Acquisition of Structure-guiding and Structure-forming Properties during Maturation from the Pro-silicatein to the Silicatein Form

作者：Schroeder, Heinz C.; Wang, Xiaohong; Manfrin, Alberto; Yu, Shu-Hong; Grebenjuk, Vlad A.; Korzhev, Michael; Wiens, Matthias; Schlossmacher, Ute; Mueller, Werner E. G.

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2012, 287(26): 22196-22205.

DOI：10.1074/jbc.M112.351486

• DEMOSPONGE SUBERITES-DOMUNCULA
• SILICEOUS SPICULES
• MECHANICAL-PROPERTIES
• BIOLOGICAL SILICA
• AXIAL FILAMENT
• CATHEPSIN-L
• SPONGE
• PROTEIN
• NANOINDENTATION
• SKELETOGENESIS

Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 degrees C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and results in the release of the 23 kDa mature silicatein, a process which very likely proceeds by autocatalysis. Almost in parallel with its formation, the mature enzyme precipitates as pure 23 kDa protein. When the precipitate is dissolved in an urea buffer, the solubilized protein displays its full enzymatic activity which is enhanced multi-fold in the presence of the silicatein interactor silintaphin-1 or of poly(ethylene glycol) (PEG). The biosilica product formed increases its compactness if silicatein is supplemented with silintaphin-1 or PEG. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa(silicatein) via 61 MPa (silicatein: silintaphin-1) to 101 MPa (silicatein: silintaphin-1 and PEG). These data show that the maturation process from the pro-silicatein state to the mature form is the crucial step during which silicatein acquires its structureguiding and structure-forming properties.

• 出版日期2012-6-22
• 单位国家地质实验测试中心; 中国科学技术大学; 中国地质科学院