Hostname: page-component-8448b6f56d-cfpbc Total loading time: 0 Render date: 2024-04-23T17:51:17.867Z Has data issue: false hasContentIssue false

Toll-interacting protein participates in immunity and development of the lepidopteran insect Antheraea pernyi

Published online by Cambridge University Press:  06 June 2023

Zhe Chen
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China
Awei Zhang
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China
Xuan Xu
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China
Lu Ding
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China
Xiaojiao Zhang
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China
Cen Qian
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China
Baojian Zhu*
College of Life Sciences, Anhui Agricultural University, Hefei 230036, China
Corresponding author: Baojian Zhu; Email:


Toll-interacting protein (Tollip) participates in multiple biological processes. However, the biological functions of Tollip proteins in insects remain to be further explored. Here, the genomic sequence of tollip gene from Antheraea pernyi (named Ap-Tollip) was identified with a length of 15,060 bp, including eight exons and seven introns. The predicted Ap-Tollip protein contained conserved C2 and CUE domains and was highly homologous to those tollips from invertebrates. Ap-Tollip was highly expressed in fat body compared with other determined tissues. As far as the developmental stages were concerned, the highest expression level was found at the 14th day in eggs or the 3rd day of the 1st instar. Ap-Tollip was also obviously regulated by lipopolysaccharide, polycytidylic acid or 20E in different tissues. In addition, the interaction between Ap-Tollip and ubiquitin was confirmed by western blotting and pull-down assay. RNAi of Ap-Tollip significantly affected the expression levels of apoptosis and autophagy-related genes. These results indicated that Ap-Tollip was involved in immunity and development of A. pernyi.

Research Paper
Copyright © The Author(s), 2023. Published by Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)



These authors contributed equally to this work.


Ahmed, M, Lai, T, Zada, S, Hwang, J, Pham, T, Yun, M and Kim, D (2018) Functional linkage of RKIP to the epithelial to mesenchymal transition and autophagy during the development of prostate cancer. Cancers 10, 273.CrossRefGoogle Scholar
Ankem, G, Mitra, S, Sun, F, Moreno, AC, Chutvirasakul, B, Azurmendi, HF, Li, L and Capelluto, DGS (2011) The C2 domain of Tollip, a Toll-like receptor signalling regulator, exhibits broad preference for phosphoinositides. Biochemical Journal 435, 597608.CrossRefGoogle ScholarPubMed
Bai, Y, Cui, XH, Gao, XL, Liu, CN, Lv, XP and Zheng, SM (2021) Poly (I: C) inhibits reticuloendothelial virus replication in chicken macrophage-like cells through the activation of toll-like receptor-3 signaling. Molecular Immunology 136, 110117.CrossRefGoogle ScholarPubMed
Bao, HB, Zhu, H, Yu, PH, Luo, GH, Zhang, R, Yue, Q and Fang, JC (2022) Time-series transcriptomic analysis reveals the molecular profiles of diapause termination induced by long photoperiods and high temperature in Chilo suppressalis. International Journal of Molecular Sciences 23, 12322.CrossRefGoogle ScholarPubMed
Burns, K, Clatworthy, J, Martin, L, Martinon, F, Plumpton, C, Maschera, B, Lewis, A, Ray, K, Tschopp, J and Volpe, F (2000) Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nature Cell Biology 2, 346351.CrossRefGoogle Scholar
Capelluto, DG (2012) Tollip: a multitasking protein in innate immunity and protein trafficking. Microbes and Infection 14, 140147.CrossRefGoogle ScholarPubMed
Cribbs, DH, Berchtold, NC, Perreau, V, Coleman, PD, Rogers, J, Tenner, AJ and Cotman, CW (2012) Extensive innate immune gene activation accompanies brain aging, increasing vulnerability to cognitive decline and neurodegeneration: a microarray study. Journal of Neuroinflammation 9, 179.CrossRefGoogle ScholarPubMed
Doi, H, Mitsui, K, Kurosawa, M, Machida, Y, Kuroiwa, Y and Nukina, N (2004) Identification of ubiquitin-interacting proteins in purified polyglutamine aggregates. FEBS Letters 571, 171176.CrossRefGoogle ScholarPubMed
Dong, DJ, Jing, YP, Liu, W, Wang, JX and Zhao, XF (2015) The steroid hormone 20-hydroxyecdysone upregulates Ste-20 Family Serine/Threonine kinase Hippo to induce programmed cell death. Journal of Biological Chemistry 290, 2473824746.CrossRefGoogle ScholarPubMed
Du, J, Zhao, P, Wang, J, Ma, S, Yao, L, Zhu, X, Yang, X, Zhang, X, Sun, Z, Liang, S, Xing, D and Duan, J (2022) Pupal diapause termination and transcriptional response of Antheraea pernyi (Lepidoptera: Saturniidae) triggered by 20-Hydroxyecdysone. Frontiers in Physiology 13, 888643.CrossRefGoogle ScholarPubMed
Feng, J, Lin, P, Wang, Y and Zhang, Z (2019) Molecular characterization, expression patterns, and functional analysis of toll-interacting protein (Tollip) in Japanese eel Anguilla japonica. Fish & Shellfish Immunology 90, 5264.CrossRefGoogle ScholarPubMed
Gao, J, Sun, Y, Sun, YX, Chen, C, Kausar, S, Tian, JW, Zhu, BJ and Liu, CL (2018) Identification and function of cAMP response element binding protein in Oak silkworm Antheraea pernyi. Journal of Invertebrate Pathology 151, 1420.CrossRefGoogle ScholarPubMed
Hillyer, JF (2016) Insect immunology and hematopoiesis. Developmental & Comparative Immunology 58, 102118.CrossRefGoogle ScholarPubMed
Ito, Y, Schaefer, N, Sanchez, A, Francisco, D, Alam, R, Martin, RJ, Ledford, JG, Stevenson, C, Jiang, D, Li, L, Kraft, M and Chu, HW (2018) Toll-interacting protein, Tollip, inhibits IL-13–mediated pulmonary eosinophilic inflammation in mice. Journal of Innate Immunity 10, 106118.CrossRefGoogle ScholarPubMed
Jiang, ZX, Nissa, MU, Guo, ZZ, Zhang, YB, Zheng, GD and Zou, SM (2022) An SNP at the target site of cid-miR-nov-1043 in the TOLLIP 3′ UTR decreases mortality rate in grass carp subjected to ENU-induced mutagenesis following grass carp reovirus infection. Fish & Shellfish Immunology 120, 451457.CrossRefGoogle ScholarPubMed
Jing, YP, Wang, D, Han, XL, Dong, DJ, Wang, JX and Zhao, XF (2016) The Steroid hormone 20-hydroxyecdysone enhances gene transcription through the cAMP response element-binding protein (CREB) signaling pathway. Journal of Biological Chemistry 291, 1277112785.CrossRefGoogle ScholarPubMed
Kang, RS, Daniels, CM, Francis, SA, Shih, SC, Salerno, WJ, Hicke, L and Radhakrishnan, I (2003) Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding. Cell 113, 621630.CrossRefGoogle ScholarPubMed
Kang, XL, Zhang, JY, Wang, D, Zhao, YM, Han, XL, Wang, JX and Zhao, XF (2019) The steroid hormone 20-hydroxyecdysone binds to dopamine receptor to repress lepidopteran insect feeding and promote pupation. PLoS Genetics 15, e1008331.CrossRefGoogle ScholarPubMed
Katayanagi, S, Setoguchi, Y, Kitagawa, S, Okamoto, T and Miyazaki, Y (2022) Alternative gene expression by TOLLIP variant is associated with lung function in chronic hypersensitivity pneumonitis. Chest 161, 458469.CrossRefGoogle ScholarPubMed
Katoh, Y, Shiba, Y, Mitsuhashi, H, Yanagida, Y, Takatsu, H and Nakayama, K (2004) Tollip and Tom1 form a complex and recruit ubiquitin-conjugated proteins onto early endosomes. Journal of Biological Chemistry 279, 2443524443.CrossRefGoogle Scholar
Kelley, LA, Mezulis, S, Yates, CM, Wass, MN and Sternberg, MJE (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols 10, 845858.CrossRefGoogle ScholarPubMed
Kumar, S, Stecher, G, Li, M, Knyaz, C and Tamura, K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35, 15471549.CrossRefGoogle ScholarPubMed
Lang, T and Mansell, A (2007) The negative regulation of Toll-like receptor and associated pathways. Immunology & Cell Biology 85, 425434.CrossRefGoogle ScholarPubMed
Li, T, Hu, J and Li, L (2004) Characterization of Tollip protein upon lipopolysaccharide challenge. Molecular Immunology 41, 8592.CrossRefGoogle ScholarPubMed
Li, K, Tian, L, Guo, ZJ, Guo, SY, Zhang, JZ, Gu, S, Palli, SR, Cao, Y and Li, S (2016 a) 20-Hydroxyecdysone (20E) Primary response gene E75 isoforms mediate steroidogenesis autoregulation and regulate developmental timing in Bombyx. The Journal of Biological Chemistry 291, 1816318175.CrossRefGoogle ScholarPubMed
Li, YB, Li, XR, Yang, T, Wang, JX and Zhao, XF (2016 b) The steroid hormone 20-hydroxyecdysone promotes switching from autophagy to apoptosis by increasing intracellular calcium levels. Insect Biochemistry and Molecular Biology 79, 7386.CrossRefGoogle ScholarPubMed
Li, WL, Zhang, ZY, Lin, L and Terenius, O (2017) Antheraea pernyi (Lepidoptera: Saturniidae) and its importance in sericulture, food consumption, and traditional Chinese medicine. Journal of Economic Entomology 110, 14041411.CrossRefGoogle ScholarPubMed
Li, YN, Liu, YB, Xie, XQ, Zhang, JN and Li, WL (2020) The modulation of trehalose metabolism by 20-Hydroxyecdysone in Antheraea pernyi (Lepidoptera: Saturniidae) during its diapause termination and post-termination period. Journal of Insect Science 20, 18.CrossRefGoogle ScholarPubMed
Lim, M, Newman, JA, Williams, HL, Masino, L, Aitkenhead, H, Gravard, AE, Gileadi, O and Svejstrup, JQ (2019) A ubiquitin-binding domain that binds a structural fold distinct from that of ubiquitin. Structure (London, England: 1993) 27, 13161325.e6.CrossRefGoogle ScholarPubMed
Liu, YQ, Li, YP, Li, XS and Qin, L (2010) The origin and dispersal of the domesticated Chinese oak silkworm, Antheraea pernyi, in China: a reconstruction based on ancient texts. Journal of Insect Science (Tucson, Ariz.) 10, 110.Google Scholar
Liu, X, Dai, F, Guo, E, Li, K, Ma, L, Tian, L, Cao, Y, Zhang, G, Palli, SR and Li, S (2015) 20-hydroxyecdysone (20E) primary response gene E93 modulates 20E signaling to promote Bombyx larval-pupal metamorphosis. Journal of Biological Chemistry 290, 2737027383.CrossRefGoogle ScholarPubMed
Liu, J, Li, Y, Zhou, X, Zhang, X, Meng, H, Liu, S, Zhang, L, He, J, He, Q and Geng, YJ (2020) CaMKIV limits metabolic damage through induction of hepatic autophagy by CREB in obese mice. Endocrinology 244, 353367.CrossRefGoogle ScholarPubMed
Livak, KJ and Schmittgen, TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods (San Diego, Calif.) 25, 402408.CrossRefGoogle ScholarPubMed
Lo, YS, Beckhouse, AG, Boulus, SL and Wells, CA (2009) Diversification of TOLLIP isoforms in mouse and man. Mammalian Genome 20, 305314.CrossRefGoogle ScholarPubMed
Lu, YL, Li, CH, Wang, DQ, Su, XR, Jin, CH, Li, Y and Li, TW (2013) Characterization of two negative regulators of the Toll-like receptor pathway in Apostichopus japonicus: inhibitor of NF-κB and Toll-interacting protein. Fish & Shellfish Immunology 35, 16631669.CrossRefGoogle ScholarPubMed
Lu, K, Psakhye, I and Jentsch, S (2014) Autophagic clearance of polyQ proteins mediated by ubiquitin-Atg8 adaptors of the conserved CUET protein family. Cell 158, 549563.CrossRefGoogle ScholarPubMed
Maekawa, T, Sano, Y, Shinagawa, T, Rahman, Z, Sakuma, T, Nomura, S, Licht, JD and Ishii, S (2008) ATF-2 controls transcription of Maspin and GADD45 alpha genes independently from p53 to suppress mammary tumors. Oncogene 27, 10451054.CrossRefGoogle ScholarPubMed
Maillard, MH, Bega, H, Uhlig, HH, Barnich, N, Grandjean, T, Chamaillard, M, Michetti, P and Velin, D (2014) Toll-interacting protein modulates colitis susceptibility in mice. Inflammatory Bowel Diseases 20, 660670.CrossRefGoogle ScholarPubMed
Mukherjee, S and Biswas, T (2014) Activation of TOLLIP by porin prevents TLR2-associated IFN-γ and TNF-α-induced apoptosis of intestinal epithelial cells. Cellular Signalling 26, 26742682.CrossRefGoogle ScholarPubMed
Niwa, YS and Niwa, R (2016) Transcriptional regulation of insect steroid hormone biosynthesis and its role in controlling timing of molting and metamorphosis. Development Growth & Differentiation 58, 94105.CrossRefGoogle ScholarPubMed
Numata, H and Shintani, Y (2023) Diapause in univoltine and semivoltine life cycles. Annual Review of Entomology 68, 257276.CrossRefGoogle ScholarPubMed
Pokatayev, V, Yang, K, Tu, X, Dobbs, N, Wu, J, Kalb, RG and Yan, N (2020) Homeostatic regulation of STING protein at the resting state by stabilizer TOLLIP. Nature Immunology 21, 158167.CrossRefGoogle ScholarPubMed
Rebl, A, Høyheim, B, Fischer, U, Köllner, B, Siegl, E and Seyfert, H (2008) Tollip, a negative regulator of TLR-signalling, is encoded by twin genes in salmonid fish. Fish & Shellfish Immunology 25, 153162.CrossRefGoogle ScholarPubMed
Rebl, A, Rebl, H, Li, S, Goldammer, T and Seyfert, H (2011) Salmonid Tollip and MyD88 factors can functionally replace their mammalian orthologues in TLR-mediated trout SAA promoter activation. Developmental & Comparative Immunology 35, 8187.CrossRefGoogle ScholarPubMed
Romanelli, D, Casati, B, Franzetti, E and Tettamanti, G (2014) A molecular view of autophagy in Lepidoptera. Biomed Research International 2014, 902315.CrossRefGoogle ScholarPubMed
Ryan, TA and Tumbarello, DA (2021) A central role for mitochondrial-derived vesicles in the innate immune response: implications for Parkinson's disease. Neural Regeneration Research 16, 17791780.CrossRefGoogle ScholarPubMed
Seok, S, Fu, T, Choi, SE, Li, Y, Zhu, R, Kumar, S, Sun, X, Yoon, G, Kang, Y, Zhong, W, Ma, J, Kemper, B and Kemper, JK (2014) Transcriptional regulation of autophagy by an FXR-CREB axis. Nature 516, 108111.CrossRefGoogle ScholarPubMed
Shan, SJ, Wang, L, Zhang, FM, Zhu, YY, An, LG and Yang, GW (2016) Characterization and expression analysis of Toll-interacting protein in common carp, Cyprinus carpio L., responding to bacterial and viral challenge. Springer Plus 5, 639.CrossRefGoogle ScholarPubMed
Shih, SC, Prag, G, Francis, SA, Sutanto, MA, Hurley, JH and Hicke, L (2003) A ubiquitin-binding motif required for intramolecular monoubiquitylation, the CUE domain. EMBO Journal 22, 12731281.CrossRefGoogle ScholarPubMed
Toruń, A, Szymańska, E, Castanon, I, Wolińska-Nizioł, L, Bartosik, A, Jastrzębski, K, Miętkowska, M, González-Gaitán, M and Miaczynska, M (2015) Endocytic adaptor protein tollip inhibits canonical wnt signaling. PLoS One 10, e0130818.CrossRefGoogle ScholarPubMed
Truman, JW (2019) The evolution of insect metamorphosis. Current Biology 29, R1252R1268.CrossRefGoogle ScholarPubMed
Walker, JM (1994) The bicinchoninic acid (BCA) assay for protein quantitation. Methods in Molecular Biology 32, 58.Google ScholarPubMed
Wang, J, Liu, B, Xu, Y, Yang, M, Wang, C, Song, M, Liu, J, Wang, W, You, J, Sun, F, Wang, D, Liu, D and Yan, H (2021) Activation of CREB-mediated autophagy by thioperamide ameliorates β-amyloid pathology and cognition in Alzheimer's disease. Aging Cell 20, e13333.CrossRefGoogle ScholarPubMed
Wang, WJ, Liu, Y, Mao, Y, Xu, YD, Wang, ZZ, Zhang, R, Liu, B, Xia, KY, Yang, MC and Yan, JP (2022) Toll-interacting protein negatively regulated innate immune response via NF-κB signal pathway in blunt snout bream, Megalobrama amblycephala. Developmental & Comparative Immunology 140, 104595.CrossRefGoogle ScholarPubMed
Wei, JG, Xu, M, Chen, XL, Zhang, P, Li, PF, Wei, SN, Yan, Y and Qin, QW (2015) Function analysis of fish Tollip gene in response to virus infection. Fish & Shellfish Immunology 47, 807816.CrossRefGoogle ScholarPubMed
Yamaguchi, N, Argueta, JGM, Masuhiro, Y, Kagishita, M, Nonaka, K, Saito, T, Hanazawa, S and Yamashita, Y (2005) Adiponectin inhibits Toll-like receptor family-induced signaling. FEBS Letters 579, 68216826.CrossRefGoogle ScholarPubMed
Yamakami, M and Yokosawa, H (2004) Tom1 (target of Myb 1) is a novel negative regulator of interleukin-1-and tumor necrosis factor-induced signaling pathways. Biological & Pharmaceutical Bulletin 27, 564566.CrossRefGoogle ScholarPubMed
Yamakami, M, Yoshimori, T and Yokosawa, H (2003) Tom1, a VHS domain-containing protein, interacts with Tollip, Ubiquitin, and Clathrin. Journal of Biological Chemistry 278, 5286552872.CrossRefGoogle ScholarPubMed
Yuan, ZM, Gong, SF, Luo, JY, Zheng, ZB, Song, B, Ma, SS, Guo, JL, Hu, C, Thiel, G, Vinson, C, Hu, CD, Wang, YZ and Li, MT (2009) Opposing roles for ATF2 and c-Fos in c-Jun-mediated neuronal apoptosis. Molecular and Cellular Biology 29, 24312442.CrossRefGoogle ScholarPubMed
Zhang, GL and Ghosh, S (2002) Negative regulation of toll-like receptor-mediated signaling by Tollip. Journal of Biological Chemistry 277, 70597065.CrossRefGoogle ScholarPubMed
Zhang, R, Li, RJ, Wang, J, Wang, SY, Zhang, MR, Hu, XL, Zhang, LL, Wang, S, Wang, RJ and Bao, ZM (2015) Identification, characterization and expression profiling of the Tollip gene in Yesso scallop (Patinopecten yessoensis). Genes & Genetic Systems 90, 99108.CrossRefGoogle ScholarPubMed
Zhu, L, Wang, LD, Luo, XL, Zhang, YX, Ding, QR, Jiang, XM, Wang, X, Pan, Y and Chen, Y (2012) Tollip, an intracellular trafficking protein, is a novel modulator of the transforming growth factor-β signaling pathway. Journal of Biological Chemistry 287, 3965339663.CrossRefGoogle ScholarPubMed
Supplementary material: File

Chen et al. supplementary material

Chen et al. supplementary material

Download Chen et al. supplementary material(File)
File 57.9 KB