Introduction

The silkworm, Bombyx mori, is one of the best-characterized silk-producing model organisms because its silk has great economic value. The composition of B. mori silk has been investigated intensively. Fibroin, the central fiber protein, is composed of heavy-chain proteins, light-chain proteins and p251,2,3. Sericins are soluble glue proteins, coating and cementing the silk fibers and three of them have been identified4,5,6. In a recent study, we revealed the complexity of the silk protein components by analysing seven silk proteomes7. In addition to the expected fibroins and sericins, we also identified various protease inhibitors, enzymes, proteins of unknown function and other proteins. Some of the identified silk proteins have definite roles in the silk, for example, protease inhibitors could protect the silk from degradation8,9. However, most proteins in the silk have unknown functions and need to be explored in depth.

Silk was spun efficiently at normal ambient temperatures and atmospheric pressure, which may be directly related to complex biochemical processes that occur in the silk gland. Silk glands expand rapidly as a result of DNA replication between day 1 and 5 in the fifth instar larvae and complete the development on day 6 of the 5th instar10. Silkworm start to spinning on day 7 of the fifth instar (V-7), which is called wandering phase. Silk gland contain three compartments according its morphology and function, including the anterior silk gland, middle silk gland and posterior silk gland. The fibroins and sericins are synthesized in the posterior and middle silk gland, respectively11,12,13,14 and then stored in the lumen of the silk gland as a concentrated aqueous silk solution15. When required for spinning, the proteins flow through the narrow anterior silk gland duct. In this process, the silk solution was converted into a solid filament by the combined action of strain and shear, together with the dehydration and induction of the ions16,17,18,19,20,21,22,23,24.

However, our knowledge on the process involved in the natural silk production is still incomplete. To extend our understanding, it is necessary to evaluate the molecular functions of various extracellular proteins in the silk gland lumen. Here, we identified the proteome in the silk gland lumen using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and demonstrated its dynamic change in five compartments of the silk gland during two developmental stages. Therefore, this is a detailed study of the natural silk ‘production line’ in the perspectives of both biology and engineering.

Results

Extraction and identification of proteins in the silk gland lumen

The silk glands were dissected out from silkworms at two different developmental stages: day 5 of the fifth instar (V-5) and day 1 of wandering (W-1). Each silk gland was cut into five compartments according its morphology (Fig. 1A), including the anterior silk gland (ASG), the anterior part of middle silk gland (A-MSG), the middle part of the middle silk gland (M-MSG), the posterior part of the middle silk gland (P-MSG) and the posterior silk gland (PSG) (Fig. 1A). Each compartment was then divided into two parts, the glandular cells and the luminal contents (Fig. 1A,B). The luminal contents from different compartments showed a wide variety of diameters. In particular, the M-MSG had the maximum diameter of 2 mm and the ASG had the minimum diameter of less than 0.1 mm (Fig. 1B). The luminal contents of the M-MSG was surrounded by a glandular cell “sheath” of a similar size, but the luminal contents of the ASG were much thinner than its glandular cell “sheath” (Fig. 1B), because the ASG contains a thick duct wall made of chitin which narrows the lumen25.

Figure 1
figure 1

Extraction and electrophoresis of proteins in the silk gland lumen.

(A) Schematic representation of the silk gland in the silkworm, Bombyx mori. S: spinneret; ASG: anterior silk gland; MSG: middle silk gland; PSG: posterior silk gland; A-MSG: anterior part of the middle silk gland; M-MSG: middle part of the middle silk gland; P-MSG: posterior part of the middle silk gland. (B) Photos of luminal contents and glandular cells of the silk gland. The silk gland was divided into five compartments according its morphology. The frozen luminal contents were extracted from the exterior silk gland cells at two stages: day 5 of the fifth instar (V-5) and day 1 of wandering (W-1). (C) SDS-PAGE of the luminal proteins in the silk gland.

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Results of SDS–PAGE revealed several similar, intensely-stained protein bands in the lumen of M-MSG, P-MSG and PSG (Fig. 1C), which mainly included the 350 kDa fibroin heavy chain26,27, the 400 kDa or 150 kDa sericin16,28, the 26 kDa fibroin light chain29 and the 27 kDa or 30 kDa fibroin p25 protein1,2,3. Fibroins and sericins were at low abundance in the lumen of ASG and A-MSG on day 5 of the fifth instar, but their level obviously increased on day 1 of wandering (Fig. 1C). A dozen protein bands seems similar between the ASG and A-MSG on day 5 of the fifth instar (Fig. 1C).

LC–MS/MS was used to determine the proteome in the lumen of five silk gland compartments during two developmental stages. With the combined analysis of triplicates samples, we identified 10,747 tryptic peptides, which assembled to 1271 proteins (Supplementary Dataset 1 and Dataset 2). The average number of peptides per protein was 8.5, leading to an average sequence coverage of 27.6% (Supplementary Dataset 2). Most proteins (94.2%) were identified by two or more unique peptides. On day 5 of the fifth instar, we identified 548, 446, 724, 797 and 645 proteins in the ASG, A-MSG, M-MSG, P-MSG and PSG, respectively (Supplementary Figure S1). On day 1 of wandering, we identified 502, 839, 402, 387 and 716 proteins in the ASG, A-MSG, M-MSG, P-MSG and PSG, respectively (Supplementary Figure S1). In contrast to day 5 of the fifth instar, substantial numbers of proteins increased in the A-MSG on day 1 of wandering, but decreased in the M-MSG and P-MSG on day 1 of wandering.

Annotation of proteins in the silk gland lumen

The Blast2GO analysis tool (version 2.6.6)30 was used to subject the luminal proteins to GO annotation (Supplementary Dataset 2) and revealed that the identified proteins were involved in metabolic process, transcription and translation, ion transport, protein transport, stress response, signal transduction, extracellular matrix, regulation of proteolysis and cytoskeleton organization. The Phobius server was used to predict the transmembrane regions and signal peptides31 and identified 868 intracellular proteins, 262 extracellular proteins and 141 transmembrane proteins (Supplementary Dataset 2). The identified intracellular proteins in the lumen of the silk gland might represent some leakage from cells of the silk gland. Although only 262 proteins were predicted to be extracellular proteins, they accounted for 87.0 ∼ 99.0% of the total protein abundance when being investigated with the intensity-based absolute quantification (iBAQ) intensity (Fig. 2A,B). Furthermore, we found that 295 proteins in the silk gland lumen were also detected in the scaffold silk and cocoon silk by previous studies7,32, making up 93.4 ∼ 99.5% of all protein molecules in the silk gland lumen (Fig. 2A,B).

Figure 2
figure 2

Identification, annotation and classification of proteins in the silk gland lumen.

(A) LC-MS/MS identified 1271 proteins in the silk gland lumen, 295 proteins of them was identified as silk proteins (Dong et al. 2013; Zhang et al. 2015) and 262 proteins were predicted as secretory proteins by Phobius website (Supplementary Dataset 2). The secretory proteins contain signal peptides but no transmembrane regions. (B) The relative abundances of silk proteins and predicted secretory proteins in the lumen of silk gland. (C) The relative abundances of the proteins in the silk gland lumen according to their functional categories. The protein abundances were calculated with intensity-based absolute quantification (iBAQ).

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Protein quantification results based on the functional classification indicated that proteins in the silk gland lumen mainly consist of fibroins, sericins, seroins, extracellular matrix proteins, protease inhibitors, enzymes and proteins of unknown function, of which fibroins were the most abundant components (Fig. 2C and Supplementary Dataset 2). Proteins from each functional category increased or decreased in different compartments of the silkworm gland lumen form day 5 of the fifth instar to day 1 of wandering (Fig. 2C).

Quantitative comparison of proteins in the silk gland lumen between two key stages

From day 5 of the fifth instar to day 1 of wandering, twelve proteins showed greatest increment based on intensities (Fig. 3 and Supplementary Table S1), including three fibroins, two sericins, three proteins of unknown functions, three protease inhibitors and one seroin. From day 5 of the fifth instar to day 1 of wandering, these twelve proteins increased in different compartments of the silk gland lumen (Fig. 3 and Supplementary Table S1): three fibroins (fibroin H, L and p25) jointly increased in the ASG, two sericins (sericin 1 and 3) increased in the ASG and A-MSG, three protease inhibitors (serine protease inhibitor BmSPI39 and BmSPI51 and carboxypeptidase inhibitor) mainly increased in the A-MSG, osifirs-9-like protein increased in ASG, A-MSG and M-MSG, glycine cell wall structral protein 1.0-like protein and fibroin p25 like protein increased in the M-MSG and P-MSG, whereas seroin 1 increased in all the silk gland. As a result, these twelve proteins in the silk gland lumen constituted the twelve most abundant cocoon proteins, accounting 94.0 ∼ 96.8% of the total protein abundance32.

Figure 3
figure 3

Constantly accumulated proteins in the silk gland lumen.

Twelve proteins showed greatest increment from day 5 of the fifth instar to day 1 of wandering (Supplementary Table S1), which also constituted the twelve most abundant cocoon proteins (Zhang et al. 2015), including three fibroins, two sericins, one seroin, three protease inhibitors and three proteins of unknown function. These proteins were compared according to their relative abundance (normalized iBAQ intensity) (Supplementary Dataset 2).

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From day 5 of the fifth instar to day 1 of wandering, eleven luminal proteins showed greatest reduction (Fig. 4 and Supplementary Table S1), which were abundant in the ASG and A-MSG lumen on day 5 of the fifth instar, but nearly disappeared on day 1 of wandering. All the eleven proteins significantly decreased (P < 0.05) in the ASG lumen, including five enzymes (juvenile hormone esterase 1, juvenile hormone epoxide hydrolase 1, ecdysone oxidase 1, beta-fructofuranosidase, 15-hydroxyprostaglandin dehydrogenase 1), two extracellular matrix proteins (cuticular protein RR-2 motif 68 and cuticular protein hypothetical 21), two protease inhibitors (BmSPI16 and BmSPI38), the sericin 2 and the uncharacterized protein (LOC101739721).

Figure 4
figure 4

Drastically reduced proteins in the silk gland lumen.

Eleven luminal proteins showed greatest reduction from day 5 of the fifth instar to day 1 of wandering (Supplementary Table S1), which were abundant in the ASG and A-MSG on day 5 of the fifth instar, but nearly disappeared on day 1 of wandering, including five enzymes, two extracellular matrix proteins, two protease inhibitors, one sericin and one protein of unknown function. These proteins were compared according to their relative abundance (normalized iBAQ intensity) (Supplementary Dataset 2).

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Hormone metabolism enzymes were identified in the silk gland lumen

Hormones play important roles in the regulation of the development and function of silk gland13. Fortunately, we identified twenty-five enzymes in the silk gland lumen that may be involved in the hormone metabolism (Supplementary Table S2). Among them, eleven juvenile hormone esterase (JHE) and JHE-like proteins, three juvenile hormone epoxide hydrolases (JHEH) and JHEH-like proteins may be involved in the juvenile hormone metabolism, while eight ecdysone oxidase (EO) and EO-like proteins, one 3-dehydroecdysone 3 alpha-reductase (3DE-3α-R) and two 3-dehydroecdysone 3 beta-reductase (3DE-3β-R) like proteins may play roles in the molting hormone metabolism.

The results of semi-quantitative RT-PCR confirmed that fifteen hormone metabolism enzymes were expressed in the silk gland (Fig. 5A). It was noteworthy that day 5 of the fifth instar and day 1 of wandering were really two key stages, because that most of hormone metabolism enzymes had obvious expression change during the two stages (Fig. 5A). Their change in mRNA level was almost the same as the variation in protein level (Fig. 5B). Furthermore, protein quantification results suggested that most of hormone metabolism enzymes were secreted into the lumen of ASG and MSG (Fig. 5B) and twelve of which had been detected in the silk (Fig. 5C)7.

Figure 5
figure 5

Hormone metabolism enzymes in the silk gland lumen.

(A) Expression patterns of seven juvenile hormone metabolism enzymes and eight molting hormone metabolism enzymes. Semi-quantitative RT-PCR was performed using gene-specific oligonucleotides (Supplementary Table S3). The silkworm housekee** gene ribosomal protein L3 (BmRpl3) was used as internal control (Liu et al. 2010). (B) The relative abundances of hormone metabolism enzymes in the silk gland lumen were compared according to the normalized iBAQ intensity (Supplementary Table S2). (C) Hormone metabolism enzymes detected in the scaffold silk and cocoon silk (Dong et al. 2013). (D) Hormone metabolism pathways in the silk gland lumen. This figure presents both the juvenile hormone metabolism pathway and molting hormone metabolism pathway according to the insect hormone biosynthesis pathways in the KEGG database and insect pathway database (iPathDB).

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According to the insect hormone biosynthesis pathways in the KEGG database and insect pathway database33,34,35, we speculated that juvenile hormone (JH) may be metabolized to inactive JH acid, JH diol and JH acid diol by JHE and JHEH, whereas ecdysone may be metabolized to 3-dehydroecdysone and 3-epiecdysone by EO and 3DE-3α-R in the silk gland lumen (Fig. 5D). It is a known fact that hormones control the development of silk gland and synthesis of silk13,36,37, whereas the hormone metabolism enzymes play important roles in the regulation of the hormonal titer38,66. The primers for semi-quantitative RT-PCR detection are listed in Supplementary Table S3. PCR amplification was performed in a total reaction volume of 25 μL using the following program: initial incubation at 94 °C for 4 min, followed by 28 cycles of 40 s at 94 °C, 40 s of annealing (52–58 °C), 30–50 s of extension (72 °C) and a final extension at 72 °C for 10 min. Aliquots of 5 μL of the PCR products were separated on 1.3% agarose gels and stained with EB.

Additional Information

How to cite this article: Dong, Z. et al. Analysis of proteome dynamics inside the silk gland lumen of Bombyx mori. Sci. Rep. 6, 21158; doi: 10.1038/srep21158 (2016).