Pham Thanh, Chuang Tyler, Lin Albert, Joo Hyun, Tsai Jerry, Crawford Taylor, Zhao Liang, Williams Caroline, Hsia Yang, Vierra Craig
Departments of †Biology and §Chemistry, University of the Pacific , Stockton, California 95211, United States.
Biomacromolecules. 2014 Nov 10;15(11):4073-81. doi: 10.1021/bm5011239. Epub 2014 Oct 9.
Dragline silk has been proposed to contain two main protein constituents, MaSp1 and MaSp2. However, the mechanical properties of synthetic spider silks spun from recombinant MaSp1 and MaSp2 proteins have yet to approach natural fibers, implying the natural spinning dope is missing critical factors. Here we report the discovery of novel molecular constituents within the spinning dope that are extruded into dragline silk. Protein studies of the liquid spinning dope from the major ampullate gland, coupled with the analysis of dragline silk fibers using mass spectrometry, demonstrate the presence of a new family of low-molecular-weight cysteine-rich proteins (CRPs) that colocalize with the MA fibroins. Expression of the CRP family members is linked to dragline silk production, specifically MaSp1 and MaSp2 mRNA synthesis. Biochemical data support that CRP molecules are secreted into the spinning dope and assembled into macromolecular complexes via disulfide bond linkages. Sequence analysis supports that CRP molecules share similarities to members that belong to the cystine slipknot superfamily, suggesting that these factors may have evolved to increase fiber toughness by serving as molecular hubs that dissipate large amounts of energy under stress. Collectively, our findings provide molecular details about the components of dragline silk, providing new insight that will advance materials development of synthetic spider silk for industrial applications.
拖牵丝被认为包含两种主要蛋白质成分，即MaSp1和MaSp2。然而，由重组MaSp1和MaSp2蛋白质纺制的合成蜘蛛丝的机械性能尚未达到天然纤维的水平，这意味着天然纺丝原液缺少关键因素。在此，我们报告了在纺丝原液中发现的新型分子成分，这些成分被挤出形成拖牵丝。对主要壶腹腺液体纺丝原液的蛋白质研究，以及使用质谱对拖牵丝纤维的分析表明，存在一个新的低分子量富含半胱氨酸的蛋白质（CRP）家族，它们与MA丝心蛋白共定位。CRP家族成员的表达与拖牵丝的产生有关，特别是与MaSp1和MaSp2 mRNA的合成有关。生化数据支持CRP分子被分泌到纺丝原液中，并通过二硫键连接组装成大分子复合物。序列分析支持CRP分子与属于胱氨酸滑结超家族的成员具有相似性，这表明这些因素可能已经进化，通过作为在压力下耗散大量能量的分子枢纽来提高纤维韧性。总的来说，我们的研究结果提供了关于拖牵丝成分的分子细节，为推进用于工业应用的合成蜘蛛丝材料开发提供了新的见解。
