Fig 1.
Five cocoon layers of B. mori.
(A) Schematic representation of cocoon layers; (B) Photos of layers; (C) Silver stain of SDS-PAGE of proteins from layers. From the inner layer towards the outer layer, the five cocoon layers were named as layer1~layer5 (L1~ L5), respectively.
Fig 2.
Relative abundance of proteins from functional categories in five cocoon layers.
Molar abundance of proteins was estimated with iBAQ intensities (A). Molar abundances of fibroins (B), sericins (C) and seroins (D) were compared among five cocoon layers by LFQ intensities.
Fig 3.
Relative abundance of protease inhibitors in five cocoon layers.
Molar abundance of proteins was estimated with iBAQ intensities (A). Six major protease inhibitors were compared among layers by LFQ intensities. Four showed the highest abundance in the outermost layer; the other two showed high abundance in outermost and innermost layers. Proteins labeled "BmSPI" are nomenclature of B. mori serine protease inhibitors identified by Zhao et al. (2012).
Table 1.
Identification of protease inhibitors in five cocoon layers.
Fig 4.
Sequence alignments of main cocoon protease inhibitors with known inhibitors: YBCL from Escherichia coli, SPI1 from Galleria mellonella, and FPI-F from Bombyx mori.
(A) PEBP-type protease inhibitors; (B) kunizt-type protease inhibitors; (C) TIL-type protease inhibitors. Alignments were by ClustalX 1.83 with default parameters and shading using GeneDoc. Black, identical residues; gray, similar residues; red, predicted P1 position; C, conserved cysteine position.
Fig 5.
Degradation of fibroins by proteases.
Sericin-free fibroins were incubated with trypsin, chymotrypsin, elastase, or protease K for 1 h at 37°C. Proteases were 0.05 μg. Fibroin was 50 μg. Products and controls were separated by SDS-PAGE and stained by coomassie brilliant blue.
Fig 6.
Inhibitory effects of cocoon protease inhibitors on proteases.
Extracted water-soluble protein samples from cocoons were incubated with protease K (A), trypsin (B), chymotrypsin (C), and elastase (D) for 15 min at room temperature and casein was added for 1 h at 37°C. Reaction products and controls were separated by SDS-PAGE and stained by coomassie brilliant blue.