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

Abstract

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.

Keywords:

• cement protein
• caddisfly larva
• molecular cloning
• phosphorylation analysis
• purification
• silk protein

Acknowledgements

This work was supported by the Global COE Programme from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), and also by Grants-in-Aid No. 22350103, No. 23651083 for KO, No. 22510028 for KH and No. 22580060 for MT from MEXT. Part of this study was performed through the Programme for Dissemination of Tenure-Track System funded by MEXT, and part was supported by the Japanese Association for Marine Biology (JAMBIO) No 23–73. The authors thank Prof. Dr Tetsuya Fujimoto and Ms Sachiko Yoshioka for the best machine tuning procedures in ³¹P-NMR spectroscopy, and Ms Shiori Ishihara and Ms Xue Bai for technical assistance in the molecular cloning experiments.