Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases |
| |
| Institution: | 1. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China;2. Vitamin D Research Institute, Shaanxi Sci-Tech University, Hanzhong 723001, Shaanxi Province, China;3. College of Education Science, Shaanxi Sci-Tech University, Hanzhong 723001, Shaanxi Province, China;1. School of Computer Science and Software Engineering, Tianjin Polytechnic University, Tianjin 300387, China;2. Department of Electrical and Computer Engineering, 2nd floor, ECERF (9107 116 Street), University of Alberta, Edmonton, AB T6G 2V4, Canada;1. Department of Physiology (Animal Physiology II), Faculty of Biology, Universidad Complutense, Madrid, Spain;2. Department of Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación Sanitaria Princesa (IP), Madrid, Spain;3. Department of Pediatrics, Universidad Autónoma de Madrid, Madrid, Spain;4. CIBER Fisiopatología de Obesidad y Nutrición, Instituto Carlos III, Madrid, Spain;1. Discipline of Medicine, The University of Adelaide, South Australia, 5000, Australia;2. NHMRC Centre of Research Excellence in Translating Nutritional Science to Good Health, The University of Adelaide, South Australia, 5000, Australia |
| |
| Abstract: | Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys2nd and Cys6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be applied in both health care and agricultural industries, and could lead to new methods for breeding fungus-resistant transgenic crops and antifungal transgenic silkworm strains. |
| |
| Keywords: | TIL-type protease inhibitors Missing cysteines Inhibitory activities Microbial proteases |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
