Abstract
Genome wide gene expression analysis, transcription analysis and mass spectrometry have revealed several uncharacterized genes and proteins from commercial silkworm Bombyx mori. Infection of B. mori larvae with microsporidian Nosema bombycis induced appearance of exclusive proteins in hemocytes of B. mori showing host response. Mass spectrometry of the exclusive proteins revealed abundance of an uncharacterized protein H9JAZ8 that displayed conserved domains of Von Willebrand Factor type D domain (VWF), C8 domain, trypsin inhibitor-like cysteine rich domain and conserved epidermal growth factor like. Protein model of H9JAZ8 confirmed presence of VWF domains, cysteine rich domains and disulphide bonds. Expression of H9JAZ8 gene showed upregulation on day 2 after the infection followed by significant down regulated expression till day 10 exhibiting infection- associated modulation of the gene. BLASTP of H9JAZ8 showed 99% amino acid sequence similarity with B. mori BGIBMGA006693 demonstrating H9JAZ8 as a paralog of B. mori hemocytin. BLASTP of H9JAZ8 revealed 18 most similar hemocytin homologues from different insect species with 99% similarity for B. mandarina hemocytin. A Maximum Parsimony phylogeny tree showed three clusters where H9JAZ8 branched from the hemocytin of B. mandarina with 100% bootstrap value. Global BLAST analysis of H9JAZ8 with hemocytin of B. mandarina (XP_028038271.1) showed deletion of two regions, a 27 amino acid stretch, RRIFFSFILIYCTFNCEAGYGIPASSD at 2–28th position and a 21 amino acid stretch, DVVVISPNHHNNSSYYSYSQG at 317-338th position. Togteher, B. mori H9JAZ8 is diverged from B. mandarina hemocytin as part of speciation and immune evolution during domestication from the wild silkworm.
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References
Ao J, Ling E, Yu XQ (2007) Drosophila C-type lectins enhance cellular encapsulation. Mol Immunol 44:2541–2548. https://doi.org/10.1016/j.molimm.2006.12.024
Arai I, Ohta M, Suzuki A, Tanaka S, Yoshizawa Y (2013) Sato R (2013) Immunohistochemical analysis of the role of hemocytin in nodule formation in the larvae of the silkworm. Bombyx mori J Insect Sci 13:125. https://doi.org/10.1673/031.013.12501
Bania J, Stachowiak D, Polanowski A (1999) Primary structure and properties of the cathepsin G/chymotrypsin inhibitor from the larval haemolymph of Apis mellifera. Eur J Biochem 262:680–687
Benkert P, Biasini M, Schwede T (2011) Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27:343–350
Cairns TM, Landsburg DJ, Whitbeck JC, Eisenberg RJ, Cohen GH (2005) Contribution of cysteine residues to the structure and function of herpes simplex virus gH/gL. Virology 332:550–562. https://doi.org/10.1016/j.virol.2004.12.006. PMID: 15680420
Cairns T, Milne RSB, Ponce-de-Leon M, Tobin DK, Cohen GH, Eisenberg RJ (2003) Structure–function analysis of herpes simplex virus (HSV-1) gD and gH/gL: clues from gD/gH chimeras. J Virol 77:6731–6742
Chen S, Zhaoming D, Xiu R, Dongchao Z, Yan Z, Muya T, Jiaxuan H, Lin Y, Ping Z (2019) Proteomic identification of immune-related silkworm proteins involved in the response to bacterial infection. J Insect Sci 9:13. https://doi.org/10.1093/jisesa/iez056
Cheng H, Yan R, Li S, Yuan Y, Liu J, Ruan C, Dai K (2009) Shear-induced interaction of platelets with von Willebrand factor results in glycoprotein Ibα shedding. Am J Physiol Heart Circ Physiol 297:2128–2135
Cierpicki T, Bania J, Otlewskim J (2000) NMR solution structure of Apis mellifera chymotrypsin/ cathepsin G inhibitor-1 (AMCI-1): structural similarity with Ascaris protease inhibitors. Protein Sci 9:976–984
David KT, Oaks JR, Halanych KM (2020) Patterns of gene evolution following duplications and speciations in vertebrates. PeerJ 8:e8813. https://doi.org/10.7717/peerj.8813
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x
Feng M, Xia J, Fei S, Peng R, Wang X, Zhou Y, Wang P, Swevers L, Sun J (2021) Identification of silkworm hemocyte subsets and analysis of their response to baculovirus infection based on single- cell RNA sequencing. Front Immunol 12:645359. https://doi.org/10.3389/fimmu.2021.645359
Gábor E, Gyöngyi C, Gábor C, Tibor T, Katalin F-M, Zsuzsanna D, István A, Éva K (2017) Hemolectin expression reveals functional heterogeneity in honey bee (Apis mellifera) hemocytes. Dev Comp Immunol 76:403–411. https://doi.org/10.1016/j.dci.2017.07.013
Galperin MY, Koonin EV (2012) Divergence and convergence in enzyme evolution. J Biol Chem 287:21–28. https://doi.org/10.1074/jbc.R111.241976
Gerardo NM, Altincicek B, Anselme C, Atamian H, Barribeau SM, de Vos M, Duncan EJ, Vilcinskas A et al (2010) Immunity and other defenses in pea aphids, Acyrthosiphon pisum. Genome Biol 11:R21. https://doi.org/10.1186/gb-2010-11-2-r21
Goto A, Kumagai T, Kumagai C, Hirose J, Narita H, Mori H, Kadowaki T, Beck K, Kitagawa Y (2001) A Drosophila haemocyte-specific protein, hemolectin, similar to human von willebrand factor. Biochem J 359:99–108
Hassan W, Nath BS, Ponnuvel KM, Mishra RK, Pradeep AR (2020) Evolutionary diversity in the intracellular microsporidian parasite Nosema sp. infecting wild silkworm revealed by IGS nucleotide sequence diversity. J Mol Evol 88:345–360. https://doi.org/10.1007/s00239-020-09936-2
Hoffmeister KM, Felbinger TW, Falet H, Denis CV, Bergmeier W, Mayadas TN, von Andrian UH, Wagner DD, Stossel TP, Hartwig JH (2003) The clearance mechanism of chilled blood platelets. Cell 112:87–97
Hu J, Ng PC (2013) SIFT Indel: predictions for the functional effects of amino acid insertions/ deletions in proteins. PLoS ONE 8(10):e77940. https://doi.org/10.1371/journal.pone.0077940
Hungund SP, Pradeep AR, Makwana P, Sagar C, Mishra RK (2020) Cellular defence and innate immunity in the larval ovarian disc and differentiated ovariole of the silkworm Bombyx mori induced by microsporidian infection. Invertebr Reprod Dev 64:10–21. https://doi.org/10.1080/07924259.2019.1669727
Kawamoto M, Jourakub A, Toyodac A, Yokoib K, Minakuchic Y, Katsumaa S, Fujiyamac A, Kiuchia T, Yamamoto K, Shimada T (2019) High-quality genome assembly of the silkworm, Bombyx mori. Insect Biochem Mol Biol 107:53–62
Kelley L, Mezulis S, Yates C et al (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858. https://doi.org/10.1038/nprot.2015.053
Koonin EV (2005) Orthologs, paralogs, and evolutionary genomics. Annu Rev Genet 39:309–338
Kotani E, Yamakawa M, Iwamoto S, Tashiro M, Mori H, Sumida M, Matsubara F, Taniai K, Kadono-Okuda K, Kato Y, Mori H (1995) Cloning and expression of the gene of hemocytin, an insect humoral lectin which is homologous with the mammalian von willebrand factor. Biochim Biophys Acta 1260:245–258
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096
Lemaitre B, Girardin SE (2013) Translation inhibition and metabolic stress pathways in the host response to bacterial pathogens. Nat Rev Microbiol 11:365–369. https://doi.org/10.1038/nrmicro3029
Lesch C, Goto A, Lindgren M, Bidla G, Dushay MS, Theopold U (2007) A role for hemolectin in coagulation and immunity in Drosophila melanogaster. Dev Comp Immunol 31:1255–1263. https://doi.org/10.1016/j.dci.2007.03.012
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real time quantitative PCR and the 2-∆∆CT method. Methods 25:402–408
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275
Luan Y, Zuo W, Li C, Gao R, Zhang H, Tong X, Han M, Hu H, Lu C, Dai F (2018) Identification of genes that control silk yield by RNA sequencing analysis of silkworm (Bombyx mori) strains of variable silk yield. Int J Mol Sci 19:3718. https://doi.org/10.3390/ijms19123718
Ma Z, Li C, Pan G, Li Z, Han B et al (2013) Genome-wide transcriptional response of silkworm (Bombyx mori) to infection by the microsporidian Nosema bombycis. PLoS ONE 8(12):e84137. https://doi.org/10.1371/journal.pone.0084137
Makwana P, Dubey H, Pradeep AR, Sivaprasad V, Ponnuvel KM, Mishra RK (2021) Dipteran endoparasitoid infestation actively suppressed host defense components in hemocytes of silkworm Bombyx mori for successful parasitism. Anim Gene 22:200118. https://doi.org/10.1016/j.angen.2021.200118
Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Bryant SH (2015) CDD: NCBI’s conserved domain database. Nucleic Acids Res 43(Database issue):D222–D226. https://doi.org/10.1093/nar/gku1221
Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics. Oxford University Press, New York
Ni W, Bao J, Mo B, Liu L, Li T, Pan G, Chen J, Zhou Z (2020) Hemocytin facilitates host immune responses against Nosema bombycis. Dev Comp Immunol 103:103495. https://doi.org/10.1016/j.dci.2019.103495
Peña C, Espeland M (2015) Diversity dynamics in Nymphalidae butterflies: effect of phylogenetic uncertainty on diversification rate shift estimates. PLoS ONE 10(4):e0120928. https://doi.org/10.1371/journal.pone.0120928
Prabhuling SH, Makwana P, Pradeep ANR, Vijayan K, Mishra RK (2021) Release of mediator enzyme β-hexosaminidase and modulated gene expression accompany hemocyte degranulation in response to parasitism in the silkworm Bombyx mori. Biochem Genet 59:997–1017. https://doi.org/10.1007/s10528-021-10046-x
Rao SN, Muthulakshmi M, Kanginakudru S, Nagaraju J (2004) Phylogenetic relationships of three new microsporidian isolates from the silkworm, Bombyx mori. J Invertebr Pathol 86:87–95
Rao SN, Nath BS, Saratchandra B (2005) Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis. Genome 48:355–366. https://doi.org/10.1139/G04-109
Rosales C (2011) Phagocytosis, a cellular immune response in insects. Invertebrate Surviv J 8:109–131
Rosengren KJ, Daly NL, Scanlon MJ, Craik DJ (2001) Solution structure of BSTI: a new trypsin inhibitor from skin secretions of Bombina bombina. Biochemistry 40:4601–4609
Satyavathi VV, Minz A, Nagaraju J (2014) Nodulation: an unexplored cellular defence mechanism. Cell Signal 26:1753–1763
Shevchenko A, Tomas H, Havli J et al (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860. https://doi.org/10.1038/nprot.2006.468
Shobahah J, Xue S, Hu D et al (2017) Quantitative phosphoproteome on the silkworm (Bombyx mori) cells infected with baculovirus. Virol J 14:117. https://doi.org/10.1186/s12985-017-0783-8
Shu Z, Zeng J, Xia L et al (2022) Structural mechanism of VWF D’D3 dimer formation. Cell Discov 8:14. https://doi.org/10.1038/s41421-022-00378-2
Subrahmanyam G, Esvaran VG, Ponnuvel KM, Hassan W, Chutia M, Das R (2019) Isolation and molecular identification of microsporidian pathogen causing nosemosis in muga silkworm, Antheraea assamensis Helfer (Lepidoptera: Saturniidae). Indian J Microbiol 59:525–529. https://doi.org/10.1007/s12088-019-00822-0
Tanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, Suzuki N, Yoshiyama M, Kaneko Y, Iwasaki T, Sunagawa T, Yamaji K, Asaoka A, Mita K, Yamakawa M (2008) A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochem Mol Biol 38:1087–1110. https://doi.org/10.1016/j.ibmb.2008.09.001
Tanaka H, Yamakawa M (2011) Regulation of the innate immune responses in the silkworm, Bombyx mori. Invertebr Surviv J 8:59–69
The International Silkworm Genome Consortium (2008) The genome of a lepidopteran model insect, the silkworm Bombyx mori insect. Biochem Mol Biol 38:1036–1045
Undeen AH, Alger NE (1971) A density gradient method for fractionating microsporidian spores. J Invertbr Pathol 18:419–420
Wang S, Li W, Liu S, Xu J (2016) RaptorX-Property: a web server for protein structure property prediction. Nucleic Acids Res 44(W1):W430–W435. https://doi.org/10.1093/nar/gkw306
Watanabe A, Miyazawa S, Kitami M, Tabunoki H, Ueda K, Sato R (2006) Characterization of a novel C-type lectin, Bombyx mori multibinding protein from the B. mori hemolymph: mechanism of wide-range microorganism recognition and role in immunity. J Immunol 177:4594–4604. https://doi.org/10.4049/jimmunol.177.7.4594
Zhang G, Ueberheide BM, Waldemarson S, Myung S, Molloy K et al (2010) Protein quantitation using mass spectrometry. Methods Mol Biol 673:211–222
Acknowledgements
The authors thank Central Silk Board, Bangalore for facilities and Department of Biotechnology, Government of India, New Delhi for financial support in the form of a research project to ARP (BT/ PR6355/PBD/19/236/2012 dated 08/01/2013).
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Kalyani, D., Varghese, A., Prabhuling, S.H. et al. Uncharacterized protein with amino acid deletions from Bombyx mori illustrates divergence from Bombyx mandarina hemocytin and showed modulated gene expression after infection by Nosema bombycis. Int J Trop Insect Sci 43, 1623–1632 (2023). https://doi.org/10.1007/s42690-023-01080-w
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DOI: https://doi.org/10.1007/s42690-023-01080-w