C-terminus processing in Tma12 is critical for its insecticidal activity

• Published in: International journal of biological macromolecules 2024-12, Vol.282 (Pt 5), p.137261, Article 137261
• Main Authors: Gupta, Bhawana, Singh, Pooja, Singh, Pradhyumna Kumar
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Biopesticide; Glycosylation; Insecticides - chemistry; Insecticides - pharmacology; KM71H; LPMO; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Protein Processing, Post-Translational; Protein Stability; Tma12; Whitefly
• ISSN: 0141-8130; 1879-0003; 1879-0003
• DOI: 10.1016/j.ijbiomac.2024.137261

Tma12 is a fern-derived biopesticide (22 kDa) whose LPMO activity is associated with its insecticidal activity. The absence of the last 9 amino acids in the crystal structure of Tma12 suggested a possibility of its C-terminus processing. In this communication, we have shown the importance of protein C-terminus in the insecticidal activity. Additionally, we have also established the role of N-linked glycosylation in protein stability. Pichia produced (His)6 tagged Tma12 in two forms. The 30 kDa protein comprising 192 amino acid residues did not show insecticidal activity. Contrary, 24 kDa protein exhibited toxicity to whiteflies with an LC50 1.38 μg/ml. Absence of (His)6 tag in 24 kDa protein indicated processing at the C-terminus which was confirmed with deletion mutagenesis. Failure in expressing glycosylation defective mutant suggested the importance of glycans in the stability of Tma12. New findings together with earlier reports suggest that along with the N-terminal catalytic center, correct C-terminus is pivotal for anti-whitefly activity of Tma12. [Display omitted] •Expression and purification of the first plant-based AA10 LPMO in P. pastoris.•C-terminus processing of Tma12 is critical for its anti-whitefly activity.•N-linked glycan is essential for protein stability.