Metametabolite-driven tanning and silk protein profiling in Saturnia japonica cocoons
Across the animal kingdom, catechol-based “tanning” reactions—enzymatic oxidation of catechol precursors to reactive quinones—are widely used to pigment and cross-link structural proteins, thereby enhancing mechanical stiffness and toughness. This strategy underlies the graded stiffness of squid beaks, the cohesive strength of mussel byssal threads, and the sclerotization of insect cuticle, where L-DOPA, dopamine and related catechols are oxidized by tyrosinases, laccases and dopachrome-converting enzymes to o-quinones that covalently cross-link proteins and chitin. In Lepidoptera, silk represents a high-performance protein scaffold, yet the sericin-rich, highly hydrophilic cocoons of Bombyx mori are mechanically and chemically labile compared with the tanned, macroporous, open-ended cocoons of the wild silkmoth Saturnia japonica, which resist standard hot-water extraction and mild degumming. In this study, we combine whole-cocoon protein profiling and metabolomics to investigate the mechanism of sericin tanning in S. japonica. Comparative analyses indicate that tanning in S. japonica sericin is primarily driven by small-molecule metabolites rather than intrinsic protein motifs. Hot-water extraction of cocoons yielded a low-molecular-weight compound that spontaneously darkened upon air oxidation; Orbitrap MS/MS total ion chromatograms revealed a dramatic intensity change of an m/z 378.1183 feature before and after tanning, and in silico CFM-ID fragmentation pointed to the catechol dimer N-(3,4-dihydroxyphenyllactic acid)-3,4-dihydroxyphenylalaninamide (DOPA-DHPLA). Subsequent purification by HPLC and structural confirmation by 2D NMR verified DOPA-DHPLA as the major tanning agent. To assess how this metabolite-mediated tanning alters sericin cohesion, we prepared sericin from S. japonica and B. mori cocoons and measured the interaction forces between sericin nanofilms using a surface forces apparatus under aqueous conditions at different pH values. While untanned S. japonica sericin exhibited adhesion comparable to B. mori sericin, tanned S. japonica sericin showed up to ~100% higher adhesive forces than B. mori sericin at pH 3 and 5. The interaction energy of S. japonica sericin increased with pH, although neither S. japonica nor B. mori sericin displayed measurable adhesion at neutral pH. These findings identify DOPA-DHPLA as a metabolite-derived catechol tanning agent that reinforces sericin cohesion in S. japonica cocoons and establish a mechanistic link between metabolite-driven tanning chemistry and the macroscopic adhesive and mechanical properties of wild silkmoth silk.
Across the animal kingdom, catechol-based “tanning” reactions—enzymatic oxidation of catechol precursors to reactive quinones—are widely used to pigment and cross-link structural proteins, thereby enhancing mechanical stiffness and toughness. This strategy underlies the graded stiffness of squid beaks, the cohesive strength of mussel byssal threads, and the sclerotization of insect cuticle, where L-DOPA, dopamine and related catechols are oxidized by tyrosinases, laccases and dopachrome-converting enzymes to o-quinones that covalently cross-link proteins and chitin. In Lepidoptera, silk represents a high-performance protein scaffold, yet the sericin-rich, highly hydrophilic cocoons of Bombyx mori are mechanically and chemically labile compared with the tanned, macroporous, open-ended cocoons of the wild silkmoth Saturnia japonica, which resist standard hot-water extraction and mild degumming. In this study, we combine whole-cocoon protein profiling and metabolomics to investigate the mechanism of sericin tanning in S. japonica. Comparative analyses indicate that tanning in S. japonica sericin is primarily driven by small-molecule metabolites rather than intrinsic protein motifs. Hot-water extraction of cocoons yielded a low-molecular-weight compound that spontaneously darkened upon air oxidation; Orbitrap MS/MS total ion chromatograms revealed a dramatic intensity change of an m/z 378.1183 feature before and after tanning, and in silico CFM-ID fragmentation pointed to the catechol dimer N-(3,4-dihydroxyphenyllactic acid)-3,4-dihydroxyphenylalaninamide (DOPA-DHPLA). Subsequent purification by HPLC and structural confirmation by 2D NMR verified DOPA-DHPLA as the major tanning agent. To assess how this metabolite-mediated tanning alters sericin cohesion, we prepared sericin from S. japonica and B. mori cocoons and measured the interaction forces between sericin nanofilms using a surface forces apparatus under aqueous conditions at different pH values. While untanned S. japonica sericin exhibited adhesion comparable to B. mori sericin, tanned S. japonica sericin showed up to ~100% higher adhesive forces than B. mori sericin at pH 3 and 5. The interaction energy of S. japonica sericin increased with pH, although neither S. japonica nor B. mori sericin displayed measurable adhesion at neutral pH. These findings identify DOPA-DHPLA as a metabolite-derived catechol tanning agent that reinforces sericin cohesion in S. japonica cocoons and establish a mechanistic link between metabolite-driven tanning chemistry and the macroscopic adhesive and mechanical properties of wild silkmoth silk.
Seoul national university