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.
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 Cys2ndand Cys6thwere 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.
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China; Vitamin D Research Institute, Shaanxi Sci-Tech University, Hanzhong 723001, Shaanxi Province, China.
Youshan
Y
Li
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
Huawei
H
Liu
College of Education Science, Shaanxi Sci-Tech University, Hanzhong 723001, Shaanxi Province, China.
Rui
R
Zhu
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
Qingyou
Q
Xia
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China. Electronic address: zhaop@swu.edu.cn.
Ping
P
Zhao
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli; D005656:Fungal Proteins; D019476:Insect Proteins / Q000737:chemistry*; D008958:Models, Molecular; D010802:Phylogeny; D000072417:Protein Domains; D055503:Protein Multimerization; D014361:Trypsin Inhibitors / Q000737:chemistry*
D005656:Fungal Proteins; D019476:Insect Proteins; D014361:Trypsin Inhibitors; D003545:Cysteine
Bombyx mori; Inhibitory activities; Microbial proteases; Missing cysteines; TIL-type protease inhibitors