Abstract
Silk cocoons are composed of fiber proteins (fibroins) and adhesive glue proteins (sericins), which provide a physical barrier to protect the inside pupa. Moreover, other proteins were identified in the cocoon silk, many of which are immune related proteins. In this study, we extracted proteins from the silkworm cocoon by Tris-HCl buffer (pH7.5), and found that they had a strong inhibitory activity against fungal proteases and they had higher abundance in the outer cocoon layers than in the inner cocoon layers. Moreover, we found that extracted cocoon proteins can inhibit the germination of Beauveria bassiana spores. Consistent with the distribution of protease inhibitors, we found that proteins from the outer cocoon layers showed better inhibitory effects against B. bassiana spores than proteins from the inner layers. Liquid chromatography-tandem mass spectrometry was used to reveal the extracted components in the scaffold silk, the outermost cocoon layer. A total of 129 proteins were identified, 30 of which were annotated as protease inhibitors. Protease inhibitors accounted for 89.1% in abundance among extracted proteins. These protease inhibitors have many intramolecular disulfide bonds to maintain their stable structure, and remained active after being boiled. This study added a new understanding to the antimicrobial function of the cocoon.
Highlights
The silkworm cocoon has been well studied as the silkworm is the model lepidopteran insect [1,2,3,4,5,6], and its cocoon has important economic value
The combination of results from three biological repeats indicated that only 4.08% proteins could be extracted from the inner cocoon layers, whereas 6.56% proteins were extracted from the outermost scaffold silk (Table 1)
The amount of protease inhibitors tended to increase from the inner to the outer cocoon layers (Fig 1B)
Summary
The silkworm cocoon has been well studied as the silkworm is the model lepidopteran insect [1,2,3,4,5,6], and its cocoon has important economic value. Some proteins with small molecular weight were found in the cocoon, including two protease inhibitors and two seroins [9,10]. The expression of protease inhibitors changed after infection by bacteria, fungi or viruses [11], indicating that they are immunity related proteins. Many protease inhibitors showed inhibitory activity against the fungal proteases, as well as the germination of Beauveria bassiana conidia [12,13,14,15]. The expression of seroins was up-regulated after infection with bacteria and virus [16,17,18]. Seroins were found showing inhibitory activity against the growth of PLOS ONE | DOI:10.1371/journal.pone.0151764. Seroins were found showing inhibitory activity against the growth of PLOS ONE | DOI:10.1371/journal.pone.0151764 March 31, 2016
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