Long-range periodic sequence of the cement/silk protein of Stenopsyche marmorata: purification and biochemical characterisation

The long-range periodic amino acid sequence of the bifunctional silk/cement protein from larvae of the caddisfly, Stenopsyche marmorata, is discussed in this study. The protein, named the S. marmorata silk protein (Smsp-1), was first purified to electrophoretic homogeneity. The results of Edman-based sequencing of Smsp-1 tryptic digests were consistent with the amino acid sequence deduced from a cDNA clone of the Smsp-1 gene. All undetected amino acids in the Edman-based sequencing were encoded as Ser, suggesting the presence of O-phospho-Ser. 31P-NMR and an O-phospho-amino acid analysis successfully showed that the O-phospho-Ser residue occurred in a clustered manner, serving a cement function for Smsp-1. Two patterns of non-phosphorylated repeats, –SLGPYGDPRGDXLGPYGG– (X = V, G or D) and –GVGPYGDGLGPYGG–, were enriched in Smsp-1 compared with the O-phospho-Ser cluster, and have fibre-forming functions.本研究针对斑纹石蛾（Stenopsyche marmorata）幼虫分泌的双功能性丝/胶蛋白的长程周期性氨基酸序列展开探讨。该蛋白被命名为斑纹石蛾丝蛋白（Smsp-1），首次纯化至电泳纯级。对Smsp-1胰蛋白酶酶解产物进行的埃德曼（Edman）测序结果，与基于Smsp-1基因的互补DNA（cDNA）克隆推导得到的氨基酸序列高度吻合。埃德曼测序中未被检出的氨基酸均被编码为丝氨酸，这提示蛋白中存在O-磷酸化丝氨酸（O-phospho-Ser）。磷-31核磁共振波谱法（31P-NMR）与O-磷酸化氨基酸分析实验均成功验证，O-磷酸化丝氨酸残基呈簇状分布，该修饰可赋予Smsp-1胶黏功能。相较于O-磷酸化丝氨酸簇区域，Smsp-1中富集有两种非磷酸化重复序列：–SLGPYGDPRGDXLGPYGG–（X为V、G或D）与–GVGPYGDGLGPYGG–，这两种序列均具备纤维形成功能。