Sericin Composition in the Silk of Antheraea yamamai

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Sericin Composition in the Silk of Antheraea yamamai Michal Zurovec, Naoyuki Yonemura, Barbara Kludkiewicz, Frantisek Sehnal, Dalibor Kodrik, Ligia Cota Vieira, Lucie Kucerova, Hynek Strnad, Peter Konik, and Hana Sehadova Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00189 • Publication Date (Web): 06 Apr 2016 Downloaded from http://pubs.acs.org on April 7, 2016

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Sericin Composition in the Silk of Antheraea yamamai Michal Zurovec a, d, Naoyuki Yonemura b, Barbara Kludkiewicz a, František Sehnal a,*, Dalibor Kodrik a, d, Ligia Cota Vieira a, Lucie Kucerova a, Hynek Strnad c, Peter Konik d and Hana Sehadova a a

Entomological Institute, Biology Centre ASCR, Branišovská 31, 370 05 České Budějovice,

Czech Republic b

National Institute of Agrobiological Sciences Tsukuba, Ibaraki 305-8634, Japan

c

Institute of Molecular Genetics ASCR, Vídeňská 1083, 142 20 Praha 4, Czech Republic

d

Faculty of Science, University of South Bohemia, Branišovská 1760, 370 05 České Budějovice,

Czech Republic

Keywords: Antheraea, Bombyx, fibroin, sericin, silk, biopolymers

ABSTRACT: The silks produced by caterpillars consist of fibroin proteins that form two core filaments, and sericin proteins that seal filaments into a fiber and conglutinate fibers in the cocoon. Sericin genes are well known in Bombyx mori (Bombycidae) but received little attention in other insects. This paper shows that Antheraea yamamai (Saturniidae) contains 5 sericin genes very different from the three sericin genes of B. mori. In spite of differences, all known sericins

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are characterized by short exons 1 and 2 (out of 3-12 exons), expression in the middle silk gland section, presence of repeats with high contents of Ser and charged amino acid residues, and secretion as a sticky silk component soluble in hot water. The B. mori sericins represent tentative phylogenetic lineages (I) BmSer1 and orthologs in Saturniidae, (II) BmSer2, and (III) BmSer3 and related sericins of Saturniidae and of the pyralid Galleria mellonella. The lineage (IV) seems to be limited to Saturniidae. Concerted evolution of the sericin genes was apparently associated with gene amplifications as well as gene loses. Differences in the silk fiber morphology indicate that the cocktail of sericins linking the filaments and coating the fiber is modified during spinning. Silks are composite biomaterials of conserved function in spite of great diversity of their composition. 1. INTRODUCTION The silk fiber produced by caterpillars is made from a pair of core filaments, one from each silk gland, that are sealed into a single fiber by coating proteins. The silk proteins were thoroughly analyzed in the domestic silkworm, Bombyx mori, and the nomenclature used for this species is now applied to other insects and often also to spiders. Proteins of the strong and flexible core of the silk filament are commonly referred to as fibroins and those of the coating are known as sericins. In caterpillars, fibroin proteins are secreted in the posterior section of the silk glands (PSG) and are pushed as a column of a jelly dope into the middle gland section (MSG) where they are over-layered by the sericin secretions. At spinning, fibroin components solidify into the filament core during dope passage through the spinneret, while the sericin sticky coating hardens with a delay. The stiffening of inner sericin layer conglutinates the pair of filaments into a single fiber and subsequent hardening of the outer sericin layers provides for fiber attachment to the substrates and for inter-fiber bonding during cocoon spinning. In sericulture, cocoons are


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processed to commercial silk by a millennia old process called degumming: the outer sericin layer is dissolved in hot alkaline water, the fiber is loosened, and fibers released from several cocoons are glued into the raw silk thread by residual sericins. Commercial silk is obtained from the domesticated silkworm, Bombyx mori (family Bombycidae), and from several wild silkmoth species of the sister family Saturniidae 1. There are dramatic differences in the fibroin composition between these two kinds of silks. The fibroin of B. mori and of nearly all other Lepidoptera consists of three proteins called heavy-chain fibroin (H-fibroin), light-chain fibroin (L-fibroin) and fibrohexamerin (Fhx), also known as P25

2-5

.

However, only H-fibroin was found in Saturniidae 6, 7; recently reported detection of a fhx cDNA in two saturniid species 8 most likely concerns structurally related fhx genes of uknown functions 9

. The structure of B. mori H-fibroin is characterized by the repeats of amino acid motif

GAGAGS

10, 11

, whereas strings of alanine characterize the H-fibroin of Saturniidae

6, 12

. Our

present study examines if the sericin components of the two kinds of silk are diversified to similar extent. As a representative of Saturniidae we use the Japanese oak silkmoth, Antheraea yamamai. The sericin proteins of B. mori are derived from three dissimilar genes. The transcript of the gene BmSer1 is alternatively spliced and yields four proteins; all of them contain 2 separate regions built of reiterated motifs 13. The BmSer2 gene generates two products that both contain a short repetitive region; the longer one further includes an additional long exon built from a reiterated motif 14, 15. BmSer3 encodes a single protein with two different repetitive regions 16. Little is known about the sericin proteins of Saturniidae. Several authors analyzed proteins obtained by different degumming procedures assuming that the major fractions represent sericins. For example, Dash et al.

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identified in the cocoon extracts from Antheraea mylitta 3 major

proteins regarded as sericins. The smallest of them was purified and taken for amino acid analysis


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and antibody preparation. The amino acid composition was different from that of B. mori sericins and the antibody recognized some antigens in the cocoon extract from Antheraea assamensis but not from B. mori, suggesting considerable differences between the sericins of B. mori and the Antheraea species. Maity et al.

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searched for sericins by screening a silk gland-specific cDNA

library from A. mylitta. They obtained 2476 EST sequences that included H-fibroin and seroin cDNAs but attempts to detect sericin-like genes failed. Such genes were subsequently detected in several species but their exclusive or preferential expression in MSG was shown only exceptionally. A recent study of the silk gland transcriptomes in 6 saturniid species concluded that several “unigenes” are homologs of the three B. mori sericin genes 8; however, our present data modify this conclusion considerably. Five genes expressed in MSG and encoding serine-rich proteins were recently identified in the saturniid Samia ricini 9. Our paper describes the occurrence of similar genes in A. yamamai. We demonstrate that these genes are expressed in MSG, encode proteins that are present in the cocoon silk and affect morphology of the silk fiber. We also define their relationship to the sericins of B. mori. 2. MATERIALS AND METHODS 2.1. Insect dissection and silk collection. Post-diapause eggs of the silkmoth Antheraea yamamai (Saturniidae) were obtained from Mr. Masashi Fukumoto of the Saku Agricultural Extension Center, Nagano prefecture, and from Dr. Koichi Suzuki of Morioka University (Japan). Hatched larvae were kept at 22-26 °C and fed leaves of the oak Quercus robur. The silk glands and other organs were dissected from the last instar larvae and either immediately frozen in liquid nitrogen or fixed for histology. The spun-out silk was examined by scanning electron microscopy. Small amount of silk was produced by the 1st and 2nd instar larvae and large volume by the post-feeding last instar larvae. Spinning began less than one day after the gut purge and


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included two distinct phases: secretion of the floss (about 6 h) that served as a scaffold for firm cocoon attachment, and subsequent cocoon construction (about 20 h). The cocoon silk was used for protein analysis. 2.2. Transcriptome preparation and analysis. Silk glands from the last instar larvae were crushed under liquid nitrogen to a powder from which total RNA was isolated with the TRIzol Reagent (Invitrogen) and purified with the NucleoSpin RNA II kit (Macherey-Nagel). The RNA integrity was checked and the concentration measured with the Bioanalyser 2100 (Agilent) and the mRNA was subsequently isolated with the aid of the Dynabeads Oligo(dT)25 (Ambion, Life Technologies). Reverse transcription was performed with the SMARTer PCR cDNA synthesis kit (Clontech) but the standard primer "3 BD SMART CDS Primer IIA" was replaced with the primer "CDS_T22" 5-AAGCAGTGGTATCAACGCAGAG(T)5G(T)7C(T)10VN-3. Sequencing library for the Roche pyrosequencer 454 (Roche, ) was prepared according to the "Rapid Library Preparation" protocol with these specifications: Rapid B adaptor sequence at the end of reads 5AGTCGTGGGAGGCAAGGCACACAGGGGATAGG-3; Sequencing key - at the start of read GACT; emPCR Primer A 5-CCATCTCATCCCTGCGTGTC-3; and emPCR Primer B 5CCTATCCCCTGTGTGCCTTG-3. The emulsion PCR and the products titration were done according to the standard Roche GS FLX+ protocol. Sequencing produced 120,000 reads that were assembled into contigs by the Newbler software package provided with the sequencer. The results were verified by blasting proteins deduced from the contig assemblies against the raw reads using the local tblastn function of BioEdit

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. Isotigs with significant BLAST hits and

longer than 250 bp were annotated. The candidate sericin sequences were eventually confirmed by the cDNA and the genomic DNA amplification and sequencing. 2.3. Preparation and analyses of chosen cDNAs. In most cases, total RNA was prepared as described above and 1 µg aliquots were taken for reverse transcription with the SuperScript II


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Reverse Transcriptase (Invitrogen) and the Oligo(dT)17 (Invitrogen) or the custom-made Trikant primer 20. The reaction was carried out at 42 °C. Desired cDNAs were amplified by PCR with the ExTaqHS polymerase (Takara) and specific primers based on sequences identified by the transcriptome analysis or by partial analyses of the newly prepared cDNAs. In some cases, the total RNA was extracted with the ISOGEN-LS solution (Nippon Gene), purified with the RNAeasy Mini Kit (Quiagene) and used for RT PCR primed by a modification of Trikant (last two T replaced with VN) and driven by the First strand cDNA synthesis kit (Fermentas). Chosen cDNA sequences were amplified with specific primers using the Advantage 2 Polymerase Mix (ClonTech). 2.4. Northern blotting. The total RNA was extracted with TRIzol Reagent (Invitrogen) and further purified with NucleoSpin RNA II kit (Macherey-Nagel) from chosen organs of single last instar larvae. Large PSG were cut to 4 pieces of similar size and gene expression was analyzed in each of them separately. The pieces were numbered P1 (adjacent to MSG), P2, P3 and P4 (terminal part). Much smaller MSG was taken as a single sample. RNA aliquots of 5 µg were taken for agarose electrophoresis, blotted onto a nylon membrane (Hybond N+, Sigma-Aldrich) and hybridized under high stringency conditions to the cDNA probes (described in the Results) labeled with α-32P[dATP]. Hybridization signals were detected by autoradiography using the storage phosphor screen of a STORM 860 Phosphorimager (Molecular Dynamics™). 2.5. Gene analysis. Whole bodies of three 1st instar larvae (about 25 mg each) were used for DNA extraction with the NucleoSpin Tissue kit (Macherey-Nagel). The larvae were homogenized in 600 µl of the lysis buffer and after 3 h at 56 °C the lysate was processed according to the protocol provided with the kit. Final solution contained 550 ng DNA per 1 µL aliquots used for PCR with the DNA polymerase KOD FX neo (Toyobo, Japan) and primers based on the cDNA sequences. Genomic DNA was circularized and sequenced to identify 5´end


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of the AySrn2 gene. PCR with a pair of primers matching regions in the 5´and 3´regions of the AySrn5 gene was used to verify a sequence of the central region of this gene. 2.6. Silk protein extraction and separation. Freshly spun cocoons were cut to small pieces and submerged (10 mg silk per 250 µl) into 10 mM TrisCl buffer pH 6.8 containing 8 M urea, 2 % SDS and 5% 2-mercaptoethanol. After 48 h at room temperature, the samples were vortexed several times and eventually centrifuged to obtain supernatants for SDS-PAGE. Both gradient (BioRad, 4-15%) and standard 10% gels were used to obtain optimal protein separation. Selected protein bands were cut out and used either for mass spectroscopy or sent for N-terminal sequencing to a service laboratory in the Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Czech Republic. The urea and 2-mercaptoethanol were in some cases removed by passing the extract diluted with PBS through a Sep-Pak C18 column (Waters). Only about 2% of the proteins were lost with this procedure. 2.7. Identification of protein fragments by mass spectroscopy. Mass spectroscopic analysis described previously

21

was slightly modified. The PAGE gels were stained with GelCode Blue

Safe Stain (Thermo Scientific) and sliced to isolate distinct bands. The slices were incubated in 200 µl 40% acetonitrile in 200 mM ammonium bicarbonate at 37 °C for 30 min. The solvent was discarded, the procedure was repeated and the snip was dried in a SpeedVac for 30 min. Solution (20 µg/ml) of proteomic grade trypsin (Sigma-Aldrich) was added (10-20 µl) until the gel became saturated The sample was incubated for 45 min at 4 °C, the surplus of trypsin solution was removed and replaced with 20 µl 9% acetonitrile in 40 mM ammonium bicarbonate. The protein digest was collected after overnight incubation at 37 °C. Peptides were isolated with the ZipTip C18 pipette tips (Millipore Corporation) and taken for LC-MS/MS using NanoAcquity UPLC coupled on-line to the ESI Q-Tof Premier mass spectrometer (the instrument, columns,


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and software were from Waters, UK). Peptides were separated by subsequent reverse-phase UPLC on the BEH300 C18 analytical column (75 mm i.d._150 mm length, particle size 1.7 mm) that was perfused at 0.4 ml/min flow rate with 0.1% formic acid containing acetonitrile in concentration increasing linearly from 3% (v/v, 1 min wash) to 40% during 30 min. Peptides eluted from the column flowed directly into the electrospray ionization source. Raw data for each sample was acquired in the data-independent MSE mode and data dependent Survey analysis mode. In both modes, peptide and fragment spectra were acquired with 2 ppm and 5 ppm tolerance, respectively. PLGS2.3 software was used for matching generated data with the entries in the species specific Uniprot and NCBI protein databases, and our private database of amino acid sequences deduced from the cDNAs specific for the silk glands of A. yamamai. N-terminal acetylation, Asn and Gln deamination, Cys carbamidomethylation, and Met oxidation were set as possible amino acid modifications. Identification of 3 consecutive y- or b-ions was required for positive peptide match.

2.8. Histology and electron microscopy. Freshly dissected silk glands were fixed, dehydrated, embedded in paraffin and sectioned at 10 µm. Sections were processed on glass slides in standard way, stained with the Mallory´s solution and photographed. Two techniques were used to prepare samples for the scanning electron microscopy. (1) Carefully cut cocoon pieces were glued to aluminium holders, sputter-coated with gold and observed in Jeol 6300 scanning electron microscope. (2) Cocoon pieces attached to special holders were rapidly frozen to -135 °C, gold coated, placed into the LT-FESEM (Low Temperature Field Emission Scanning Electron Microscope) Jeol JSM-7401F, fractured inside the microscope, allowed the surface coating to sublime for 5-10 minutes, coated again at -95 °C and observed.


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2.9. Phylogenetic analysis. We used following cDNA sequences for the construction of a phylogenetic tree: Samia ricini (LC001866, LC001867, LC001868 partial sequence, LC001869 partial sequence, LC001870), Bombyx mori Ser1 (NM_001044041), Ser2l (NM_001172816), Ser2s (NM_001172817), Ser3 (NM_001114644), Galleria mellonella MG1 (KC478777) and MG2 (KC478778). Coding sequences of the identified sericin genes were aligned with the MUSCLE program 22 and evolutionary analyses were conducted in MEGA6 23. Best model for calculating evolutionary distances was chosen according to the lowest BIC score (Bayesian Information Criterion) and AICc score (Akaike Information Criterion, corrected). Phylogenetic analysis was performed using the Minimum Evolution method. All positions with less than 90% site coverage were eliminated.

3. RESULTS 3.1. Transcriptome analysis. Transcriptome based on the silk glands of the fully grown A. yamamai larvae contained 107 thousands high-quality reads that covered 36.6 Mb of the DNA sequence. Contigs assembled with the aid of the Newbler v2.5 assembler (Roche) incorporated in average 352 bp. Four hundred largest (> 250 bp) isotigs were analyzed by comparison with the GenBank data using BLASTx software. Annotated isotigs were assigned to diverse functional groups (Figure S1) that were represented in the following ratios: gene products involved in transcription or translation (31.05%), enzymes (23.08%), genes involved in signal transduction (10.82%), genes encoding counterparts of cytoskeleton (4.56%), silk proteins (1.99%), protease inhibitors (3.13%), other genes (13.39%) and unknown genes (11.97%). 3.2. Identification of sericin cDNAs. Several candidate sericin contigs containing serine-rich repetitive regions were detected in our transcriptome database with the BioEdit software and the sequences of sericin genes of B. mori (GenBank Accession Nos. NM_001044041.1,


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NM_001172816.1, NM_001114644.1) and Galleria mellonella (Nos. KC478777, KC478778, KC478778) used as queries. The contigs required manual checking because the in silico assembly often reduced the repetitive regions; the resulting contigs contained faithful 3’ and 5’ ends encoding unique N- and C-termini, but varying number of repeats that had to be verified by PCR. The contigs could be separated into two groups based on the comparisons of their DNA sequences.

Figure 1. Schematic maps of the AySrn genes of Antheraea yamamai. Transcribed regions are depicted as boxes: empty in the 5’ and 3’ non-coding sequences, hatched in the coding repetitive regions and gray in the coding non-repetitive regions (gap in the hatched bar of AySrn5 indicates incomplete knowledge of this gene). Numbers of nucleotides are shown above each gene section. The positions of key primers A through T, which were used for the gene analysis, are marked with arrowheads and the positions and the lengths of probes used in the Northern blots analysis are presented as line segments above each gene map. The first group contained a single contig, which encoded 5145 bp long ORF. Putative translation product included N-terminal signal peptide of 22 amino acid residues, followed by a long nonrepetitive N-terminus, long central region composed of repeats, and a nonrepetitive Cterminus (Figure 1). The C-terminus included a short region resembling B. mori Ser1 that is


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characterized by the presence of two cysteine residues

13

. Based on this conserved feature, the

similarity of the first two exons and of the amino acid composition, this A. yamamai gene was designated as AySrn1. The second group of contigs contained sequences that were obviously derived from four closely related genes called AySrn2, AySrn3, AySrn4 and AySrn5. We use “Srn” designation to avoid impression that AySrn2 and AySrn3 are the orthologs of BmSer2 and BmSer3. Identification of several sericin-like genes in A. yamamai was reported recently 8. We found that one of them (unigene 23495) was identical with part of the AySrn1 we describe. Two other putative sericin fragments described in A. yamamai as unigenes 10600 and 11042

8

were not

closely related to any of the Srn genes. We ascertained that both unigenes in question were expressed in the silk glands (unpublished data) but neither of them encoded a signal peptide. BLAST search in the GenBank showed that proteins closely related (more than 80% identity) to the product of unigene 10600 are known from diverse insects as titin (or intracellular connectin) and proteins closely related to the unigene 11042 are conserved lepidopteran proteins named “serine/arginine repetitive matrix proteins 2-like”. We conclude that neither of the unigenes 10600 and 11042 represents a sericin gene and that A. yamamai harbors five sericin genes we have identified and refer to as the Srn genes. 3.3. Structural analysis of the putative sericin genes. Primers designed from the cDNAs of the five AySrn sequences were used to verify our computer assemblies and to determine the exonintron structure of the corresponding genes. The amplified genomic regions confirmed that the assembled cDNA sequences (GenBank Accession numbers from LC085887 to LC085891) are correct and represent five distinct genes. Each of them contains a complete ORF with the initiation codon and a termination codon (TAA or TGA), and a non-coding tail with the polyadenylation signal AATAAA. All Srn genes consist of two conserved short exons and a long


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exon 3 (Figure 1). Analysis of the internal sequences of exon 3 was difficult due to the presence of similar repeats. Since PCR amplifications sometimes yielded more than one product, we had to verify the sequences with different combinations of primers (the positions of key primers are indicated in Figure 1). Considerable similarities were found in the coding sequences of genes AySrn2, AySrn3, AySrn4 and AySrn5; the central region of all of them is composed of at least 10 (AySrn5) to 35 (AySrn2) repeats, each of 114 bp. The second halves of the AySrn3 and AySrn4 sequences are nearly identical (Figure S2). The coding sequence of AySrn1 is unique and includes 11 copies of a 153 bp long repeat. The introns occupy about half of each gene; the 1st and 2nd introns are of different lengths in AySrn2 and AySrn3 and of similar lengths in AySrn4 and AySrn5. Intron sequences are gene specific and differ from one another to similar extent as from those of the fibroin gene that was analyzed for comparison. 3.4. Expression of the Srn genes. The frequency of Srn reads detected by local BLAST in our transcriptome was high (e-value lower than e-100) and differed significantly for individual genes (Table 1). The data suggest that the expression of AySrn2 and AySrn4 is about five times higher than that of AySrn1, ten times higher than AySrn3, and hundred times higher than AySrn5. The expression level of the fibroin gene, which was analyzed as a positive control, seemed similar to AySrn3.


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Table 1. Characteristics of A. yamamai silk genes detected in the transcriptome database*. Gene

Number of reads*

Gene length

mRNA predicted

mRNA in Northerns

PAGE band (proteomics)

GenBank Acc. #

AySrn1

1230

8549 bp

5.4 kb

5.5 kb

1+2+3

LC08587

AySrn2

6126

9413 bp

5.2 kb

5.3 kb

2+3

LC08588

AySrn3

2959

6691 bp

3.5 kb

3.8 + 5.0 kb

3

LC08589

AySrn4

6394

8317 bp

5.1 kb

5.3 kb

2+3

LC08590

AySrn5

75

>4510 bp

>2.3 kb

5.0 + 3.8 kb

Not detected

LC08591

Fibroin

2788

9419 bp**

9.3 kb**

9.5 kb

2

AB542805

*Reads were identified at the e-100 threshold value; predicted mRNA length was determined from the cDNA sequences and confirmed by the Northern blots; encoded silk proteins were detected in indicated bands of the PAGE analysis (see Figure 4). **Data from Sezutsu and Yukuhiro 12. The tissue specificity and the developmental pattern of gene expression were examined by Northern blot analysis. Hybridization probes were prepared from the cDNAs by PCR with specific primers (Table S1) based on thorough comparison of the gene sequences. In spite of this effort, unique probes were obtained only for AySrn1 and Fibroin, whereas probes used to detect transcripts of other Srn genes were similar (Figure S3). The probes for AySrn3 and AySrn5 showed nearly 65% identity. The use of high stringency conditions minimized but apparently did not completely eliminate unwanted cross-hybridizations. All probes hybridized to specific bands of RNA isolated from MSG (lane B in Figure 2) or from the whole silk gland (lanes G, H, I). Only the probe specific for AySrn1 recognized its target also in the RNA sample from the PSG region adjacent to MSG (lane C). As expected, the transcript hybridizing with the control fibroin probe occurred in all four parts of PSG (lanes C, D, E, F) and was absent in MSG. None of the


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probes hybridized with the control RNAs prepared from the carcass without silk glands (Lane A). No signal was also obtained with RNA prepared from the silk glands of larvae one day after ecdysis into the last instar. This result and the enormous increase of the silk gland size suggest that intensive silk synthesis occurs during voracious feeding of the last instar larvae. The positions of most hybridizing bands in Figure 2 correspond to the transcript sizes deduced from the respective genes (Table 1). However, the probes for AySrn3 and AySrn5 each recognized two RNA bands, one around 3.8 kb and another one ca 5 kb. The smaller transcript hybridized strongly with the AySrn3 probe, and the larger one with the AySrn5 probe. From the size of the larger transcript we deduce that we failed to identify about half of the central repetitive region of the AySrn5 gene. The actual size of AySrn5 transcript probably matches the hybridizing band of ca 5 kb, while the AySer3 transcript most probably represents the 3.8 kb gene product. The AySrn1 probe cross-hybridized weakly with the fibroin transcript (top panel in Figure 2). The differences in hybridization intensities between lanes B, G, H and I indicate that the transcription rates of the examined genes change in course of the last larval instar.


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Figure 2. Northern blots of total RNA extracted from the body carcass without silk glands (lane A), MSG (lane B), PSG regions P1 to P4 (lanes C, D, E and F, respectively), and whole silk glands from a fully grown larva (lane G), larva in thin cocoon (lane H) and larva at the start of cocoon spinning (lane I). Probes designed for AySrn1, AySrn2, AySrn3, AySrn4 and AySrn5 hybridized with specific bands in the blots of the MSG RNA; the AySrn1 probe recognized a


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target also in the RNA extract from the P1 region which is adjacent to MSG (lane C). Transcript hybridizing with the fibroin probe (blot FibH) occurred in all four parts of PSG and was absent in MSG. Table 2. Size comparisons of the AySrn proteins Parameter*

AySrn1

AySrn2

AySrn3

AySrn4

AySrn5

Number of AA residues

1721

1567

1061

1573

>625

Molecular mass

176.7492 kDa

157.7444 kDa

104.48849 kDa

153.8604 kDa

>64.9675 kDa

Residues of signal peptide

22

18

18

18

18

Non-repetitive N-terminus

1041

52

165

61

94

Repetitive region

583

1330

836

1294

>412

Length/number of repeats

51 residues, 11 copies




38 residues, >10 copies

Non-repetitive C-terminus

75

180

80

199

101

Full sequence of AySrn5 is not known

3.5. Predicted Srn proteins. Basic features of proteins deduced from the AySrn genes are compared in Table 2 and their amino acid sequences are shown in Figure S4. Ser, Thr and Gly dominate and Glu and Tyr are present in considerable proportions in all putative Srn proteins. AySrn1 is exceptional by having high content of Pro and His, while Asn is the fourth most frequent residue in the remaining Srn proteins. AySrn5 is distinguished by a 5% content of Lys that is present in smaller ratio in the other proteins. The pI values ranging from 4.93 (AySrn3) to 5.92 (AySrn5) reflect the high content of charged residues. The AySrn proteins are highly hydrophilic, except for their signal peptide regions (Figure S5), and contain several potential glycosylation sites (Figure S4). All AySrn proteins start with a predicted signal peptide that is


16

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followed by a nonrepetitive N-terminus, a central region composed of repeats and a nonrepetitive C-terminus (Figure S6). The comparison of amino acid sequences confirms the singularity of AySrn1 (Figure 3). This protein with a signal peptide of 22 residues includes a long (1071 residues) nonrepetitive Nterminal region that is relatively low in Ser and rich in the charged amino acid residues and in Pro and Thr. Short motifs such as SGPYPG, SGPRS, GPHSGPH and TSTT occur in a few copies dispersed in this non-repetitive region. The repetitive sequence of AySrn1 encompasses 11 full copies of a highly conserved repeat of 51 residues; internal 38 residues of the repeat resemble the repeats conserved in other Srn proteins. The non-repetitive C-terminus of AySrn1 contains 75 residues and is characterized by the presence of two Cys residues at -18 and -20 (counted backwards from terminal Leu).

Figure 3. Alignments of selected regions of deduced AySrn proteins. Upper panel: amino termini with indicated exons; note the Ser rich regions in AySrn3, 4 and 5. The positions of signal peptide cleavage are indicated by arrowheads in exon 2. Central panel: Unique repeats in AySrn1 in contrast to the repeats in other AySrn proteins. Lower panel: Carboxy termini (66 amino acid residues are shown; note nearly identical sequences of AySrn3 and AySrn4 and their similarity with AySrn2.


17

Biomacromolecules

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The other Srn proteins form a distinct group.

Page 18 of 39

Their N-termini begin with conserved

sequences of 11 and 10 amino acids (Figure 3) derived from the exons 1 and 2; presumptive signal peptide cleavage site is located between Ala and Leu in positions 18 and 19. The sequence similarities are dramatically reduced beginning by position 22, i.e. with the first residue encoded by the 3rd exon. However, starting by residue 32, the AySrn3 and AySrn5 proteins contain identical sequence HTESYSTNSSSSSHDESH that is shortened to SSSSSH in AySrn4. The nonrepetitive N-terminal sequences of the AySrn proteins 2 to 5 are from 52 to 165 residues long and are relatively serine-rich (Figure 3). Characteristic features of the long N terminus in AySrn3 include groups of charged residues and a string of 5 serine residues. The repetitive region of AySrn1 is shorter than in the other AySrn proteins (Table 2); the detected repetitive region of AySrn5 (380 residues) is probably incomplete. The length of the repetitive region depends solely on the number of repeats, not on their size; all repeats in Srn2, 3, 4 and 5 consist of 38 amino acid residues and exhibit considerable similarity between the proteins (Figure 3). In AySrn2, there are 35 reiterations of a repeat that is slightly different between the uneven and even repeat positions (Figure S6). The repetitive regions of AySrn3 (786 residues) and AySrn4 (1294 residues) contain 21 and 34 nearly identical repeats. AySrn3 differs from AySrn4 by longer non-repetitive N-terminus and much shorter non-repetitive C-terminus. Putative AySrn5 (625 residues) contains 94 and 101 residues in the non-repetitive N-terminus and Cterminus, respectively, and 412 residues in 11 diversified repeats. Based on the Northern analysis we assume that we know only about half of the AySrn5 repeats. The repeats are diversified but the spacing of residues with similar properties is often conserved. Except for AySrn1, the C-termini of A. yamamai sericins are characterized by strings of Ser and by short motifs containing charged residues (Figure 3). The sequence of 71 terminal residues of AySrn2 is similar to that of AySrn3, and the corresponding regions of AySrn3 and AySrn4


18

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differ only by 6 residues. The C-terminal sequence EEKSESHSENSSYSSKTATYGSAQDC and the motif SRTAQS in position -79 are fully conserved in these two proteins. 3.6. The identification of AySrn proteins in the cocoon silk. PAGE separation of proteins extracted from the cocoons of A. yamamai yielded 8 bands that were excised and analyzed by LC-MS. Thirteen proteins were identified, including four AySrn proteins (Figure 4). Band 1 contained AySrn1, band 2 included AySrn1, AySrn2 and AySrn4, and all AySrn proteins except AySrn5 were detected in band 3.. Peptide fragments detected in the protein hydrolysate and matching the deduced AySrn sequences are listed in Figure S6. Independent N terminal sequencing of proteins extracted from the PAGE gel confirmed the presence of AySrn4 in bands 2 and 3 and the presence of other proteins in bands 4 – 8 (Table S2).

Figure 4. Silk proteins separation by PAGE (A) and identification by mass spectroscopy (B). A: Protein size markers are shown on the left and numbers of analyzed protein bands on the right. B: Reference codes (GenBank or our AYAM transcriptome database) of identified silk proteins and numbers of matching peptide fragments detected by LC-MS with a data independent (dI) and data dependent (dda) modes of analysis (see supplementary information for complete data).


19

Biomacromolecules

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Protein markers indicated that AySrn1 migrated in PAGE in the region of ca 230 kDa and all four detected Srn proteins were present within 180-230 kDa. Since the sizes deduced from the cDNAs sequences were smaller than these values, we assume that the proteins were glycosylated, possibly to different extent. Relatively high serine content in these proteins could also slow down protein mobility 24. Our failure to detect AySrn5 was probably due to a relatively low level of its expression indicated by the low number of reads identified in our transcriptome by local BLAST search (Table 1). In addition, protein extraction from the cocoons is only partial (insoluble remains may contain Srn proteins) and may be associated with protein degradation – Srn never occurred as neatly separated bands. 3.7. Silk production and appearance. Histological sections demonstrated the presence of one type of secretion in PSG and small amounts of a different secretion in MSG (Figure 5). Consistently with the fibroin gene expression in PSG and the Srn genes expression in MSG (Figure 2), we regard the PSG secretion as fibroin and the MSG secretion as sericin. Large secretory vesicles released from the PSG cells disperse as tiny droplets within fibroin, and subsequently enlarge as they penetrate the fibroin column. This observation is in agreement with the suggestion that lysosomes released from the silk glands cells into liquid fibroin cause the formation of pores that are typical for the fibroin of Saturniidae 25. The vesicles were not seen in MSG that was characterized by a loose layer of sericin secretion between the cells and the fibroin column. The tiny droplets persisting in the periphery of fibroin column were presumably involved in pore creation.


20

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Biomacromolecules

Figure 5. Transverse histological sections through A. yamamai PSG (A, C) and MSG (B, D). Description: c, cells; n, nuclei; f, fibroin; v, large secretory vesicles; bar, 10 µm. Arrows indicate blue-stained sericin layer in the MSG (B, D). We used the freeze fracturing technique of scanning electron microscopy (SEM) to get some information on the structure of the secreted silk (Figure 6). The sericin coating of silk filaments appeared as a smooth envelope (Figure 6B) from which the fibroin core was pulled out in some fractures (Figure 6C). More often, however, the envelope was broken to plates (Figure 6D) and the fibroin core composed of microfibrils was exposed (Figures 6D, E). Microfibrils appeared as microvilli in some transverse filament fractures (Figure 6E, F). The longitudinal fiber fractions produced considerable number of microfibrils of uneven diameter ranging from 10 to 500 nm (Figure 6 G, H, I). Some regions of the fibroin filaments resisted separation into microfibrils by the freeze fracturing. In some cases it was possible to detect ca 10 nm thick links connecting separated microfibrils (Figure S7).


21

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Figure 6. Scanning electron microscopy (SEM) of the freeze fractured cocoon wall (A) and transversally (B, C, D, E, F) or longitudinally (G, H, I) fractured individual silk fibers. Description: f, fibroin; mf, microfibrils; s, sericin; m, microvilli.


22

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Figure 7. SEM of A. yamamai silk fibers produced by the 1st instar larvae (A, B) and postfeeding last instar larvae: floss fibers (C), fibers from the outer and middle cocoon layers (D, E and F, silk filaments connected by thin sericin bridges only), flat and ribbon-like fibers in the inner cocoon layers (G, H). Calcium oxalate crystals are common (I). SEM was also used to compare silk fibers spun out at different developmental times. We found that the 1st instar larvae spin very fine (1.7 µm thick, invisible by naked eye) anchoring and guiding fibers to bridge free spaces between the branches and leaves. Similarly to the cocoon silk, these fibers consist of 2 filaments conglutinated by small amount of sericin (Figure 7A, B). Intensive silk secretion occurs in the last larval instar. PSG rapidly enlarge when the larvae cease


23

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feeding, the MSG increase occurs later and is much less dramatic. Small amount (ca 16 mg in our culture) of white floss silk (Figure 7C) is produced for about 6 h prior to cocoon spinning and provides a scaffold for cocoon attachment. Silk filaments of the floss silk are often not sealed into fibers but seem to form a meshwork whereby sericin provides random crosslinking between single filaments. The floss silk gradually merges into the yellow or yellow-green silk that makes the relatively soft outer cocoon layer (about 50 mg). The pairs of adjacent silk filaments are sealed into compact fibers that are occasionally cross-linked by sericin coating into interlacing meshwork in the cocoon wall (Figures 6A and 7D). More inner cocoon layers, which represent the cocoon shell of about 240 mg, consist of white silk with reduced amount of coating; the filaments are in some places separated from one another (Figure 7E) and are hold together by fine sericin links (Figure 7F). The cocoon shell is rigid and can be pealed into thin flakes. In the most inner cocoon layers, the silk fibers are flat and appear as ribbons (Figure 7G, H). The maximal fiber cross-section grows during spinning from 28.1 ± 6.1 µm on the cocoon surface to 37.7±5.2 µm in the middle cocoon layer, and decreases to 26.7 ± 4.1 µm in the inner-most layer. All cocoon layers contain numerous 1.5-2.5 µm crystals (Figure 7I) that are known from different saturniid species and have been identified as calcium oxalate

26

. The amount and the spatial

distribution of crystals in the cocoon vary. The crystals easily fall off from the silk as a white powder. 4. DISCUSSION 4.1. Identification of sericin genes in Antheraea yamamai. Commercial silks of the closely related families Saturniidae (silkmoths) and Bombycidae (silkworms) possess similar physical properties

27

, but their protein compositions differ. Differences in the fibroin proteins have been

well documented 12 and the sericins of the model saturniid A. yamamai are analyzed in this paper.


24

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Biomacromolecules

In our search for sericin contigs in the database of silk gland transcriptome we looked for the following features: gene expression in MSG, similarity of the putative translation product with known sericins, presence of signal peptide and of repetitive sequence regions rich in Ser, and protein detection in the cocoon silk. This approach allowed identification of the AySrn1, AySrn2 and AySrn4 cDNAs; the remaining genes were detected in the transcriptome database on the basis of similarity with AySrn2. The sequences were verified by RT PCR and by amplification of the genomic fragments. All five Srn genes of A. yamamai include short exons 1 and 2 and a long exon 3. The first two exons are of similar length as those in the sericin genes of B. mori and G. mellonella

28

yamamai

and

. In addition, there is a clear similarity of peptides encoded by these exons in A. G.

mellonella

(MKFSLVLLLAV

+

IFAAVQALPN

in

AySrn2

and

MKFTVALLVIA + AFVAVQAAPRA in GmMG2). All these genes are expressed in MSG region (AySrn1 also in adjacent quarter of PSG) and encode proteins rich in serine (22.6-27.5% of all amino acid residues) and in charged amino acid residues. Identification of five genes in the genomic DNA, results of the Northern blots and proteins detection in the silk provide conclusive evidence that the Srn genes represent genuine sericins of A. yamamai. The gene designated AySrn1 encodes a protein that has higher proportion of non-repetitive sequence than the repetitive sequence, while almost entire sequence of proteins deduced from the genes AySrn2, AySrn3, AySrn4 and AySrn5 is repetitive and the repeats of these four sericins are similar. 4.2. The diversity and phylogeny of sericins. The silkworm genes BmSer1, BmSer2 and BmSer3 contain 9, 11 and 3 exons, respectively. The BmSer1 gene, which generates 4 mRNAs by alternative splicing

29

, encodes repetitive amino acid sequences in long exon 6 (sequenced only

partially), short exon 7 and the medium-length exon 8 13. Repeats encoded in exon 8 each contain 38 amino acid residues like the repeats of the AySrn genes 2 - 4 but the sequence is different: SSHRGGSVSSTGSSSNTDSSTKNAGSSTSGGTSTYGYS (underlined residues match the


25

Biomacromolecules

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Page 26 of 39

repeat of AySrn5). The gene BmSer2 produces two transcripts by alternative splicing

15

and

contains 11 exons, of which exons 9 and 10 are partial duplications 14. The translation products of BmSer2

include

several

types

of

repeats:

KFENLDKDNVCE

from

exon

3,

TEKAKPNDSSPSHKD from exon 8, and RSPSHKDTEKAKPND from exon 9 14. The BmSer3 gene resembles the sericin genes of A. yamamai by the gene structure with a long 3rd exon that encodes 10 repeats of 86 amino acid residues (with 45 % Ser) and 18.5 reiterations of a variable octapeptide motif SSSSKQAS 16. The known sericins of Galleria mellonella are derived from 3 related genes MG1, MG2 and only partly analyzed MG3 28, 30. MG1 and MG2 contain four exons, of which exons 1 and 2 are of similar length as the first two exons of the sericin genes of B. mori and A. yamamai. Sericin proteins of G. mellonella are characterized by high content of 3 amino acids (Ser 52.4 – 59.9%, Asn 11.1-17.5% and Gly 14.6-23.1%) that dominate in the repeats NGSSGSSSSN (GmMG1), NSSGSSSSNNSSGSSSSN (GmMG2) and NGSSSSSGSSSS (GmMG3). The length of the 1st and 2nd exons, presence of serine-rich repeats, relatively high content of charged residues, the secretion in MSG and the solubility in hot water are features in common to all known sericins. However, every species analyzed so far is characterized by specific structure of the sericin genes (3 exons in A. yamamai, 4 in G. mellonella, and 3, 9 and 11 in B. mori) and specific composition of the Sericin proteins (Table 3). We assume that the simple structure of sericin genes in G. mellonella and A. yamamai indicates similarity with the ancestral gene, whereas the structure of sericin genes of B. mori grossly diversified, particularly in BmSer1 and BmSer2. The exon/intron diversification seems to be independent of changes in the amino acid composition of the encoded sericin proteins. To probe the extent of such changes, we constructed a phylogenetic tree based on the sericin coding sequences of B. mori, G. mellonella, A. yamamai and Samia ricini (Figure 8). The data on S. ricini were obtained by Tsubota et al. 9 who analyzed


26

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EST libraries prepared separately from the PSG and A/MSG (anterior plus middle silk gland regions). They identified five sericin-like genes that were represented more in the A/MSG library than in the PSG library. Our analysis of these genes confirmed their homology with B. mori sericins as well as with the Srn sequences of A. yamamai.

Figure 8. The phylogeny tree of sericins inferred from 3 BmSer cDNAs (B. mori, Bombycidae), 5 AySrn cDNAs (A. yamamai, Saturniidae), 5 Samia ricini cDNAs (Saturniidae, GenBank Nos. LC001866-70) and 2 GmMG cDNAs (Galleria mellonella, Pyralidae) using the Minimum Evolution method. Bootstrap values (10000 replicates) above 50% are shown next to the branches. The evolutionary distances were computed with the Maximum Composite Likelihood method, rate variation among sites was modeled with a gamma distribution (shape parameter = 17.39). Evolutionary analyses were conducted with the MEGA6 software.


27

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Table 3. Comparison of sericin properties in G. mellonella; B. mori and A. yamamai Protein

Per cent amino acid residues present at ≥ 5%

Number of repeats x residues/repeat

Number of residues Total

Charged

Total charge

Ip

GmMG1

1

54.5 S; 23.1 G; 11.1 N

17 × 12

567

2/0

+ 1.9

11.0

GmMG2

2

59.9 S; 17.5 N; 8.9 G

>71 × 8

˃ 925

6/15

+ 9.1

4.0

GmMG3

2

52.4 S; 14.6 N; 14.1 G

>39 × 6

>347

8/13

- 4.9

4.5

BmSer1

3

30.7 S; 10.8 G; 9.4 T; 7.1 D; 7.1 A; 5.0 N

12 × 38

1217

115/115

+2.8

7.5

BmSer2

4

17.1 K; 15.1 S; 11.7 D; 11.1 E; 5.8 T; 5.6 P

44 × 15; 7 × 11

1758

415/413

+17.3

8.5

BmSer3

5

43.6 S; 12.0 G; 6.7 N; 5.5 Q; 5.3 D

10 x 86; 8.5 x 8

1271

113/124

+9.5

5.7

AySrn1

6

22.6 S; 17.9 T; 12.3 G; 9.0 P; 7.4 D; 5.3 H; 5.3 Y

11 x 51

1721

81/177

-80.7

5.18

AySrn2

7

24.8 S; 15.4 T; 18.7 G; 10.0 N; 5.6 D; 7.5 Y

35 x 38

1567

82/123

-31.7

5.69

AySrn3

8

26.5 S; 16.2 T; 18.2 G; 9.2 N; 7.2 D; 5.0 Y

21 x 38

1061

61/101

-35.6

4.93

AySrn4

9

27.5 S; 14.9 T; 20.4 G; 8.1 N; 6.7 D; 5.0 Y

34 x 38

1573

86/137

-45.2

4.96

AySrn5

10

24.5 S; 13.3 T; 12.6 G; 13.0 N; 6.2 D; 6.6 Y; 5.0 K

>10 x 38

>625

47/56

-6.6

5.92

Data were taken from the following GenBank deposits: 1 KC478777; 2 KC478777; 3 NM_001044041.1; 4

NM_001172816.1;

5

NM_001114644.1;

6

LC085887;

7

LC08588; 8LC08589;

9

LC08590 and

10

LC085891. Single letter abbreviations are used for the amino acid residues. Full sequences of GmMG2, GmMG3 and AySrn5 are not known. Numbers of positively (Arg; His; Lys) and negatively (Tyr; Asp; Glu; Cys) charged amino acid residues are provided in the format +/-. Protein characteristics were established for the largest protein product of the respective gene with http://web.expasy.org/protparam.

The phylogenetic tree (Figure 8) suggests early splits of a hypothetical ancestral sericin gene into four separate lineages that are tentatively called types I, II, III, and IV. The lineage (I) includes BmSer1, AySrn1 and the S. ricini gene registered in the GenBank as LC001869. It seems that the precursor of lineage I was the first to split off from the ancestral gene before it repeatedly duplicated to yield the other lineages. The lineage (II) has lower bootstrap support and so far includes only the gene BmSer2, which is differentially spliced and yields 2 highly adhesive proteins secreted before cocoon spinning 14. The lineage (III) contains sericins from B. mori, G. mellonella and S. ricini but not from A. yamamai. The lineage (IV) apparently diversified in Saturniidae. More data on additional lepidopteran taxa are needed to reconstruct the sericin gene history.


28

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Biomacromolecules

4.3. Sericin properties deduced from the amino acid sequences. The high content of serine is probably crucial for sericin function, notably for the conversion of viscous sticky secretion into the solid surface of silk filaments and fibers. It has been shown that Ser residues can interact with some other amino acids via hydrogen bridges to form beta sheet protein conformation

24

. Three

dominating amino acid residues (Ser, Asn and Gly) in the sericins of G. mellonella can form typical beta sheets in most of the molecule but the sericins of B. mori and A. yamamai are more complex. Those of A. yamamai contain 13.3-17.9 % Thr that can function similarly to Ser (22.627.5%) by participating in the beta sheet formation with other dominating amino acid residues (Gly, Asn). Notable is the high content of Asp (5.6-7.4%) and Tyr (5.3-7.5%) that is responsible for the total negative charge of the Ay sericins. This is in contrast to B. mori sericins that are close to neutral in spite of the relatively high content of charged amino acid residues. We ponder on the possible role of electrostatic interactions in the conversion of liquid sericins into hardened glue but no experimental data are available. The high content of Ser and Thr also invites speculation on possible phosphorylation of some of these residues. Heavy phosphorylation was shown, along with other post-translational modifications, for B. mori H-fibroin

31

but no similar

data have been published on the sericin proteins. Phosphorylated residues in the H-fibroin are cross-linked by calcium ions whereby calcium concentration controls the conformation transition of the H-fibroin 32. Finally, the conserved positions of 2 Cys residues in the type I sericins, and of 1 Cys residue in AySrn3 and AySrn4 indicate their importance for sericin conformation or for their covalent bonding to other proteins, but evidence is wanting. In B. mori it has been shown repeatedly that each sericin type is secreted in distinct MSG region and at specific times

16, 29, 33, 34

. Developmental regulation of the sericin genes expression

was also demonstrated in G. mellonella 28. The time and the site of secretion determine when will be the respective sericin added to the surface of the polymerizing fibroin filaments and fibers.


29

Biomacromolecules

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The BmSer1 gene is expressed in the posterior MSG region during larval development and with much increased intensity in the last larval instar in preparation for cocoon spinning

35

. In this

study we show that all Srn genes are expressed in MSG and AySrn1 also in the PSG section adjacent to MSG in the post-feeding larvae of A. yamamai. We surmise that type I sericins are located close to fibroin and are involved in filament conaglutination in both species. Formation of covalent linkage with fibroin via the Cys residues is not excluded. The sericins of B. mori consist of four to six major and several minor polypeptides with apparent molecular masses ranging in PAGE from 65 to 400 kDa

33, 36

. In A. yamamai we

detected AySrn1 in protein fraction of apparent size ca 240 kDa and together with AySrn2 and AySrn4 also in the 200 kDa fraction. The fraction of ca 180 kDa contained all sericin proteins except AySrn5. Molecular masses deduced from the putative amino acid sequences are much lower. We assume that the difference is due to glycosylation. Sericin glycosylation is probably common 37 but remains to be examined. 4.4. Silk texture. Sericins provide silk filaments with stickiness and affect morphology of the native silk fiber. Silk components soluble in hot water are usually regarded as sericins, although they certainly include some additional minor silk components. The water soluble fraction of the cocoon silk makes up 31.6 % of the cocoon silk in B. mori and 15.7% in S. ricini 38. Judging from the silk gland histology and the silk appearance in SEM, the proportion of sericin in the silk of A. yamamai is similar to that in S. ricini. The dominance of fibroin over sericin can also be deduced from the large size of PSG that is up to ten times larger than MSG in the fully grown A. yamamai larvae. The big PSG lumen is filled with fibroin secretion, while only thin layer of sericins fills the space between fibroin column and the MSG epithel (Figure 5). Northern blots confirmed the expression of fibroin gene in PSG and of all Srn genes in MSG (AySrn1 also in the PSG quarter adjacent to MSG). It seems that fibroin accumulates in PSG and partly also in MSG where


30

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sericins are secreted but probably not stored. This is in contrast to B. mori and most other Lepidoptera with smaller PSG than the MSG, the site of sericin secretion and of both fibroin and sericin storage. In A. yamamai and other saturniids, the gelatinous fibroin column in the silk glands as well as the fibroin core of the solid silk filaments, are characterized by porous structure that was studied by Akai

25

who proposed that the pores are formed by lysozymes released from the PSG cells as

vacuolar droplets. The round pores become elongated during fibroin passage through the anterior silk gland section and the spinneret. Our results are consistent with this explanation and indicate that owing to the long pores, longitudinal freeze fracturing breaks the fibroin filaments into fibrils that are about 1 mm long and may be only 10 nm thick. In case of transverse freeze fracturing, the broken microfibrils appear as microvilli, possibly separated by spaces left after the pores. For A. pernyi it was shown that the shape and the mechanical properties of silk change in the course of cocoon spinning and are correlated with the amount of sericin 39. We observed similar difference in silk morphology in A. yamamai. In this species, the fibers of the floss silk are often formed by single fibroin filaments enveloped and occasionally cross-linked with other filaments by the sericin proteins. The amount of sericin is relatively large at this time, probably to ensure firm floss attachment to diverse substrates. This is different from the typical larval silk or the cocoon silk, in which a pair of fibroin filaments is sealed into a single fiber by a relatively small amount of sericin. In some parts of the cocoon, the filaments are separate and interconnected only by fine sericin bridges. These diverse morphologies obviously depend on the amounts and properties of the blends of sericins secreted in the course of spinning.

5. CONCLUSIONS


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Transcriptome based on the Antheraea yamamai silk glands contains 107 thousands highquality reads that were assembled into 341 isotigs larger than 250 bp. They were annotated to diverse functional groups; 1.99% isotigs encoded the silk proteins. Major silk proteins separated from the cocoon extracts by PAGE and analyzed by mass spectroscopy and N-terminal sequencing matched the relevant isotigs. Five cDNAs were recognized as putative sericin genes that were fully characterized and named AySrn1-5. All of them contain 3 exons, of which the short exons 1 and 2 are conserved in the sericins of other species. The AySrn proteins are rich in Ser, Thr, Gly and charged amino acid residues (especially Glu and Tyr), and include regions with repeats composed of 51 residues in AySrn1 and 38 residues in AySrn2, AySrn3, AySrn4 and AySrn5. Comparison with other species allowed to define the key features of sericins: expression in the middle silk gland region: short exons 1 and 2; open reading frame encoding proteins with a signal peptide and repetitive region(s) with high contents of Ser and charged amino acid residues; presence of deduced proteins in silk; similarity with proven sericins of other Lepidoptera. Constructed phylogenetic tree suggests early splits of a hypothetical ancestral sericin gene into four lineages (three of them are exemplified by Bombyx mori sericins). Northern analysis revealed that all AySrn genes are specifically expressed in the middle silk gland region (AySrn1 also in adjacent section of the posterior region). The posterior gland region produces large amounts of fibroin, while the secretion of sericins is relatively small. Vesicles produced in the posterior region create pores in the fibroin dope. The pores are elongated during dope passage through the spinneret and persist in the cocoon silk. The volume and the composition of sericins affect the appearance of the silk fibers that is altered in course of spinning.

ASSOCIATED CONTENT


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Supporting Information listed below is available free of charge via the Internet at “http://pubs.acs.org.” •

Analysis of silk gland specific transcriptome, comparison of 5 sericin genes and proteins, light microscopy of silk glands, electron microscopy of silk and results of N-terminal amino acid sequences and LC-MS analysis (pdf).

AUTHOR INFORMATION (* corresponding author) Michal Zurovec, Entomological Institute, Biology Centre ASCR, Branišovská 31, 370 05 České Budějovice, Czech Republic, email [email protected] Naoyuki Yonemura, National Institute of Agrobiological Sciencesm Tsukuba, Ibaraki 305-8634, Japan, email [email protected] Barbara Kludkiewicz, Entomological Institute, Biology Centre ASCR, Branišovská 31, 370 05 České Budějovice, Czech Republic, email [email protected] * František Sehnal, Entomological Institute, Biology Centre ASCR, Branišovská 31, 370 05 České Budějovice, Czech Republic, email [email protected] Dalibor Kodrík, Entomological Institute, Biology Centre ASCR, Branišovská 31, 370 05 České Budějovice, Czech Republic, email [email protected] Ligia Cota Vieira, Entomological Institute, Biology Centre ASCR, Branišovská 31, 370 05 České Budějovice, Czech Republic, email [email protected] Lucie Kucerova, Entomological Institute, Biology Centre ASCR, Branišovská 31, 370 05 České Budějovice, Czech Republic, email [email protected]


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Hynek Strnad, Institute of Molecular Genetics ASCR, Vídeňská 1083, 142 20 Praha 4, Czech Republic, email [email protected] Peter Konik, Faculty of Science, University of South Bohemia, Branišovská 1760, 370 05 České Budějovice, Czech Republic, email [email protected] Hana Sehadova, Entomological Institute, Biology Centre ASCR, Branišovská 31, 370 05 České Budějovice, Czech Republic, email [email protected] ACKNOWLEDGEMENTS We appreciate the gifts of Antheraea yamamai eggs from Mr. Masashi Fukumoto of the Saku Agricultural Extension Center, Nagano prefecture, and from Dr. Koichi Suzuki of the Morioka University (Japan). We further thank to Dr. Zdeněk Voburka of the Institute of Organic Chemistry and Biochemistry CAS, Prague, for the N-terminal sequencing, to our colleagues Dr. František Weyda and Mrs. Jitka Pflegerová for the help with electron microscopy and to Mgr. Tereza Koniková for silk extraction and electrophoresis. This study used some of the research infrastructure

built

in

frame

of

the

EC

project

MODBIOLIN,

GA

316304,

FP7/REGPOT/2012/2013/1.

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TABLE OF CONTENTS GRAPHIC


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Sericin Composition in the Silk of Antheraea yamamai

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