Activity identification and proteomics analysis of water-soluble proteins in cocoon coat from different silkworm...

The cocoon coat of Bombyx mori contains abundant bioactive proteins that contribute to its protective functions, yet varietal differences remain largely unexplored. In this study, water-soluble proteins were extracted from N4 (yellow), D9L (green), and 305 (white) cocoon coats using PBS (pH 7.0), followed by analyses of antioxidant capacity, antibacterial activity, and proteomic composition. All extracts exhibited strong antioxidant activity with DPPH scavenging rates of 71-81%. Antibacterial assays revealed distinct inhibition patterns: 305 proteins were most effective against Escherichia coli and Beauveria bassiana, D9L proteins strongly inhibited Trichophyton rubrum, while N4 showed intermediate effects. DIA-based proteomics identified 462 proteins, approximately 60% of which had catalytic or binding functions, enriched in protease and protease-inhibitor categories. Major components included Ser3 and Ser1-like sericins, low-abundance Ser2, and fibroin subunits (Fib-H, Fib-L). High-abundance proteins mainly comprised serine and carboxypeptidase inhibitors and several TIL domain-containing zonadhesin-like proteins. Differential protein analysis linked varietal antimicrobial differences to these bioactive components. Structural prediction of the zonadhesin-like protein using AlphaFold 3 revealed a unique convex-loop conformation formed by interactions among tandem TIL domains, suggesting potential roles in pathogen recognition or defense. This study establishes a comprehensive proteomic atlas of cocoon coats from diverse silkworm varieties, identifies BmSPI39 and seroin as key drivers of strain-specific defenses, and elucidates an adaptive defense mechanism mediated by soluble silk proteins, thereby providing specific molecular targets for disease-resistant silkworm breeding.