Characterization of the splicing isoform TuSp1-sv provides novel insights into spidroin functional diversification

Spider silk exhibits extraordinary mechanical properties, with its functional diversity largely attributed to the sequence variation of spidroin genes. However, the contribution of alternative splicing to the functional diversity of spidroin, particularly in the case of tubuliform spidroin 1 (TuSp1), which is critical for tubuliform silk construction, remains poorly understood. In this study, we identified a novel splicing isoform of TuSp1 (TuSp1-sv) from the orb-weaving spider Araneus ventricosus and confirmed its expression in silk glands through RT-PCR and mass spectrometry. The TuSp1-sv retains only partial N-terminal and complete C-terminal domains, entirely lacking the repetitive core. Unlike the recombinant TuSp1-NC, which contains complete terminal domains, the recombinant TuSp1-sv protein forms stable dimers and self-assembles into amyloid-like fibrils with high β-sheet content, while also exhibiting unique shear-induced fiber formation. These results suggest that alternative splicing reprograms spidroin function by truncating the entire repetitive domain and a partial N-terminal domain, thereby shifting the role of TuSp1 variant from a primary structural component to a potential silk assembly regulator. Overall, our study provides new insights into the functional diversification of spider silk proteins mediated by alternative splicing, with significant implications for the design of bioinspired materials.