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The silk gland proteome of Stenopsyche angustata provides insights into the underwater silk secretion

ABSTRACT: Caddisworms (Trichoptera) spin adhesive silks to construct a variety of underwater composite structures. Many studies have focused on the fibroin heavy chain of caddisworm silk and found that it contains heavy phosphorylation to maintain a stable secondary structure. Besides fibroins, recent studies have also identified some new silk proteins within caddisworm silk. To better understand the silk composition and its secretion process, this study reports the silk gland proteome of a retreat-building caddisworm, Stenopsyche angustata. Using LC-MS/MS, 2389 proteins were identified in the silk gland of S. angustata, among which 192 were predicted as secreted silk proteins. Twenty-nine proteins were found to be enriched in the front silk gland, while 109 proteins were enriched in the caudal silk gland. The fibroin heavy chain and nine uncharacterized silk proteins were identified as phosphorylated proteins. By analyzing the sequence of the fibroin heavy chain, we found it contains 13 Gly/Thr/Pro-rich regions, 12 Val/Ser/Arg-rich regions, and a Gly/Arg/Thr-rich region. Three uncharacterized proteins were identified as sericin-like proteins due to having larger molecular weights, signal peptides, and repetitive motifs rich in serine. This study provides valuable information for further clarifying the secretion and adhesion of underwater caddisworm silk.

ORGANISM(S): Stenopsyche Angustata

SUBMITTER: Qingyou Xia

PROVIDER: PXD042275 | iProX | Tue May 16 00:00:00 BST 2023

REPOSITORIES: iProX

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Project description:To identify functions that distinguish the posterior and median cells producing fibroin and sericin in the silk gland of Bombyx mori, serial analysis of gene expression (SAGE) profiles from both silk gland regions were analyzed and compared. The construction of a B. mori reference tag collection extracted from a set of 38000 Bombyx EST sequenced from the 3’ side, helped annotating the SAGE libraries. Most of the tags appeared at similar relative concentration in the two libraries except for those corresponding to silk proteins that were found region-specific and highly abundant. Strikingly, besides tags from silk protein mRNAs, 19 tags were found in the class of high abundance in the median cell library, which were absent in the posterior cell tag collection. Except tags from SP1 mRNA, no PSG specific tags were found in the same class of abundance. The analysis of MSG-specific different transcripts led to suggest that middle silk gland cell realizes more diversified functions as those already known, of synthesis and secretion of the silk sericins. EST libraries from 11 silkworm tissues were 3’-sequenced to ensure the identification of the most terminal tag. 37,920 sequences were analyzed on ABI 3700 or 3730XL sequencers. Electrophoregrams were processed with KB Basecaller (3730XL traces) or with PHRED (3700 traces) to obtain the .phd files from which were extracted text sequences and their corresponding quality files in Fasta format (Phd2Fasta, Green and Ewing, 1995; 2002). Vector sequences and bad quality regions were removed with an home made software after identification by Lucy (Chou and Holmes, 2001). Chimera were removed by an home made software and retrotransposon sequences were masked by RepeatMasker (http://repeatmasker.org). The cleaned sequences were clusterized with TGICL package (TIGR) and assembled into contigs with CAP3 (Huang and Madan, 1999). Contigs were identified with Blastn and Blastx on GenBank and SwissProt/TREMBL, respectively. Some clusters, splitted by CAP3 procedure, have been regrouped on the basis of Blastx identity. Identitag (Keime et al., 2004) was used to extract all possible tags (forward and reverse) to create a reference database. Moreover, a quality index is attached to each tag depending to the presense of poly-A, polyadenylation signal and its proximity to 3-prime extremity in the original mRNA sequence. This database was supplemented with the tags extracted from public B. mori sequences (GenBank, Silkbase) with the same software. Tags were extracted from MSG and PSG libraries (from 2304 and 3072 sequenced inserts respectively) with the Sagenhaft software (Beissbarth et al., 2004) then identified and compared with Identitag. The assessment of significant differences among the two libraries was performed by using the Z-test used for comparison of SAGE libraries of different size (Kal et al., 1999). For graphic purpose and to avoid division by zero we used a tag value of 1 for tags that were not detected in MSG or PSG libraries. Since the two SAGE libraries showed up different TAG number, we used the Z-test for comparing the two mRNA populations.

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The silk gland proteome of Stenopsyche angustata provides insights into the underwater silk secretion

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