Identification and Molecular Characterization of a Serine Protease Inhibitor Gene in the Khapra Beetle Trogoderma granarium

Abstract

Insect serine protease inhibitors (ISPIs) are essential for regulating various protease-mediated activities and play crucial roles in metabolism, metamorphosis, reproduction, and immunity. As a member of the ISPIs, serpins are recognized as the most essential protease inhibitor family in higher eukaryotes, encompassing a diverse array of biological functions. They are involved in the Toll pathway, the prophenoloxidase cascade, development, immunity, and reproduction in all insects. In this study, a serpin from the khapra beetle, Trogoderma granarium (Everts) (Coleoptera: Dermestideae) was identified and characterized using both transcriptomic and bioinformatics methodologies. The BGISEQ-500 platform was used to construct a cDNA library from T. granarium, which led to the identification and characterization of a novel Serine Protease Inhibitor gene (TgSPI). Sequence analysis confirmed TgSPI's classification within the serine protease inhibitor (SPI) superfamily. It has conserved features, including a Reactive Center Loop (RCL) close to the C-terminal end, which is essential for protease inhibition. Phylogenetic analysis and 3D structure modeling of TgSPI were performed using MEGA6 software and the Phyre2 Protein Fold Recognition Server, respectively. The phylogenetic analysis positioned TgSPI within a cluster of coleopteran insect SPIs (ISPIs), supporting its evolutionary lineage. Predicted tertiary structure modeling of TgSPI revealed similarity to conserpin in the latent state. This study provides foundational information on the evolutionary patterns and structural-functional aspects of TgSPI in the khapra beetle and highlights probable role of TgSPI as a promising target for further genetic and functional studies aimed at sustainable pest control strategies.

Keywords

• Gene characterization
• Insect serine protease inhibitor
• Khapra beetle
• Serpin

References

1. Chamankhah M, Braun L, Visal-Shah S, O’Grady M, Baldwin D, Shi X, Hegedus DD. 2003. Mamestra configurata serpin-1 homologues: cloning, localization and developmental regulation. Insect Biochem Mol Biol, 33(3): 355-369.
2. Charron Y, Madani R, Combepine C, Gajdosik V, Hwu Y, Margaritondo G, Vassalli JD. 2008. The serpin Spn5 is essential for wing expansion in Drosophila melanogaster. Int J Dev Biol, 52(7): 933-942.
3. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M. 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21(18): 3674-3676. https://doi.org/10.1093/BIOINFORMATICS/BTI610
4. Dageri A, Kadir ML, Guz N, Ogreten A, Arshad M. 2023. The involvement of Antifreeze protein maxi-like and Cold-shock domain-containing protein genes in cold-induced larval diapause and cold-shock treatment of khapra beetle. J Stored Prod Res, 101: 102074.
5. Dageri A. 2024. Molecular characterization and expression analysis of six small heat shock protein genes in Trogoderma granarium during cold and starvation-induced larval diapause. J Stored Prod Res, 108: 102368.
6. EPPO. 2024. European and Mediterranean plant protection organization. EPPO global data base. Trogoderma granarium. URL=https://gd.eppo.int/taxon/TROGGA/distribution (accessed date: July 01, 2024).
7. Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791.
8. Han P, Fan J, Liu Y, Cuthbertson AG, Yan S, Qiu BL, Ren S. 2014. RNAi-mediated knockdown of serine protease inhibitor genes increases the mortality of Plutella xylostella challenged by destruxin A. PloS One, 9(5): e97863.

9. Hedstrom L. 2002. Serine protease mechanism and specificity. Chem Rev, 102(12): 4501-4524.
10. Huntington JA, Read RJ, Carrell RW. 2000. Structure of a serpin–protease complex shows inhibition by deformation. Nature, 407(6806): 923-926.
11. Huntington JA, Carrell RW. 2001. The serpins: nature's molecular mousetraps. Sci Prog, 84(2): 125-136.
12. Irving JA, Pike RN, Lesk AM, Whisstock JC. 2000. Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res, 10(12): 1845-1864.
13. Jiang R, Zhang B, Kurokawa K, So YI, Kim EH, Hwang HO, Lee BL. 2011. 93-kDa twin-domain serine protease inhibitor (Serpin) has a regulatory function on the beetle Toll proteolytic signaling cascade. J Biol Chem, 286(40): 35087-35095.
14. Li B, Yu HZ, Ye CJ, Ma Y, Li X, Fan T, Xu JP. 2017. Bombyx mori Serpin6 regulates prophenoloxidase activity and the expression of antimicrobial proteins. Gene, 610: 64-70.
15. Li GY, Yang L, Xiao KR, Song QS, Stanley D, Wei SJ, Zhu JY. 2022. Characterization and expression profiling of serine protease inhibitors in the yellow mealworm Tenebrio molitor. Arch Insect Biochem Physiol, 111(3): e21948.
16. Liu T, Chu J, Wang Q, Wang Y, Zhang X, Liu D, Wang. 2024. Role of serpin-25 in prophenoloxidase activation and expression of antimicrobial peptide genes in the silkworm Bombyx mori. J Asia-Pac Entomol, 27(2): 102222.
17. Loebermann H, Tokuoka R, Deisenhofer J, Huber R. 1984. Human α1-proteinase inhibitor: crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function. J Mol Biol, 177(3): 531-557.
18. Madeira F, Pearce M, Tivey AR, Basutkar P, Lee J, Edbali O, Lopez R. 2022. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res, 50(W1): W276-W279.
19. Meekins DA, Kanost MR, Michel K. 2017, February. Serpins in arthropod biology. Sem Cell Devel Biol, 62: 105-119.
20. Saadati F. Bandani AR. 2011. Effects of serine protease inhibitors on growth and development and digestive serine proteinases of the Sunn pest, Eurygaster integriceps. J Insect Sci, 11(1): 72.
21. Saitou N, Nei M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406-425.
22. Sanrattana W, Sefiane, T, Smits S, van Kleef ND, Fens MH, Lenting PJ, de Maat S. 2021. A reactive center loop–based prediction platform to enhance the design of therapeutic SERPINs. Proc Natl Acad Sci, 118(45): e2108458118.
23. Shakeel M, Xu X, De Mandal S, Jin F. 2019. Role of serine protease inhibitors in insect- host-pathogen interactions. Arch Insect Biochem Physiol, 102(3): 1-8. https://doi.org/10.1002/arch.21556
24. Shakeel M. 2021. Molecular identification, characterization, and expression analysis of a serine protease inhibitor gene from cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). Braz J Biol, 81(3): 516-525.
25. Stratikos E, Gettins PG. 1999. Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 Å and full insertion of the reactive center loop into β-sheet A. Proc Natl Acad Sci, 96(9): 4808-4813.
26. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol, 30: 2725-2729.
27. Tang Y, Wang Y, Pei Z, Li W, Zhang D, Liu L, Kong L, Liu S, Jiang X, Ma H. 2016. A serine protease inhibitor from Musca domestica larva exhibits inhibitory activity against elastase and chymotrypsin. Biotechnol Lett, 38(7): 1147-1153. https://doi.org/10.1007/s10529-016-2089-0
28. Yang L, Mei Y, Fang Q, Wang J, Yan Z, Song Q, Lin Z, Ye G. 2017. Identification and characterization of serine protease inhibitors in a parasitic wasp, Pteromalus puparum. Sci Rep, 7(1): 1-13. https://doi.org/10.1038/s41598-017-16000-5
29. Zhang C, Wei J, Naing ZL, Soe ET, Tang J, Liang G. 2022. Up-regulated serpin gene involved in Cry1Ac resistance in Helicoverpa armigera. Pestic Biochem Physiol, 188: 105269.
30. Zuckerkandl E, Pauling L. 1965. Evolutionary divergence and convergence in proteins. Edited in Evolving Genes and Proteins by V. Bryson and HJ. Vogel. Academic Press, New York, US, pp: 97-166.