SerP4 | MW603456 | QWS65013 | 250/225 | 24,140 | 15 | R|IVGG | - |
| SerP6 | MW603457 | QWS65014 | 258/226 | 23,414 | 17 | R|IVGG | - |
| SerP11 | MW603486 | QWS65043 | 447/231 | 26,306 | 19 | K|IIGG | Thrombospondin type 1 repeats (132-170) |
| SerP30 | MW603458 | QWS65015 | 249/226 | 24,862 | 16 | K|IIGG | - |
| SerP40 | MW603459 | QWS65016 | 392/241 | 26,535 | 21* | G|NPGG | Clip_15 (68-112) |
| SerP48 | MW603460 | QWS65017 | 321/295 | 32,018 | 22 | R|IVGG | - |
| SerP55 | MW603461 | QWS65018 | 1,672/245 | 26,947 | 21 | R|VVRG | CBM_14 (138-189, 212-263, 303-354), Ldl_recept_a (969-1003, 1089-1122, 1240-1279), SRCR (1134-1236, 1288-1345), PAN_1 (1003-1084) |
| SerP76 | MW603462 | QWS65019 | 387/362 | 39,417 | - | K|IIGG | - |
| SerP84 | MW603463 | QWS65020 | 332/308 | 33,286 | 18 | K|VVGG | - |
| SerP86 | MW603464 | QWS65021 | 458/258 | 28,226 | 22 | R|ILDG | Clip_25 (28-78) |
| SerP113 | MW603465 | QWS65022 | 386/255 | 28,255 | 23 | R|IING | Clip_2 (32-87) |
| SerP119 | MW603466 | QWS65023 | 387/253 | 28,333 | 26 | L|IVGG | Clip_1 (34-80) |
| SerP125 | MW603467 | QWS65024 | 278/254 | 27,535 | 19 | R|IVGG | - |
| SerP127 | MW603468 | QWS65025 | 376/247 | 27,075 | 22 | R|IVNG | Clip_2 (27-80) |
| SerP131 | MW603469 | QWS65026 | 375/247 | 26,799 | 22 | R|VVNG | Clip_2 (30-83) |
| SerP135 | MW603470 | QWS65027 | 292/246 | 26,850 | 22 | G|IIGG | - |
| SerP141 | MW603471 | QWS65028 | 444/259 | 28,844 | - | R|IFGG | Clip_2 (102-152) |
| SerP145 | MW603472 | QWS65029 | 370/241 | 26,781 | 22 | H|IVGG | Clip_1 (31-76) |
| SerP163 | MW603473 | QWS65030 | 354/254 | 28,041 | 21* | V|IAFG | Clip_1 (28-73) |
| SerP173 | MW603474 | QWS65031 | 347/249 | 27,495 | 19 | F|VFGG | Clip_1 (26-71) |
| SerP1832 | AB363980 | BAG14262 | 383/265 | 29,203 | 18 | R|IYGG | Clip_2 (21-74) |
| SerP193 | MW603475 | QWS65032 | 375/247 | 27,564 | 22* | R|ILGG | Clip_2 (48-97) |
| SerP209 | MW603476 | QWS65033 | 258/227 | 22,943 | 16 | R|IIGG | - |
| SerP227 | MW603477 | QWS65034 | 376/251 | 27,969 | 23 | L|IVGG | Clip_1 (31-76) |
| SerP228 | MW603478 | QWS65035 | 374/250 | 27,849 | 20 | L|IVGG | Clip_1 (28-74) |
| SerP247 | MW603479 | QWS65036 | 379/257 | 28,343 | 18 | T|IISM | Clip_1 (26-71) |
| SerP266 | MW603480 | QWS65037 | 281/256 | 27,895 | 18 | K|IVGG | - |
| SerP272 | MW603481 | QWS65038 | 404/297 | 32,710 | 17* | K|IYGG | Clip_2 (21-74) |
| SerP282 | MW603482 | QWS65039 | 328/270 | 29,212 | 17* | G|ITGG | Clip_1 (27-53) |
| SerP345 | MW603483 | QWS65040 | 359/234 | 26,360 | 22 | L|IVGG | Clip_1 (30-76) |
| SerP370 | MW603484 | QWS65041 | 407/257 | 28,048 | 23* | K|ISNG | Clip_2 (27-69, 78-121) |
| SerP409 | MW603485 | QWS65042 | 447/234 | 26,142 | 22* | K|IGKG | Clip_1? (58-92), Clip_2 (148-195) |
| SerPH70 | MW419917 | QRE01765 | 280/249 | 27,206 | 19 | K|IVGG | - |
| SerPH216 | MW419918 | QRE01766 | 348/254 | 28,298 | 16 | K|IGND | Clip PPAF-2 (21-66) |
| SerPH2233 | AJ400904 | CAC12696 | 400/264 | 29,412 | 21 | R|ITGN | Clip PPAF-2 (64-104) |
| SerPH235 | MW419919 | QRE01767 | 407/263 | 28,787 | 16 | K|ITGN | Clip PPAF-2 (53-96) |
| SerPH4154 | AB084067 | BAC15605 | 444/261 | 28,711 | 15 | N|LIGG | Clip PPAF-2 (68-114) |
Prabhakar et al., 2007
Kim et al., 2008
Kwon et al., 2000
Lee et al., 2002
Clip domains were grouped manually (Clip_1 and Clip_2) and position curated according to [7].
Signal peptide cleavage site probability is less than 0.5.
Table 2.
Non-gut trypsin sequences found only in the combined transcriptome of Tenebrio molitor and absent in the larval gut transcriptome. All active sites consist of H D S, and S1 subsites are D G G.
| Identification | NCBI Nucleotide Accession | NCBI Protein Accession | Preproenzyme/Mature Enzyme (aa) | Mature Enzyme (Da) | Signal Peptide (aa) | Proenzyme Cleavage Site | Regulatory Domains (aa position) |
|---|---|---|---|---|---|---|---|
| NGSerP3 | MW628465 | QWS65044 | 259/228 | 24,386 | 16 | K|IVGG | - |
| NGSerP5 | MW628466 | QWS65045 | 333/236 | 26,035 | 24 | R|IVGG | - |
| NGSerP14 | MW628467 | QWS65046 | 1293/286 | 31,448 | - | R|IVGG | Ldl_recept_a (774–808, 842–878, 1260–1289), SRCR (900–995) |
| NGSerP15 | MW628468 | QWS65047 | 516/235 | 25,699 | 23 | R|IVGG | EBNA-3C (201–246) |
| NGSerP20 | MW628469 | QWS65048 | 361/238 | 26,395 | 17* | R|IVGG | - |
| NGSerP21 | MW628470 | QWS65049 | 276/228 | 24,732 | 22 | R|IVGG | - |
| NGSerP22 | MW628471 | QWS65050 | 290/242 | 25,975 | 17* | R|VVGG | - |
| NGSerP24 | MW628472 | QWS65051 | 810/243 | 27,555 | 19 | R|IVGG | Family of unknown function (DUF5585) (259–544) |
| NGSerP26 | MW628476 | QWS65055 | 254/227 | 24,214 | 23 | R|IVGG | - |
| NGSerP27 | MW628473 | QWS65052 | 369/242 | 26,704 | 19 | R|IVGG | - |
| NGSerP28 | MW628477 | QWS65056 | 310/241 | 27,033 | 26 | R|IVGG | - |
| NGSerP35 | MW628478 | QWS65057 | 260/231 | 24,884 | 21* | R|IVGG | - |
| NGSerP37 | MW628479 | QWS65058 | 298/251 | 27,327 | 19 | R|IVNG | - |
| NGSerP65 | MW628474 | QWS65053 | 619/240 | 26,001 | 20 | R|IVGG | - |
| NGSerP66 | MW628480 | QWS65059 | 523/245 | 27,560 | - | R|VVNG | Clip_11 (164–207) |
| NGSerP77 | MW628481 | QWS65060 | 288/252 | 27,164 | 17 | G|IIGG | - |
| NGSerP104 | MW628482 | QWS65061 | 332/300 | 32,646 | 18 | R|IFGG | - |
| NGSerP109 | MW628483 | QWS65062 | 964/247 | 26,987 | 17 | H|IVGG | 104 kDa microneme/rhoptry antigen (291–529) |
| NGSerP116 | MW628484 | QWS65063 | 381/257 | 28,382 | 16 | V|IAFG | Clip_2 (25–80) |
| NGSerP166 | MW628485 | QWS65064 | 376/259 | 28,449 | 15 | F|VFGG | Clip_2 (23–78) |
| NGSerP275 | MW628486 | QWS65065 | 430/257 | 28,969 | 23 | R|IYGG | Clip_21 (32–75, 80–135) |
| NGSerP297 | MW628487 | QWS65066 | 350/255 | 28,238 | 18 | R|ILGG | Clip_1 (25–70) |
| NGSerP317 | MW628475 | QWS65054 | 389/246 | 27,195 | 16 | R|IIGG | - |
| NGSerP347 | MW628488 | QWS65067 | 367/256 | 28,001 | 25* | R|IIGG | Clip_1 (36–81) |
Clip domains were grouped manually (Clip_1 and Clip_2) and position curated according to [7].
Signal peptide cleavage site probability is less than 0.5.
From each S1 peptidase sequence, we analyzed the length of the predicted preproenzyme and mature enzyme form, molecular mass of the mature enzyme, active site and S1 substrate binding subsite residues, position of signal peptide and proenzyme cleavage sites, and the presence of a transmembrane domain. We also identified regulatory domains: clip_1 and clip_2 [7], carbohydrate-binding module family 14 (CBM_14) domain, low density lipoprotein receptor gene family (ldl_recept_a), scavenger receptor cysteine-rich (SRCR) domain, PAN_1 - (1) the N-terminal N domains of members of the plasminogen/hepatocyte growth factor family, (2) the apple domains of the plasma prekallikrein/coagulation factor XI family, and (3) domains of various nematode proteins referred to as the PAN module, thrombospondin type 1 repeats, Epstein-Barr virus nuclear antigen 3C (EBNA-3C), Family of unknown function (DUF5585), 104 kDa microneme/rhoptry antigen.
2. Experimental Design, Materials and Methods
2.1. Preparation of biological material and RNA isolation
The midgut transcriptome data from T. molitor larvae were obtained at The Center for Grain and Animal Health Research (CGAHR, Manhattan, KS USA), where laboratory colonies of T. molitor are maintained on a diet of 50% oat flakes, 2.5% brewer's yeast, and 47.5% wheat flour at 28 °C, 75% R.H., in darkness. Approximately five-week old larvae with an average weight of 5.1 mg from three independent biological replicates were fasted overnight and were placed on a diet consisting of 85% stabilized wheat germ, 10% wheat flour, and 5% brewer's yeast for 12 h. For each replicate, the midgut was extracted from 4 to 7 larvae and placed in room temperature RNAlater (Ambion, Austin TX USA). For RNA isolation, excess RNAlater was blotted, and pooled midguts were ground with a plastic pestle in 1.5 ml microfuge tube containing liquid nitrogen. Total RNA was isolated using the Absolutely RNA Kit with DNase on-column treatment (Agilent, La Jolla, CA USA).
Whole-body transcriptomes from different stages of the life cycle of T. molitor - eggs, larvae of the 1st instar (the first week after hatching), larvae of the 4th instar (in the fifth week after hatching), pupae, young males and females with non-chitinized integuments, and two weeks old adult males and females were obtained from the laboratory colony at the Lomonosov Moscow State University (Moscow, Russia). All stages were maintained on milled oat flakes at 26 ± 0.5 °C and 75% relative humidity. Two replicates were obtained for eggs, 1st and 4th instar larvae, pupae, adult males and young females; one replicate was obtained for young males and adult females. RNA was extracted using the RNEasy Mini kit (Qiagen, Hilden, Germany). Immediately prior to isolation, the samples were homogenized by trituration in liquid nitrogen. The concentration of isolated RNA was measured on a Qubit (Thermofisher, Waltham, MA USA) fluorimeter using a set of reagents for high-sensitivity RNA analysis. The integrity of the RNA was checked by capillary electrophoresis on a Bioanalyzer 2100 (Agilent). The NEBNext RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA USA) was used to prepare the libraries according to recommended protocol with a fragmentation time of 5 min.
2.2. Sequencing of cDNA
The resulting midgut total RNA was sent to a sequencing facility (National Center for Genome Resources - NCGR, Santa Fe, NM, USA), where mRNA was isolated by polyA, standard libraries were made, and paired-end sequencing was performed on a Illumina HiSeq 2000 (San Diego, CA, USA) using standard protocols from the manufacturer. Approximately 240 million sequence reads were obtained, with an approximate 250 bp insert.
The libraries of different T. molitor developmental stages were sequenced on a Illumina HiSeq 2000 (Lomonosov Moscow State University, Moscow, Russia) using the TruSeq SBS Kit v3 reagent kit (200 cycles) with the following settings: read length 101, index read length 7, reverse reading length 101. The preprocessed samples contained from 7 million to 24 million reads.
2.3. Assembly of contigs and final sequences
Assembly of T. molitor larval midgut sequences was performed de novo with SeqManNGen (v. 4.0.1.4, DNAStar, Madison, WI USA) and described in [8]. It included NCGR assembly from all replicates, resulting in 197,800 contigs (N50 = 2232), previous databases of Sanger sequencing [3] and pyrosequencing [9] of mRNA from the larval gut.
The whole T. molitor transcriptome assembly was performed with SeqManNGen (v 15.0.0.160, default parameters) and included the midgut assembly described above combined with the Illumina sequencing data obtained for T. molitor developmental stages. There were 342,592,161 total reads assembled, with 143,807,206 reads not assembled and 382,435,025 removed during sampling due to read depth. Reads were assembled into 130,559 contigs, with 36,463 contigs >1 kb.
Potential coding sequences, starting at methionine and covering at least 20% of the mRNA sequence, were found in the T. molitor contigs using custom software. BLAST [10] and custom scripts were used to identify ORFs homologous to those encoding serine peptidases from the S1 chymotrypsin family and their inactive homologs. The sequence of human trypsin 2 (UniProt ID P07478) was used as a query. Multiple sequence alignment with BioEdit (v. 7.0.5) was used to refine and build consensus sequences, and in the case of SNPs, the amino acid chosen was the highest percentage and more than 50% of the total. ORFs that were grouped into blocks with identity of at least 95% and that overlapped with another block of at least 10 amino acid residues were considered as a unique peptidase.
2.4. Analysis of sequences
The molecular mass of the mature enzyme of the predicted protein was computed using ExPASy Server (https://web.expasy.org/compute_pi/). Signal peptide was predicted with SignalP 5.0 server (http://www.cbs.dtu.dk/services/SignalP/index.php) [11]. The start of the mature enzyme, positions of proenzyme cleavage site, active site and S1 substrate binding subsite residues were predicted by sequence homology through alignment with mature human trypsin 2 (UniProt ID P07478) using BioEdit (v. 7.0.5) and Clustal Omega multiple sequence alignment tool (https://www.ebi.ac.uk/Tools/msa/clustalo/) [12]. The presence of a transmembrane domain was predicted with TMHMM Server (v. 2.0) (http://www.cbs.dtu.dk/services/TMHMM/) [13].
Regulatory domains: clip_1 and clip_2, CBM_14, ldl_recept_a, SRCR, PAN_1, thrombospondin type 1 repeats, EBNA-3C, Family of unknown function (DUF5585), 104 kDa microneme/rhoptry antigen, were searched with HMMER web server (https://www.ebi.ac.uk/Tools/hmmer/) [14], and using NCBI Conserved Domains search service (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [15]. Clip domains were manually curated and annotated according to [7].
Ethics statement
Institutional Review Board Statement: This research was performed in accordance with Kansas State University Research Compliance Office, Institutional Biosafety Committee registration number 1191, “Functional Genomics of Stored Product Insects”.
CRediT author statement
Nikita I. Zhiganov: Investigation, Software. Valeriia F. Tereshchenkova: Writing - Original Draft, Validation. Brenda Oppert: Supervision. Irina Y. Filippova: Writing - Review & Editing. Nataliya V. Belyaeva: Visualization. Yakov E. Dunaevsky: Data Curation. Mikhail A. Belozersky: Formal analysis. Elena N. Elpidina: Conceptualization, Methodology, Writing - Review & Editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.
Acknowledgments
This study was supported by Russian Foundation for Basic Research grant № 20-54-56044 Iran_t (collection of active serine peptidases data) and by Russian Science Foundation grant № 19-74-00080 (collection of inactive serine peptidases homologs data). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.
Footnotes
Supplementary material associated with this article can be found in the online version at doi:10.1016/j.dib.2021.107301.
Appendix. Supplementary materials
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