{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T11:52:03Z","timestamp":1787313123288,"version":"build-2736575974"},"reference-count":47,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2022,4,22]],"date-time":"2022-04-22T00:00:00Z","timestamp":1650585600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Genes encoding snake venom toxins have been studied extensively. However, genes involved in the modification and functioning of venom proteins are little known. Protobothrops is a genus of pit vipers, which are venomous and inhabit the Nansei (Southwest) islands of Japan, Taiwan China, Vietnam, Thailand, Myanmar, Nepal, Bhutan, and India. Our previous study decoded the genome of Protobothrops flavoviridis, a species endemic to the Nansei Islands, Japan, and revealed unique evolutionary processes of some venom genes. In this study, we analyzed genes that are highly expressed in venom glands to survey genes for candidate enzymes or chaperone proteins involved in toxin folding and modification. We found that, in addition to genes that encode venom proteins and ribosomal proteins, genes that encode protein disulfide isomerase (PDI) family members (orthologs of human P4HB and PDIA3), Selenoprotein M (SELENOM), and Calreticulin (CALR) are highly expressed in venom glands. Since these enzymes or chaperones are involved in protein modification and potentially possess protein folding functions, we propose that P4HB, SELENOM, CALR, and PDIA3 encode candidate enzymes or chaperones to confer toxic functions upon the venom transcriptome.<\/jats:p>","DOI":"10.3390\/toxins14050300","type":"journal-article","created":{"date-parts":[[2022,4,24]],"date-time":"2022-04-24T00:45:21Z","timestamp":1650761121000},"page":"300","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Active Expression of Genes for Protein Modification Enzymes in Habu Venom Glands"],"prefix":"10.3390","volume":"14","author":[{"given":"Akiko","family":"Isomoto","sequence":"first","affiliation":[{"name":"Division of Genomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan"},{"name":"Department of Biological Chemistry, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eiichi","family":"Shoguchi","sequence":"additional","affiliation":[{"name":"Marine Genomics Unit, Okinawa Institute of Science, Technology Graduate University, Onna 904-0495, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6313-3340","authenticated-orcid":false,"given":"Kanako","family":"Hisata","sequence":"additional","affiliation":[{"name":"Marine Genomics Unit, Okinawa Institute of Science, Technology Graduate University, Onna 904-0495, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4954-6373","authenticated-orcid":false,"given":"Jun","family":"Inoue","sequence":"additional","affiliation":[{"name":"Marine Genomics Unit, Okinawa Institute of Science, Technology Graduate University, Onna 904-0495, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yinrui","family":"Sun","sequence":"additional","affiliation":[{"name":"Division of Genomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8229-0467","authenticated-orcid":false,"given":"Kenji","family":"Inaba","sequence":"additional","affiliation":[{"name":"Institute of Multidisciplinary Research for Advanced Material, Tohoku University, Sendai 980-8577, Japan"},{"name":"Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4480-3572","authenticated-orcid":false,"given":"Noriyuki","family":"Satoh","sequence":"additional","affiliation":[{"name":"Marine Genomics Unit, Okinawa Institute of Science, Technology Graduate University, Onna 904-0495, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8700-6330","authenticated-orcid":false,"given":"Tomohisa","family":"Ogawa","sequence":"additional","affiliation":[{"name":"Department of Biological Chemistry, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7623-7969","authenticated-orcid":false,"given":"Hiroki","family":"Shibata","sequence":"additional","affiliation":[{"name":"Division of Genomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,4,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.ympev.2016.04.027","article-title":"The taxonomic position and the unexpected divergence of the Habu viper, Protobothrops among Japanese subtropical islands","volume":"101","author":"Shibata","year":"2016","journal-title":"Mol. Phylogenet. Evol."},{"key":"ref_2","first-page":"1245","article-title":"Dokujyakousyoh","volume":"53","author":"Miyagi","year":"2004","journal-title":"Clin. All-Round"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Aird, S.D., Watanabe, Y., Villar-Briones, A., Roy, M.C., Terada, K., and Mikheyev, A.S. (2013). Quantitative high-throughput profiling of snake venom gland transcriptomes and proteomes (Ovophis okinavensis and Protobothrops flavoviridis). BMC Genom., 14.","DOI":"10.1186\/1471-2164-14-790"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Aird, S.D., Aggarwal, S., Villar-Briones, A., Tin, M.M., Terada, K., and Mikheyev, A.S. (2015). Snake venoms are integrated systems, but abundant venom proteins evolve more rapidly. BMC Genom., 16.","DOI":"10.1186\/s12864-015-1832-6"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1093\/gbe\/evr043","article-title":"Discovery of highly divergent repeat landscapes in snake genomes using high-throughput sequencing","volume":"3","author":"Castoe","year":"2011","journal-title":"Genome Biol. Evol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1016\/S0079-6603(08)61036-3","article-title":"Molecular evolution of snake toxins: Is the functional diversity of snake toxins associated with a mechanism of accelerated evolution?","volume":"59","author":"Ohno","year":"1998","journal-title":"Prog. Nucleic Acid Res. Mol. Biol."},{"key":"ref_7","unstructured":"M\u00e9nez, A. (2002). Accelerated and Regional Evolution of Snake Venom Gland Isozymes, John Wiley & Sons. Perspectives in Molecular Toxinology."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1016\/j.toxicon.2003.11.003","article-title":"Molecular evolution of myotoxic phospholipases A2 from snake venom","volume":"42","author":"Ohno","year":"2003","journal-title":"Toxicon"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"11300","DOI":"10.1038\/s41598-018-28749-4","article-title":"The habu genome reveals accelerated evolution of venom protein genes","volume":"8","author":"Shibata","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Ogawa, T., Oda-Ueda, N., Hisata, K., Nakamura, H., Chijiwa, T., Hattori, S., Isomoto, A., Yugeta, H., Yamasaki, S., and Fukumaki, Y. (2019). Alternative mRNA Splicing in Three Venom Families Underlying a Possible Production of Divergent Venom Proteins of the Habu Snake. Toxins, 11.","DOI":"10.3390\/toxins11100581"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1939","DOI":"10.1007\/s00018-015-1844-z","article-title":"Privileged frameworks from snake venom","volume":"72","author":"Reeks","year":"2015","journal-title":"Cell Mol. Life Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"849","DOI":"10.1016\/j.toxicon.2010.06.018","article-title":"NMR methods for determining disulfide-bond connectivities","volume":"56","author":"Mobli","year":"2010","journal-title":"Toxicon"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1016\/j.bbapap.2011.04.009","article-title":"Snake venom metalloproteinases: Structure, function and relevance to the mammalian ADAM\/ADAMTS family proteins","volume":"1824","author":"Takeda","year":"2012","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Rokyta, D.R., Lemmon, A.R., Margres, M.J., and Aronow, K. (2012). The venom-gland transcriptome of the eastern diamondback rattlesnake (Crotalus adamanteus). BMC Genom., 13.","DOI":"10.1186\/1471-2164-13-312"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"460","DOI":"10.1016\/j.jprot.2013.10.026","article-title":"Activation of Bothrops jararaca snake venom gland and venom production: A proteomic approach","volume":"94","author":"Luna","year":"2013","journal-title":"J. Proteom."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1093\/molbev\/msy226","article-title":"ORTHOSCOPE: An Automatic Web Tool for Phylogenetically Inferring Bilaterian Orthogroups with User-Selected Taxa","volume":"36","author":"Inoue","year":"2019","journal-title":"Mol. Biol. Evol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1186\/1479-7364-6-6","article-title":"The human protein disulfide isomerase gene family","volume":"6","author":"Galligan","year":"2012","journal-title":"Hum. Genom."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3330","DOI":"10.1038\/emboj.2010.222","article-title":"Crystal structures of human Ero1\u03b1 reveal the mechanisms of regulated and targeted oxidation of PDI","volume":"29","author":"Inaba","year":"2010","journal-title":"EMBO J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2456","DOI":"10.1038\/srep02456","article-title":"Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding","volume":"3","author":"Sato","year":"2013","journal-title":"Sci. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1016\/j.freeradbiomed.2015.02.010","article-title":"Structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum","volume":"83","author":"Okumura","year":"2015","journal-title":"Free Radic. Biol. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"790","DOI":"10.1089\/ars.2011.4418","article-title":"Structure, mechanism, and evolution of Ero1 family enzymes","volume":"16","author":"Araki","year":"2012","journal-title":"Antioxid. Redox Signal."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2826","DOI":"10.1016\/j.febslet.2012.07.023","article-title":"Protein disulfide isomerases in neurodegeneration: From disease mechanisms to biomedical applications","volume":"586","author":"Andreu","year":"2012","journal-title":"FEBS Lett."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"787","DOI":"10.1016\/j.molcel.2010.11.010","article-title":"Oxidative protein folding by an endoplasmic reticulum-localized peroxiredoxin","volume":"40","author":"Zito","year":"2010","journal-title":"Mol. Cell"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4185","DOI":"10.1038\/emboj.2010.273","article-title":"Recycling of peroxiredoxin IV provides a novel pathway for disulphide formation in the endoplasmic reticulum","volume":"29","author":"Tavender","year":"2010","journal-title":"EMBO J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2262","DOI":"10.1111\/j.1742-4658.2012.08593.x","article-title":"Disulfide bond formation network in the three biological kingdoms, bacteria, fungi and mammals","volume":"279","author":"Sato","year":"2012","journal-title":"FEBS J."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1016\/j.str.2013.12.013","article-title":"Radically different thioredoxin domain arrangement of ERp46, an efficient disulfide bond introducer of the mammalian PDI family","volume":"22","author":"Kojima","year":"2014","journal-title":"Structure"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"33066","DOI":"10.1074\/jbc.M505023200","article-title":"Tissue-specific expression and dimerization of the endoplasmic reticulum oxidoreductase Ero1 beta","volume":"280","author":"Gubbens","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1038\/s41588-019-0559-8","article-title":"The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins","volume":"52","author":"Suryamohan","year":"2020","journal-title":"Nat. Genet."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"23685","DOI":"10.1074\/jbc.M003061200","article-title":"Endoplasmic reticulum oxidoreductin 1-lbeta (ERO1-Lbeta), a human gene induced in the course of the unfolded protein response","volume":"275","author":"Pagani","year":"2000","journal-title":"J. Biol. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1042\/BJ20101357","article-title":"The endoplasmic reticulum sulfhydryl oxidase Ero1\u03b2 drives efficient oxidative protein folding with loose regulation","volume":"434","author":"Wang","year":"2011","journal-title":"Biochem. J."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3904","DOI":"10.1021\/pr1012976","article-title":"Specialisation of the venom gland proteome in predatory cone snails reveals functional diversification of the conotoxin biosynthetic pathway","volume":"10","author":"Siero","year":"2011","journal-title":"J. Proteome Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1038\/nbt.1883","article-title":"Full-length transcriptome assembly from RNA-Seq data without a reference genome","volume":"29","author":"Grabherr","year":"2011","journal-title":"Nat. Biotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1494","DOI":"10.1038\/nprot.2013.084","article-title":"De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis","volume":"8","author":"Haas","year":"2013","journal-title":"Nat. Protoc."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2114","DOI":"10.1093\/bioinformatics\/btu170","article-title":"Trimmomatic: A flexible trimmer for Illumina sequence data","volume":"30","author":"Bolger","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1038\/nbt.3519","article-title":"Near-optimal probabilistic RNA-seq quantification","volume":"34","author":"Bray","year":"2016","journal-title":"Nat. Biotechnol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"3059","DOI":"10.1093\/nar\/gkf436","article-title":"MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform","volume":"30","author":"Katoh","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1972","DOI":"10.1093\/bioinformatics\/btp348","article-title":"trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses","volume":"25","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"W609","DOI":"10.1093\/nar\/gkl315","article-title":"PAL2NAL: Robust conversion of protein sequence alignments into the corresponding codon alignments","volume":"34","author":"Suyama","year":"2006","journal-title":"Nucleic Acids Res."},{"key":"ref_39","first-page":"406","article-title":"The neighbor-joining method: A new method for reconstructing phylogenetic trees","volume":"4","author":"Saitou","year":"1987","journal-title":"Mol. Biol. Evol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1536","DOI":"10.1093\/bioinformatics\/bts184","article-title":"ape 3.0: New tools for distance-based phylogenetics and evolutionary analysis in R","volume":"28","author":"Popescu","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2798","DOI":"10.1093\/molbev\/msv150","article-title":"FastME 2.0: A Comprehensive, Accurate and Fast Distance-Based Phylogeny Inference Program","volume":"32","author":"Lefort","year":"2015","journal-title":"Mol. Biol. Evol."},{"key":"ref_42","first-page":"512","article-title":"Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees","volume":"10","author":"Tamura","year":"1993","journal-title":"Mol. Biol. Evol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1007\/BF00160154","article-title":"Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: Approximate methods","volume":"39","author":"Yang","year":"1994","journal-title":"J. Mol. Evol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"691","DOI":"10.1093\/oxfordjournals.molbev.a003851","article-title":"A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach","volume":"18","author":"Whelan","year":"2001","journal-title":"Mol. Biol. Evol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1792","DOI":"10.1093\/nar\/gkh340","article-title":"MUSCLE: Multiple sequence alignment with high accuracy and high throughput","volume":"32","author":"Edgar","year":"2004","journal-title":"Nucleic Acids Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2947","DOI":"10.1093\/bioinformatics\/btm404","article-title":"Clustal W and Clustal X version 2.0","volume":"23","author":"Larkin","year":"2007","journal-title":"Bioinformatics"},{"key":"ref_47","first-page":"418","article-title":"Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions","volume":"3","author":"Nei","year":"1986","journal-title":"Mol. Biol. Evol."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/14\/5\/300\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:59:15Z","timestamp":1760137155000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/14\/5\/300"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,4,22]]},"references-count":47,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2022,5]]}},"alternative-id":["toxins14050300"],"URL":"https:\/\/doi.org\/10.3390\/toxins14050300","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,4,22]]}}}