New insight into the mechanism underlying fibroin secretion in silkworm, Bombyx mori Dingpei Long, Weijian Lu, Yang Zhang, Qing Guo, Zhonghuai Xiang and Aichun Zhao State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of the Agricultural Ministry, Southwest University, Chongqing, China
Keywords EGFP/H-chain fusion protein; fibroin secretion mechanism; piggyBac; silk; transgenic silkworm Correspondence A. Zhao, State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of the Agricultural Ministry, Southwest University, BeiBei, Chongqing 400716, China Fax: +86 23 68251128 Tel: +86 23 68251803 E-mails: [email protected]; [email protected] (Received 4 June 2014, revised 21 August 2014, accepted 6 October 2014)
In order to investigate the role of different parts of the fibroin heavy chain (H-chain) in the secretion of fibroin in the silk gland of the silkworm (Bombyx mori) in vivo, two enhanced green fluorescent protein (EGFP)/Hchain fusion genes with deduced protein sequences containing an identical N-terminal region and different C-terminal regions of the H-chain were introduced into the B. mori genome using a piggyBac-mediated germline transformation. EGFP fluorescence and molecular analysis showed the products of two different EGFP/H-chain fusion proteins were secreted into the posterior silk gland lumen and aggregated in the middle silk gland and spun into cocoons. The results revealed that only the non-repetitive N terminus of the H-chain is essential for secretion of the H-chain into the posterior silk gland lumen. In addition, our results also indicated that the most likely post-translational modification of the H-chain is at the C-terminal domain. Here, our results not only provide a theoretical basis for the genetic modification of silk fiber as a functional biomaterial but also are of great significance to establishing a new silk gland bioreactor to mass-produce exogenous proteins in an active form.
doi:10.1111/febs.13105
Introduction Silk produced by the silkworm Bombyx mori is a macromolecular protein well known in the textile industry for its luster and mechanical properties. Silk is a very good model material for examination of protein structure, function and mechanisms of secretion and it has been studied in attempts to understand the processing mechanisms and to exploit the properties of the protein for use as biomaterials [1]. As a high-performance biomaterial, silk produced by B. mori can be used in the textile industry and in the fields of biotechnology, biomedicine and the chemical industry [2].
The silkworm synthesizes abundant silk proteins and spins them into silk threads, which are used to build a cocoon shell. The major components of cocoon silk in the normal silkworm are fibroin and sericin, which represent approximately 75% (w/w) and 25% (w/w) of a cocoon, respectively [3]. Fibroin is synthesized in the posterior silk gland (PSG) and accumulates in the middle silk gland (MSG) lumen, where sericin is synthesized. Fibroin, which is secreted via the anterior silk gland (ASG), contains three different proteins: fibroin heavy chain (H-chain), fibroin light chain (L-chain)
Abbreviations ASG, anterior silk gland; CTD, C-terminal domain; EGFP, enhanced green fluorescent protein; fhx/P25, fibrohexamarin; H-chain, fibroin heavy chain; LBS, L-chain binding site; L-chain, fibroin light chain; 2-ME, b-mercaptoethanol; MSG, middle silk gland; NTD, N-terminal domain; poly (A), polyadenylation signal sequence of the H-chain gene; PSG, posterior silk gland; PTM, post-translational modification; TSL, transgenic strain containing the EGFP/H-chain fusion gene with LBS sequence; TSP, transgenic strain containing the EGFP/H-chain fusion gene with poly(A) sequence; TSL-FP, the EGFP/H-chain fusion proteins derived from TSL silkworms; TSP-FP, the EGFP/H-chain fusion proteins derived from TSP silkworms.
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
89
D. Long et al.
The mechanism of fibroin secretion in silkworm
and fibrohexamarin (fhx), named P25 [4]. In the PSG, these proteins form a large protein complex designated the elementary unit of fibroin, which consists of six sets of H-chainL-chain (H-L) disulfide-linked heterodimers and one molecule of the glycoprotein fhx/P25 [4], which interacts non-covalently with six sets of H-L heterodimers and forms the elementary unit in the endoplasmic reticulum and is then secreted into the Golgi apparatus [4,5]. The H-chain, which can form up to 92% (w/w) of the elementary unit of fibroin, is the major component (w/w) of the elementary unit. The H-chain consists of three domains: an N-terminal domain (NTD), a repeated region and a C-terminal domain (CTD) [6,7]. The NTD is considered to work as a signal peptide [8] and to contribute to the solubility of the H-chain [9]. The repeated region consists of a hydrophobic crystalline region (GAGAGS/Y)n and a hydrophilic noncrystalline region [6,7] which is the main component of the H-chain and contributes to the physical properties of fibroin [10]. The CTD contains three cysteine residues that form intramolecular and intermolecular disulfide bonds; a single disulfide bond is formed between Cys172 of the L-chain and Cys-c20 (the twentieth residue from the CTD) of the H-chain [11]. The H-L heterodimer is suggested to be essential for efficient secretion of fibroin from the PSG cells on the basis of the results of studies on the B. mori fibroin secretion-deficient naked pupa mutants Nd-s and Nd-sD [12–14]. Earlier, we constructed and introduced three enhanced green fluorescent protein (EGFP)/Hchain fusion genes with different 50 -flanking sequences and identical 30 -flanking sequences into the normal B. mori strain N4 to assess the ability to produce recombinant protein. The results suggested that the NTD of the H-chain was important for the secretion of the Hchain into the PSG lumen [15]. The roles of three regions of the H-chain in the secretion of fibroin are not entirely clear, however, especially whether the CTD is essential for the efficient secretion of fibroin from PSG cells. In this study, we constructed two EGFP/H-chain fusion genes with identical 50 -flanking sequence and different 30 -flanking sequences. These EGFP/H-chain fusion genes were introduced into the silkworm genome using a piggyBac-mediated silkworm germline transformation [16]. Two different EGFP/H-chain fusion genes were expressed in the PSG of transgenic silkworms, the products of recombinant proteins were secreted into the PSG lumen, aggregated in the MSG and spun into cocoons. In accord with our earlier studies, we confirmed that only the NTD of the Hchain is essential for the secretion of the H-chain into the PSG lumen. Here, we provide new insight into the mechanism underlying fibroin secretion; our results 90
suggest that the PSG cells of transgenic silkworms can secrete the elementary unit of fibroin into the PSG lumen and can direct secretion of the H-chain fusion protein into the PSG lumen and spin into cocoons without forming an H-L heterodimer.
Results Histological distribution of EGFP in the silk glands of transgenic silkworms Figure 1 shows the structures of two different ecombinant piggyBac transformation vectors pBac {3 9 P3-DsRed}-R3 and pBac{3 9 P3-DsRed; Fib-H P3-EGFP-poly(A)}. Finally, one G1 transgenic strain containing an EGFP/H-chain fusion gene with the L-chain binding site (LBS) sequence was produced and designated TSL, and four G1 transgenic strains containing an EGFP/H-chain fusion gene with the polyadenylation signal sequence of the H-chain gene (poly (A)) were produced (TSPs) and designated TSP-1–TSP4 as described in Materials and methods. EGFP fluorescence was detected in TSL and TSP individuals. Silk glands dissected from day 2 of the fifth instar were observed using a fluorescence stereomicroscope as described in Materials and methods. The PSGs and MSGs of TSL and TSP silkworms on day 2 of the fifth instar exhibited strong EGFP fluorescence (Fig. 2A). We observed the trend of EGFP secreted from PSG to MSG in the silk glands of TSL and TSP silkworms, and there was no difference between TSL and TSP individuals (Fig. 2A, e,f). In addition, PSGs and MSGs of TSL and TSP strains were sectioned and viewed under a light microscope (Fig. 3A). Cross-sections of PSGs and MSGs from TSL and TSP silkworms showed that both EGFPs were expressed and the expressed product was secreted effectively into the PSG lumen and the fibroin layers of MSGs but not the sericin layers of MSGs (Fig. 3A, e–h). Moreover, EGFP fluorescence was not detected in other tissues of TSL and TSP individuals, suggesting that the fibroin H-chain promoter sequence determined the tissue-selective expression of these EGFP/H-chain fusion genes very efficiently [15,17]. Distribution of EGFP in the cocoon silks of transgenic silkworms Cocoons from different transgenic silkworms were observed under a fluorescence stereomicroscope (Fig. 2B). The cocoons from the TSL and TSP silkworms displayed strong green fluorescence (Fig. 2B, e, f), indicating that a large amount of recombinant EGFP was synthesized in the silk glands and spun into FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
The mechanism of fibroin secretion in silkworm
A
B
Fig. 1. Construction of piggyBac-derived vectors and different EGFP/H-chain fusion genes. (A) Structures of the transformation vector pBac {3 9 P3-DsRed}-R3 and EGFP/H-chain fusion gene including the LBS. (B) Structures of the transformation vector pBac{3 9 P3-DsRed; FibH P3-EGFP-poly(A)} and EGFP/H-chain fusion gene including only the polyadenylation sequence. pBacL, left arm of piggyBac transposon; pBacR, right arm of piggyBac transposon; 3 9 P3, 3 9 P3 promoter; DsRed, red fluorescence protein gene (red box); SV40, SV40 polyadenylation signal sequence. The numbers of nucleotide sequences of fibroin H-chain gene in the parentheses are consistent with the position of the sequences from Accession No. AF226688. The different DNA fragments are indicated by an abbreviated form: 50 -flanking, 50 -flanking sequence of the H-chain gene; E1, exon1 of the H-chain gene; Intron, intron 1 of the H-chain gene; 50 -E2, 50 -terminal sequence of exon2 of the H-chain gene; EGFP, enhanced green fluorescence protein gene (green box); 30 -E2, 30 -terminal sequence of exon2 of the H-chain gene; poly(A), polyadenylation signal sequence of the H-chain gene.
cocoons. In addition, paraffin sections of silk from the cocoons of TSL and TSP silkworms were viewed under a laser scanning confocal microscope (Fig. 3B). The results showed that EGFP was located in the fibroin layer, but not the sericin layer, of cocoon silk from TSL and TSP silkworms (Fig. 3B, b,d). Analysis of EGFP/H-chain fusion transcripts in the PSGs of transgenic silkworms To determine the different constructs of EGFP/Hchain fusion genes in TSL and TSP individuals, PCR was done on genomic DNAs from TSL, TSP and wild-type strain 871 adults with the primer pair EGFP-MF/poly(A)-MR (Fig. 4A). As shown in Fig. 4B, the PCR products were a 306 bp DNA fragment for the TSL individuals, a 134 bp DNA fragment for TSP-1–TSP-4 individuals and no amplified frag-
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
ment for wild-type strain 871 silkworm control, which is consistent with the putative pattern. RT-PCR was performed as described in Materials and methods to further confirm the expression of these EGFP/H-chain fusion genes in the PSGs of TSL and TSP silkworms. As shown in Fig. 4C, the expected 1418 bp EGFP/H-chain fusion gene and 717 bp EGFP gene PCR products were observed for cDNA from transcripts in the PSG of TSL individuals, and the expected 1246 bp EGFP/H-chain fusion gene and 717 bp EGFP gene PCR products were observed for cDNA from transcripts in the PSG of TSP-1–TSP-4 individuals by using the primer pair FibH-50 UTR-F/poly(A)-MR. The cDNA from transcripts in the PSG of wild-type 871 strain and fat body of TSP-1 were used as controls. All PCR products were sequenced and, as expected, no structural change was detected (data not shown). These results
91
D. Long et al.
The mechanism of fibroin secretion in silkworm
A a
b
c
d
e
f
a
b
c
d
e
f
B
Fig. 2. Distribution of EGFP in the silk glands and cocoons from transgenic silkworms. (A) Silk glands were dissected from one individual of wild-type strain 871 (a, d), TSL (b, e) and TSP-1 (c, f) of 2-day-old fifth instar larvae, and were viewed under white light (a–c) and GFP fluorescence (d–f). (B) Cocoons produced by one individual of wild-type 871 (a, d), TSL (b, e) and TSP-1 (c, f) were observed under white light (a–c) and GFP fluorescence (d–f). White scale bar represents 5 mm.
confirmed that the different EGFP/H-chain fusion transcripts of EGFP/H-chain fusion genes were synthesized specifically in the cells from PSGs of TSL and TSP silkworms. Analysis of EGFP/H-chain fusion proteins in the cocoon silk from transgenic silkworms The cocoon silk proteins from TSL, TSP-1 and wild-type 871 silkworms were dissolved in 60% (w/v) 92
lithium thiocyanate without or with b-mercaptoethanol (2-ME) and separated by SDS/PAGE to further investigate whether the different EGFP/H-chain fusion proteins were linked with the L-chain via a disulfide bond in the cocoon silk from TSL and TSP silkworms. The same diluted samples were subjected to western blotting with anti-GFP antibody (Fig. 5). The results of SDS/PAGE suggested that the EGFP/H-chain fusion proteins derived from TSL silkworms (TSL-FP) were produced as a TSL-FP FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
The mechanism of fibroin secretion in silkworm
A a
b
c
d
e
f
g
h
b
c
d
B a
Fig. 3. Distribution of EGFP in PSGs, MSGs and cocoon silks of transgenic silkworms. (A) Paraffin sections were prepared from PSGs (a, b, e, f) and MSGs (c, d, g, h) of the TSL (a, c, e, g) and TSP-1 (b, d, f, h) silkworms, and observed under white light (a–d) and GFP fluorescence (e–h) using a fluorescence microscope. White scale bar represents 25 lm. (B) Paraffin sections were prepared from cocoon silks of the TSL (a, b) and TSP-1 (c, d), and observed under white light (a, c) and GFP fluorescence (b, d) using a laser scanning confocal microscope. Scale bar represents 10 lm.
and L-chain disulfide-linked (TSL-FP-L) heterodimer of 82 kDa without treatment with 2-ME, and a single TSL-FP of 57 kDa with 2-ME treatment, which could break the disulfide bond between TSL-FP and the L-chain (Fig. 5A). The EGFP/H-chain fusion proteins derived from TSP-1 silkworms (named TSPFP) were produced as a single TSP-FP of 43 kDa whether without or with 2-ME treatment (Fig. 5A). Western blotting with an anti-GFP antibody showed these bands at the same position and size as SDS/ PAGE in the TSL and TSP-1 samples (Fig. 5B). The results confirmed that TSL-FP was linked with the L-chainvia a disulfide bond in the fibroin of cocoon silks from TSL silkworms, but TSP-FP was
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
secreted directly into the fibroin of cocoon silk from TSP silkworms without forming an H-L heterodimer.
Discussion The essential H-chain domain for the secretion of fibroin Silk has long attracted attention as a biodegradable fiber of considerable strength, elasticity and durability. The properties of B. mori silk fiber depend on the amino acid repeats that interact during fibroin H-chain polymerization [18]; however, the true mechanism
93
D. Long et al.
The mechanism of fibroin secretion in silkworm
A
B
C
Fig. 4. Analysis of EGFP/H-chain fusion transcripts in the PSGs of transgenic silkworms. (A) Schematic maps of the transgene constructs in genomes of TSL (top) and TSP (bottom) individuals. The EGFP-MF/poly(A)-MR primer pair was used for PCR analysis of genomic DNAs from TSL and TSP individuals. FibH-50 UTR-F/poly(A)-MR and EGFP-F/EGFP-R primer pairs were used for RT-PCR analysis of cDNA from EGFP/H-chain fusion transcripts in the PSGs of TSL and TSP individuals. (B) PCR analysis of genomic DNA using primer pair EGFP-MF/poly (A)-MR. The expected 306 bp PCR products were observed for TSL, and the expected 134 bp PCR products were observed for TSP-1–TSP4. WT, wild-type 871 strain was used as a control; lane M, trans2K Plus DNA marker. (C) RT-PCR confirmation of EGFP/H-chain fusion gene expression in the PSGs. The expected 1418 bp PCR products were observed for TSL, and the expected 1246 bp PCR products were observed for TSP-1–TSP-4 by using primer pair FibH-50 UTR-F/poly(A)-MR (top). The expected 717 bp EGFP gene PCR products were observed for TSL and TSP-1–TSP-4 by using primer pair EGFP-F/EGFP-R (bottom). The cDNA from transcripts in the PSG of wild-type 871 strain (WT) and fat body (FB) of TSP-1 was used as control. The amplification of BmActin A3 using primer pair BmActin3-f/BmActin3-r served as an internal control.
underlying the secretion of silk fibroin is not entirely clear. In an earlier study, we demonstrated that the NTD of the H-chain was important in the secretion of fibroin, and the signal peptide from amino acid residues 1–21 of the NTD, but not the repeated region of the H-chain, was essential for the secretion of the H-chain into the PSG lumen [15]. In this experiment, the EGFP/H-chain fusion protein TSL-FP (named R2R3-FP in our earlier work 94
[15]) lacks the repeated region of the H-chain, and fusion protein TSP-FP lacks the repeated region and CTD of the H-chain (Fig. 6), but TSL-FP and TSPFP were both secreted into the PSG lumen, aggregated in the MSG and spun into cocoons. By analyzing the secretion of TSL-FP and TSP-FP in the PSGs of transgenic silkworms, the present study demonstrated the repeated region and CTD of the H-chain were not necessary for secretion of the H-chain into the PSG FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
A
The mechanism of fibroin secretion in silkworm
B
Fig. 5. SDS/PAGE and western blotting analysis of transgenic silkworm cocoon silk proteins using anti-GFP antibody. (A) Cocoon silk protein samples from wild-type 871, TSL and TSP-1 silkworms were subjected to SDS/10% PAGE without or with b-galactosidase (2-ME) treatment. (B) Western blotting of proteins in the gel as in (A) with the anti-GFP antibody. M, molecular mass markers. Sizes are indicated on the left of the panels. Triangles indicate TSL-FP-L heterodimer, asterisks indicate TSL-FP, and arrowheads indicate TSP-FP. and + indicate the protein samples without or with 2-ME treatment, respectively.
Fig. 6. Domain structure of the normal Hchain and three different EGFP/H-chain fusion proteins, TSL-FP, TSP-FP and R1FP. The repeated region of the normal Hchain comprises 12 hydrophobic crystalline domains separated by 11 hydrophilic noncrystalline domains [6,7]. The TSL-FP is also named R2R3-FP in our earlier work [15].
lumen. In accord with our earlier studies, we confirmed that only the NTD of the H-chain was essential for the secretion of the H-chain into the PSG lumen. Different functions for the H-chain domains in the assembly and secretion of fibroin Some earlier studies found the repeated region of the normal H-chain comprises 12 low-complexity hydrophobic crystalline domains separated by 11 hydrophilic noncrystalline domains (Fig. 6) [6,7,9]. Twelve hydrophobic crystalline domains composed of Gly-X repeats account for 94% of the H-chain; X is Ala in 65%, Ser in 23% and Tyr in 10% of the repeats [6,7]. The main function of the repeated region was suggested to be a
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
contribution to maintenance of the physical properties of fibroin [9,10], but not for the secretion of fibroin, which was confirmed by the results of this study. On the basis of what was discussed above, we suggest the most important function of the NTD of the H-chain is that it contains a signal peptide sequence (21 amino acids long) essential for the secretion of fibroin (Fig. S1). The CTD of the H-chain has a cysteine residue (Cys-c20) that links with the L-chain (Cys172 in the mature L-chain) via a disulfide bond and the other two cysteine residues (Cys-c4 and Cysc1) form an intramolecular disulfide bond [11,19]. The H-L heterodimers and N-glycosylated fhx/P25 assemble into an elementary unit (6 : 6 : 1 molar ratio of H-chain/L-chain/fhx/P25) through hydrophobic inter-
95
D. Long et al.
The mechanism of fibroin secretion in silkworm
actions between fhx/P25 and H-chains and by hydrogen bonding between N-linked oligosaccharide chains of fhx/P25 and H-chains [4]. Some earlier studies suggested that the H-L disulfide bond was critical for secretion of the fibroin molecule into the cell lumen on the basis of the evidence that L-chain-deficient mutant silkworms (Nd-s and Nd-sD mutants) are unable to secrete silk fiber [12,13], and it has been reported that the mutant chimeric Nd-sD L-chain lacks Cys172, which was encoded by the original sixth exon and consequently could not form a disulfide bond with the H-chain [14]. In this study, the TSP-FP lacks the CTD (and Cys-c20) in the PSG cells of TSP silkworms, so the TSP-FP could not link with the L-chain via a disulfide bond forming an H-L heterodimer in the PSG cells of TSP silkworms; however, we observed secretion of the TSP-FP into the lumen of the PSG cells of TSP silkworms. This result revealed that formation of the H-L heterodimer was not essential for secretion of the H-chain into the PSG lumen. We suggested that the CTD of the H-chain was necessary only for forming the H-L heterodimer in the PSG cells. In this study, the molecular mass of the TSL-FP and TSL-FP-L heterodimers was estimated by SDS/ PAGE to be ~ 57 kDa and ~ 82 kDa, respectively, which is 6.6 kDa larger than the predicted molecular mass of the TSL-FP (49.4 kDa) and TSL-FP-L heterodimers (75.4 kDa). This might be owing to the abnormal mobility of the H-chain fusion proteins in SDS/ PAGE or the presence of post-translational modifications (PTMs), such as glycosylation or phosphorylation, as reported in our earlier study and elsewhere [15,17,20,21]. In general, the presence of a cluster of basic amino acid residues causes a delay in protein migration during SDS/PAGE [22]. An earlier report suggested there were basic amino acids (R, K and H) in the cluster at the CTD of the H-chain fusion protein (Fig. S1), and this might have been the cause of the molecular mass difference determined by SDS/ PAGE [17]. However, the possibility of PTMs (phosphorylation or glycosylation) cannot be excluded. Phosphorylation of fiber proteins has been reported in spider silk [23] and in the L-chain and fhx/P25 of silkworms [24] and glycosylation of fibroin proteins has been reported in fhx/P25 [11,19], and we reported the presence of potential PTMs on the H-chain [15]. The identity and function of these PTMs on the H-chain are unclear but there is the possibility of PTMs on the normal H-chain, TSL-FP and TSP-FP, since the NTD and CTD of the normal H-chain, TSL-FP and TSPFP, contain many amino acid residue sites that could be phosphorylated and glycosylated (Fig. S1). Actu96
ally, we observed that the molecular mass of TSP-FP without the CTD of the H-chain was estimated to be about 43 kDa by SDS/PAGE, which was consistent with the predicted molecular mass of TSP-FP (43.6 kDa), so we speculated that the likeliest cause of the abnormal migration is the presence of a C-terminal basic amino acid cluster and the most likely PTM of the H-chain is also at the CTD. New insights into the fibroin secretion mechanism in the PSG cells of B. mori Inoue et al. reported the use of the piggyBac transposon for germline transformation of the Nd-sD mutant with a normal L-chain-GFP fusion gene to construct a transgenic mutant [25]. Normal development of the PSG in the transgenic mutant was restored and it formed a normal cocoon. Inoue et al. had confirmed that the molar ratio of the H-chain/L-chain-GFP fusion protein/fhx/P25 in the secreted fibroin of the transgenic mutant was strictly 6 : 6 : 1 [25]. Combined with the present study, we propose two new insights about the secretion of normal H-chain and H-chain fusion protein in the PSG cells of the silkworm: (a) H-L linkage could contribute to the maintenance of the elementary unit but the formation of the H-L heterodimer is not essential for secretion of the H-chain into the PSG lumen; (b) the H-chain and the H-chain fusion protein could be secreted directly into the PSG lumen and spun into cocoons without forming the HL heterodimer, especially for the small molecules and hydrophilic H-chain fusion proteins. Most of the H-chains were secreted into the silkworm PSG in the form of the elementary unit of fibroin [4]. Because the normal H-chain has a high molecular mass (~ 350 kDa) [26] and contains 12 hydrophobic crystalline domains of high molecular mass which account for 94% of the H-chain [6,7], the form of the elementary unit could contribute to secretion of the H-chains. When the normal H-chain or H-chain fusion protein (such as the TSL-FP) containing the CTD of the H-chain is first linked with the normal L-chain (containing Cys172) to form the H-L heterodimer, six H-L heterodimers are linked with one molecule of fhx/P25 to form the elementary unit for the secretion of fibroin [4]. It has been reported that fhx/P25 interacts with the H-chain in the absence of H-L linkage but its content of oligosaccharides was reduced when the H-L linkage was not formed [19], so H-L linkage could contribute to maintenance of the elementary unit. The disulfide bond between the L-chain and Cysc20 of the H-chain does not appear to be essential for secretion of the H-chains, however; actually, a FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
small amount of the L-chain-free H-chain (< 1% compared to the normal level fibroin-producing silkworms) could be secreted from the naked pupa mutants [4]. In addition, the present study demonstrated that TSP-FP from TSP silkworms not containing the CTD of the H-chain could be secreted into the PSG lumen and spun into cocoons. These results strongly suggested that direct secretion of the H-chain or H-chain fusion proteins without forming the H-L heterodimer into the PSG lumen of the silkworms was a pathway for secretion of the H-chain into PSG cells. In this study, we used a small molecule and the hydrophilic EGFP domain to replace these hydrophobic crystalline domains of the normal H-chain in two EGFP/H-chain fusion proteins (Fig. 6). Because TSL-FP could be linked preferentially with the L-chain through a disulfide bond, the TSL-FP-L heterodimer further assembled with fhx/ P25 to form the elementary unit for secretion of large amounts of the TSL-FP, the same as secretion of the normal H-chain. On the other hand, TSP-FP could not be linked to the L-chain to form the H-L heterodimer and it could be secreted into the PSG lumen directly; understanding the real reasons requires further study. Our results showed that TSLFP and TSP-FP were expressed highly efficiently in the silk glands and cocoons of TSL and TSP silkworms using fluorescence detection; further, there was no obvious difference in fluorescence intensity between the two types of cocoons from TSL and TSP silkworms, respectively. Advantages and prospects of the new fibroin H-chain expression system Since Kurihara et al. [27] first reported using the Hchain gene for the production of an active feline interferon in the cocoon of transgenic silkworms, the fibroin H-chain expression system has been used widely to produce exogenous proteins in the silk gland and cocoon of transgenic silkworms [15,17,21,28–30]. Earlier studies suggested production of the recombinant proteins mediated by the fibroin H-chain expression system required both the NTD and CTD of the H-chain [31]. However, this study revealed that production of the TSP-FP using the new fibroin H-chain expression system required only the NTD or a signal peptide sequence of the H-chain, which would make the construction of this system simpler compared to using the earlier system containing both the NTD and CTD of the H-chain. In addition, the expression efficiency of the H-chain fusion protein mediated by this new system was comparable to the earlier system. FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
The mechanism of fibroin secretion in silkworm
More importantly, PTMs of recombinant proteins often have a critical effect on the function of the exogenous target proteins [32]. Because both the NTD and CTD of the H-chain contained potential N-glycosylation and generic phosphorylation sites (Fig. S1), and the H-chain fusion proteins (such as the TSL-FP) containing the CTD of the H-chain could be linked to the normal L-chain, the activity of the target proteins might be affected by the possible PTMs of the NTD and CTD of the H-chain fusion proteins and the formation of the H-L heterodimer. Kurihara et al. [27] reported that the NTD and CTD of the H-chain in the fusion protein reduced the biological activity of the target protein from transgenic silkworms. Our new fibroin H-chain expression system has only a short NTD with an essential signal peptide for the encoded fusion proteins. Therefore, this new system would give a greater chance to express the targeted protein in an active form, which would enable production of protein drugs or novel biomaterials on the basis of transgenic silk. In this study, we confirmed that only the non-repetitive N terminus with a signal peptide of the H-chain is essential for the secretion of the H-chain into the PSG lumen, through observation and analysis of the secretion of different EGFP/H-chain fusion proteins in the PSG and cocoon of transgenic silkworms. Our results suggested that PSG cells of transgenic silkworms could secrete the elementary unit of fibroin into the PSG lumen and could direct secretion of the H-chain fusion protein into the PSG lumen and spinning into cocoons without forming the H-L heterodimer. A new fibroin Hchain expression system with only a shorter N-terminal sequence of the H-chain for fusion with the exogenous genes could give a greater chance to express the targeted protein in an active form. In addition, exploitation of this new system in commercial silkworm strains would contribute to the development of silkworm resources and the utilization of silkworm bioreactors and show great potential to increase the value of the commercial production of exogenous proteins.
Materials and methods Silkworm strain used for germline transformation The Chinese lineage diapause B. mori strain 871 (white cocoon, commercial strain) was maintained at the Gene Resource Bank of Domesticated Silkworms (Southwest University, Chongqing, China). The 15 °C-IMES germline transformation strategy [33] was used to change the diapause character of strain 871 eggs for DNA pre-blastoderm
97
D. Long et al.
The mechanism of fibroin secretion in silkworm
microinjection. Microinjection with non-diapause strain 871 embryos and screening of transgenic silkworms were done as described elsewhere [34,35].
Construction of vectors and generation of transgenic silkworms The piggyBac-derived vector pBac{3 9 P3-DsRed}-R3 [15] (Fig. 1A) was maintained in our laboratory. pBac{3 9 P3DsRed}-R3 contains an EGFP/H-chain fusion gene with the LBS, which consists of the fibroin H-chain gene 30 -terminal sequence including the deduced Cys-c20 of the H-chain and the polyadenylation signal sequence of the H-chain gene (Fig. 1A). To construct the vector pBac {3 9 P3-DsRed; Fib-H P3-EGFP-poly(A)} (Fig. 1B), the poly(A) sequence was amplified by PCR using the primer pair poly(A)-F-BamHI/LBS-R-SalI (Table S1) with the pBac{3 9 P3-DsRedaf}-R3 plasmid as a template. The 182 bp PCR product was inserted into the BamHI/SalI site of the plasmid pSL-LBS-EGFP+Fib-H P3 [15] to generate plasmid pSL{Fib-H P3-EGFP-poly(A)}. pBac{3 9 P3DsRed; Fib-H P3-EGFP-poly(A)} was constructed by cloning a 3.2 kb AscI fragment from pSL{Fib-H P3-EGFPpoly(A)} into AscI-cut pBac{3 9 P3-DsRedaf} [36]. pBac {3 9 P3-DsRed; Fib-H P3-EGFP-poly(A)} contains an EGFP/H-chain fusion gene with the H-chain gene 30 -terminal sequence, which includes only the poly(A) (Fig. 1B). The piggyBac-derived vectors were injected with the helper plasmid pHA3PIG [16] into G0 non-diapause 871 eggs at the pre-blastoderm stage. G0 adults were backcrossed with wild-type strain 871. We obtained a total of five G1 transgenic strains, including one TSL strain from the pBac{3 9 P3-DsRed}-R3 vector insertion and four TSP strains from the pBac{3 9 P3-DsRed; Fib-H P3EGFP-poly(A)} vector insertion (Table S2).
Inverse PCR The chromosomal insertion sites of the piggyBac-derived vectors on the chromosomes were determined by inverse PCR. About 10 lg of genomic DNA was digested with HaeIII overnight at 37 °C and circularized by overnight ligation at 16 °C. The ligated product was PCR-amplified with the transposon-specific primer pairs PLF/PLR (for piggyBac left arm) and PRF/PRR (for piggyBac right arm) (Table S1). The PCR fragments were cloned and sequenced. Localization of the silkworm genomic insertion sites of the piggyBac-derived vectors were completed using SILKMAP software (www.silkdb.org/silksoft/silkmap.html). The results showed that the sequence of the two piggyBac arms was bordered by the characteristic TTAA site in all transgenic strains, and the piggyBac inserts in the genome of TSL and TSP strains were located on different chromosomes (Table S3).
98
EGFP fluorescence observation of the silk glands and cocoons of transgenic silkworms The silk glands of transgenic silkworms on day 2 of the fifth instar were dissected and placed onto glass slides to determine EGFP expression using a fluorescence stereomicroscope [Olympus MacroViewMVX10-AUTO (Olympus, Tokyo, Japan) equipped with a GFP filter]. Cocoons from transgenic silkworms were selected at random and photographed using the same microscope.
Histological fluorescence detection of expression of EGFP in transgenic silkworms The fresh silk glands from transgenic silkworms on day 6 of the fifth instar were washed and then fixed in Bouin fixative [0.9% (v/v) picric acid, 5% (v/v) acetic acid and 9% (v/v) formaldehyde] for 24 h at room temperature. After washing with PBS, the silk glands were dehydrated by passage through a graded series of ethanol, added to a 1 : 1 (v/v) mixture of dimethylbenzene and ethanol for 1 h and then into dimethylbenzene for 20 min before embedding in paraffin wax and were cut into 5-lm-thick slices, which were transformed to gelatin-coated slides and dried at 40 °C overnight. Subsequently, sections were observed under a light microscope (Olympus BX51TRF). Transgenic silk fibers were collected and twisted into a narrow bundle, and then embedded directly into paraffin wax and cut into 7lm-thick slices before observation under a laser scanning confocal microscope (Leica TCS-SP2; Leica, Mannheim, Germany).
Genomic PCR The primer pair EGFP-MF/poly(A)-MR (Table S1) was used to confirm transgenic individuals. Genomic DNA extracted from transgenic and wild-type strain 871 adults was used as a template for PCR. The purified PCR fragments were cloned and sequenced.
RT-PCR Total RNA was extracted from the PSG and the fat body of day 4 of the fifth instar using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as described previously [15]. cDNA was synthesized using the Perfect Real Time version of the PrimerScriptTM RT reagent kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China) according to the manufacturer’s instructions. PCR amplifications were done using the primer pairs FibH-50 UTR-F/poly(A)-MR, EGFP-F/EGFP-R and BmActin3-f/BmActin3-r (Table S1). Amplification of the BmActin A3 (AAC47446) served as an internal control.
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
SDS/PAGE and western blotting About 25 mg of each transgenic silkworm cocoon shell was dissolved in 1 mL of 60% (w/v) lithium thiocyanate by vortex-mixing for 10 min, incubation at room temperature for 1 h followed by centrifugation at 10 000 g in a microcentrifuge for 5 min. Then, 4-lL samples were subjected to SDS/PAGE [10% (w/v) polyacrylamide slab gel] by dissolving in an equal volume of sample loading buffer [5% (v/v) glycerol, 1% (w/v) SDS, 0.05% (w/v) bromophenol blue, 0.0625 M Tris/HCl, pH 6.8] without or with 2% (v/v) 2-ME and boiled for 5 min. After electrophoresis, the gel was stained with 0.1% (w/v) Coomassie Brilliant Blue R250, 10% (v/v) acetic acid, 50% (v/v) methanol. About 2 lL of each sample was diluted five-fold with 10 mM Tris/ HCl (pH 6.8) and then 2 lL of the diluted sample was dissolved in sample loading buffer without or with 2% 2-ME and subjected to SDS/PAGE. The gel was transferred directly onto a polyvinylidene difluoride membrane (Roche, Mannheim, Germany), rinsed briefly and blocked for 1 h at room temperature with TBST (0.136 M NaCl, 20 mM Tris/HCl pH 7.6, 0.1% (w/v) Tween 20) containing 5% (v/ v) skim milk. After rinsing twice and washing three times with TBST, the membrane was incubated at room temperature for 1 h in TBST containing 2500-fold diluted GFP antibody (Beyotime, Jiangsu, China). The membrane was rinsed twice and washed three times (1 9 15 min and 2 9 5 min) with TBST, and then incubated at room temperature for 1 h in TBST containing 10 000-fold diluted horseradish-peroxidase-labeled rabbit IgG secondary antibody (Beyotime). After rinsing and washing with TBST, the antibody was detected with ECL plus Western Blotting Detection Reagents according to the manufacturer’s instructions (Beyotime). The signals were obtained by a chemiluminescence imaging system (Clinx ChemiScope series, Shanghai, China).
Acknowledgements This work was supported by grants from the Hi-Tech Research and Development (863) Program of China (No. 2013AA102507) and the China Agriculture Research System (No. CARS-22-ZJ0102).
Author contributions A.Z. and Z.X. planned the experiments; D.L., W.L. and Y.Z. performed the experiments; D.L. and Q.G. analyzed the data; D.L. and A.Z. wrote the paper.
References 1 Vepari C & Kaplan DL (2007) Silk as a biomaterial. Prog Polym Sci 32, 991–1007.
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
The mechanism of fibroin secretion in silkworm
2 Omenetto FG & Kaplan DL (2010) New opportunities for an ancient material. Science 329, 528–531. 3 Grzelak K (1995) Control of expression of silk protein genes. Comp Biochem Physiol B Biochem Mol Biol 110, 671–681. 4 Inoue S, Tanaka K, Arisaka F, Kimura S, Ohtomo K & Mizuno S (2000) Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6: 6: 1 molar ratio. J Biol Chem 275, 40517–40528. 5 Inoue S, Tanaka K, Tanaka H, Ohtomo K, Kanda T, Imamura M, Quan GX, Kojima K, Yamashita T & Nakajima T (2004) Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L-chain for protection of a1, 2-mannose residues in Nlinked oligosaccharide chains of fibrohexamerin/P25. Eur J Biochem 271, 356–366. 6 Zhou C, Confalonieri F, Medina N, Zivanovic Y, Esnault C, Yang T, Jacquet M, Janin J, Duguet M & Perasso R (2000) Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res 28, 2413– 2419. 7 Zhou C, Confalonieri F, Jacquet M, Perasso R, Li ZG & Janin J (2001) Silk fibroin: structural implications of a remarkable amino acid sequence. Proteins 44, 119–122. 8 Wang SP, Guo TQ, Guo XY, Huang JT & Lu CD (2006) In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector. Biochem Biophys Res Commun 341, 1203–1210. 9 Jin HJ & Kaplan DL (2003) Mechanism of silk processing in insects and spiders. Nature 424, 1057– 1061. 10 Mita K, Ichimura S & James TC (1994) Highly repetitive structure and its organization of the silk fibroin gene. J Mol Evol 38, 583–592. 11 Tanaka K, Kajiyama N, Ishikura K, Waga S, Kikuchi A, Ohtomo K, Takagi T & Mizuno S (1999) Determination of the site of disulfide linkage between heavy and light chains of silk fibroin produced by Bombyx mori. Biochim Biophys Acta 1432, 92–103. 12 Takei F, Oyama F, Kimura K, Hyodo A, Mizuno S & Shimura K (1984) Reduced level of secretion and absence of subunit combination for the fibroin synthesized by a mutant silkworm, Nd (2). J Cell Biol 99, 2005–2010. 13 Takei F, Kikuchi Y, Kikuchi A, Mizuno S & Shimura K (1987) Further evidence for importance of the subunit combination of silk fibroin in its efficient secretion from the posterior silk gland cells. J Cell Biol 105, 175–180. 14 Mori K, Tanaka K, Kikuchi Y, Waga M, Waga S & Mizuno S (1995) Production of a chimeric fibroin lightchain polypeptide in a fibroin secretion-deficient naked
99
D. Long et al.
The mechanism of fibroin secretion in silkworm
15
16
17
18
19
20
21
22
23
24
25
100
pupa mutant of the silkworm Bombyx mori. J Mol Biol 251, 217–228. Zhao A, Zhao T, Zhang Y, Xia Q, Lu C, Zhou Z, Xiang Z & Nakagaki M (2010) New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm. Transgenic Res 19, 29–44. Tamura T, Thibert C, Royer C, Kanda T, Eappen A, Kamba M, K^ omoto N, Thomas J-L, Mauchamp B & Chavancy G (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposonderived vector. Nat Biotechnol 18, 81–84. Kojima K, Kuwana Y, Sezutsu H, Kobayashi I, Uchino K, Tamura T & Tamada Y (2007) A new method for the modification of fibroin heavy chain protein in the transgenic silkworm. Biosci Biotechnol Biochem 71, 2943–2951. Fedic R, Zurovec M & Sehnal F (2003) Correlation between fibroin amino acid sequence and physical silk properties. J Biol Chem 278, 35255–35264. Tanaka K, Inoue S & Mizuno S (1999) Hydrophobic interaction of P25, containing Asn-linked oligosaccharide chains, with the H-L complex of silk fibroin produced by Bombyx mori. Insect Biochem Mol Biol 29, 269–276. Teule F, Miao Y, Sohn B, Kim Y, Hull JJ, Fraser MJ, Lewis RV & Jarvis DL (2012) Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties. Proc Natl Acad Sci USA 109, 923–928. Iizuka T, Sezutsu H, Tatematsu KI, Kobayashi I, Yonemura N, Uchino K, Nakajima K, Kojima K, Takabayashi C & Machii H (2013) Colored fluorescent silk made by transgenic silkworms. Adv Funct Mater 23, 5232–5239. Takano E, Maki M, Mori H, Hatanaka M, Marti T, Titani K, Kannagi R, Ooi T & Murachi T (1988) Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis. Biochemistry 27, 1964–1972. Michal CA, Simmons AH, Chew B, Zax DB & Jelinski LW (1996) Presence of phosphorus in Nephila clavipes dragline silk. Biophys J 70, 489–493. Zhang P, Aso Y, Yamamoto K, Banno Y, Wang Y, Tsuchida K, Kawaguchi Y & Fujii H (2006) Proteome analysis of silk gland proteins from the silkworm, Bombyx mori. Proteomics 6, 2586–2599. Inoue S, Kanda T, Imamura M, Quan G, Kojima K, Tanaka H, Tomita M, Hino R, Yoshizato K & Mizuno S (2005) A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins. Insect Biochem Mol Biol 35, 51– 59.
26 Shimura K (1983) Chemical composition and biosynthesis of silk proteins. Cell Mol Life Sci 39, 455– 461. 27 Kurihara H, Sezutsu H, Tamura T & Yamada K (2007) Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system. Biochem Biophys Res Commun 355, 976–980. 28 Shimizu K, Ogawa S, Hino R, Adachi T, Tomita M & Yoshizato K (2007) Structure and function of 50 flanking regions of Bombyx mori fibroin heavy chain gene: Identification of a novel transcription enhancing element with a homeodomain protein-binding motif. Insect Biochem Mol Biol 37, 713–725. 29 Zhu Z, Kikuchi Y, Kojima K, Tamura T, Kuwabara N, Nakamura T & Asakura T (2010) Mechanical properties of regenerated Bombyx mori silk fibers and recombinant silk fibers produced by transgenic silkworms. J Biomater Sci Polym Ed 21, 395–411. 30 Nagano A, Tanioka Y, Sakurai N, Sezutsu H, Kuboyama N, Kiba H, Tanimoto Y, Nishiyama N & Asakura T (2011) Regeneration of the femoral epicondyle on calcium-binding silk scaffolds developed using transgenic silk fibroin produced by transgenic silkworm. Acta Biomater 7, 1192–1201. 31 Tatemastu K, Sezutsu H & Tamura T (2012) Utilization of transgenic silkworms for recombinant protein production. J Biotechnol Biomaterial S9, 1–8. 32 Walsh CT, Garneau-Tsodikova S & Gatto GJ Jr (2005) Protein posttranslational modifications: the chemistry of proteome diversifications. Angew Chem Int Ed Engl 44, 7342–7372. 33 Zhao A, Long D, Ma S, Xu L, Zhang M, Dai F, Xia Q, Lu C & Xiang Z (2012) Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains. Insect Sci 19, 172–182. 34 Long D, Zhao A, Chen X, Zhang Y, Lu W, Guo Q, Handler AM & Xiang Z (2012) FLP recombinasemediated site-specific recombination in silkworm, Bombyx mori. PLoS One 7, e40150. 35 Long D, Zhao A, Xu L, Lu W, Guo Q, Zhang Y & Xiang Z (2013) In vivo site-specific integration of transgene in silkworm via phiC31 integrase-mediated cassette exchange. Insect Biochem Mol Biol 43, 997– 1008. 36 Horn C & Wimmer EA (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210, 630–637.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site:
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
Fig. S1. Amino acid sequence alignment between three different EGFP/H-chain fusion proteins, TSL-FP, TSP-FP and R1-FP. Table S1. Summary of primers used in the present study.
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
The mechanism of fibroin secretion in silkworm
Table S2. Injection of piggyBac-derived vectors in G0 silkworm embryos of the strain 871. Table S3. Identification of genomic insertion sites of piggyBac vectors by inverse PCR.
101
Keywords EGFP/H-chain fusion protein; fibroin secretion mechanism; piggyBac; silk; transgenic silkworm Correspondence A. Zhao, State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of the Agricultural Ministry, Southwest University, BeiBei, Chongqing 400716, China Fax: +86 23 68251128 Tel: +86 23 68251803 E-mails: [email protected]; [email protected] (Received 4 June 2014, revised 21 August 2014, accepted 6 October 2014)
In order to investigate the role of different parts of the fibroin heavy chain (H-chain) in the secretion of fibroin in the silk gland of the silkworm (Bombyx mori) in vivo, two enhanced green fluorescent protein (EGFP)/Hchain fusion genes with deduced protein sequences containing an identical N-terminal region and different C-terminal regions of the H-chain were introduced into the B. mori genome using a piggyBac-mediated germline transformation. EGFP fluorescence and molecular analysis showed the products of two different EGFP/H-chain fusion proteins were secreted into the posterior silk gland lumen and aggregated in the middle silk gland and spun into cocoons. The results revealed that only the non-repetitive N terminus of the H-chain is essential for secretion of the H-chain into the posterior silk gland lumen. In addition, our results also indicated that the most likely post-translational modification of the H-chain is at the C-terminal domain. Here, our results not only provide a theoretical basis for the genetic modification of silk fiber as a functional biomaterial but also are of great significance to establishing a new silk gland bioreactor to mass-produce exogenous proteins in an active form.
doi:10.1111/febs.13105
Introduction Silk produced by the silkworm Bombyx mori is a macromolecular protein well known in the textile industry for its luster and mechanical properties. Silk is a very good model material for examination of protein structure, function and mechanisms of secretion and it has been studied in attempts to understand the processing mechanisms and to exploit the properties of the protein for use as biomaterials [1]. As a high-performance biomaterial, silk produced by B. mori can be used in the textile industry and in the fields of biotechnology, biomedicine and the chemical industry [2].
The silkworm synthesizes abundant silk proteins and spins them into silk threads, which are used to build a cocoon shell. The major components of cocoon silk in the normal silkworm are fibroin and sericin, which represent approximately 75% (w/w) and 25% (w/w) of a cocoon, respectively [3]. Fibroin is synthesized in the posterior silk gland (PSG) and accumulates in the middle silk gland (MSG) lumen, where sericin is synthesized. Fibroin, which is secreted via the anterior silk gland (ASG), contains three different proteins: fibroin heavy chain (H-chain), fibroin light chain (L-chain)
Abbreviations ASG, anterior silk gland; CTD, C-terminal domain; EGFP, enhanced green fluorescent protein; fhx/P25, fibrohexamarin; H-chain, fibroin heavy chain; LBS, L-chain binding site; L-chain, fibroin light chain; 2-ME, b-mercaptoethanol; MSG, middle silk gland; NTD, N-terminal domain; poly (A), polyadenylation signal sequence of the H-chain gene; PSG, posterior silk gland; PTM, post-translational modification; TSL, transgenic strain containing the EGFP/H-chain fusion gene with LBS sequence; TSP, transgenic strain containing the EGFP/H-chain fusion gene with poly(A) sequence; TSL-FP, the EGFP/H-chain fusion proteins derived from TSL silkworms; TSP-FP, the EGFP/H-chain fusion proteins derived from TSP silkworms.
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
89
D. Long et al.
The mechanism of fibroin secretion in silkworm
and fibrohexamarin (fhx), named P25 [4]. In the PSG, these proteins form a large protein complex designated the elementary unit of fibroin, which consists of six sets of H-chainL-chain (H-L) disulfide-linked heterodimers and one molecule of the glycoprotein fhx/P25 [4], which interacts non-covalently with six sets of H-L heterodimers and forms the elementary unit in the endoplasmic reticulum and is then secreted into the Golgi apparatus [4,5]. The H-chain, which can form up to 92% (w/w) of the elementary unit of fibroin, is the major component (w/w) of the elementary unit. The H-chain consists of three domains: an N-terminal domain (NTD), a repeated region and a C-terminal domain (CTD) [6,7]. The NTD is considered to work as a signal peptide [8] and to contribute to the solubility of the H-chain [9]. The repeated region consists of a hydrophobic crystalline region (GAGAGS/Y)n and a hydrophilic noncrystalline region [6,7] which is the main component of the H-chain and contributes to the physical properties of fibroin [10]. The CTD contains three cysteine residues that form intramolecular and intermolecular disulfide bonds; a single disulfide bond is formed between Cys172 of the L-chain and Cys-c20 (the twentieth residue from the CTD) of the H-chain [11]. The H-L heterodimer is suggested to be essential for efficient secretion of fibroin from the PSG cells on the basis of the results of studies on the B. mori fibroin secretion-deficient naked pupa mutants Nd-s and Nd-sD [12–14]. Earlier, we constructed and introduced three enhanced green fluorescent protein (EGFP)/Hchain fusion genes with different 50 -flanking sequences and identical 30 -flanking sequences into the normal B. mori strain N4 to assess the ability to produce recombinant protein. The results suggested that the NTD of the H-chain was important for the secretion of the Hchain into the PSG lumen [15]. The roles of three regions of the H-chain in the secretion of fibroin are not entirely clear, however, especially whether the CTD is essential for the efficient secretion of fibroin from PSG cells. In this study, we constructed two EGFP/H-chain fusion genes with identical 50 -flanking sequence and different 30 -flanking sequences. These EGFP/H-chain fusion genes were introduced into the silkworm genome using a piggyBac-mediated silkworm germline transformation [16]. Two different EGFP/H-chain fusion genes were expressed in the PSG of transgenic silkworms, the products of recombinant proteins were secreted into the PSG lumen, aggregated in the MSG and spun into cocoons. In accord with our earlier studies, we confirmed that only the NTD of the Hchain is essential for the secretion of the H-chain into the PSG lumen. Here, we provide new insight into the mechanism underlying fibroin secretion; our results 90
suggest that the PSG cells of transgenic silkworms can secrete the elementary unit of fibroin into the PSG lumen and can direct secretion of the H-chain fusion protein into the PSG lumen and spin into cocoons without forming an H-L heterodimer.
Results Histological distribution of EGFP in the silk glands of transgenic silkworms Figure 1 shows the structures of two different ecombinant piggyBac transformation vectors pBac {3 9 P3-DsRed}-R3 and pBac{3 9 P3-DsRed; Fib-H P3-EGFP-poly(A)}. Finally, one G1 transgenic strain containing an EGFP/H-chain fusion gene with the L-chain binding site (LBS) sequence was produced and designated TSL, and four G1 transgenic strains containing an EGFP/H-chain fusion gene with the polyadenylation signal sequence of the H-chain gene (poly (A)) were produced (TSPs) and designated TSP-1–TSP4 as described in Materials and methods. EGFP fluorescence was detected in TSL and TSP individuals. Silk glands dissected from day 2 of the fifth instar were observed using a fluorescence stereomicroscope as described in Materials and methods. The PSGs and MSGs of TSL and TSP silkworms on day 2 of the fifth instar exhibited strong EGFP fluorescence (Fig. 2A). We observed the trend of EGFP secreted from PSG to MSG in the silk glands of TSL and TSP silkworms, and there was no difference between TSL and TSP individuals (Fig. 2A, e,f). In addition, PSGs and MSGs of TSL and TSP strains were sectioned and viewed under a light microscope (Fig. 3A). Cross-sections of PSGs and MSGs from TSL and TSP silkworms showed that both EGFPs were expressed and the expressed product was secreted effectively into the PSG lumen and the fibroin layers of MSGs but not the sericin layers of MSGs (Fig. 3A, e–h). Moreover, EGFP fluorescence was not detected in other tissues of TSL and TSP individuals, suggesting that the fibroin H-chain promoter sequence determined the tissue-selective expression of these EGFP/H-chain fusion genes very efficiently [15,17]. Distribution of EGFP in the cocoon silks of transgenic silkworms Cocoons from different transgenic silkworms were observed under a fluorescence stereomicroscope (Fig. 2B). The cocoons from the TSL and TSP silkworms displayed strong green fluorescence (Fig. 2B, e, f), indicating that a large amount of recombinant EGFP was synthesized in the silk glands and spun into FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
The mechanism of fibroin secretion in silkworm
A
B
Fig. 1. Construction of piggyBac-derived vectors and different EGFP/H-chain fusion genes. (A) Structures of the transformation vector pBac {3 9 P3-DsRed}-R3 and EGFP/H-chain fusion gene including the LBS. (B) Structures of the transformation vector pBac{3 9 P3-DsRed; FibH P3-EGFP-poly(A)} and EGFP/H-chain fusion gene including only the polyadenylation sequence. pBacL, left arm of piggyBac transposon; pBacR, right arm of piggyBac transposon; 3 9 P3, 3 9 P3 promoter; DsRed, red fluorescence protein gene (red box); SV40, SV40 polyadenylation signal sequence. The numbers of nucleotide sequences of fibroin H-chain gene in the parentheses are consistent with the position of the sequences from Accession No. AF226688. The different DNA fragments are indicated by an abbreviated form: 50 -flanking, 50 -flanking sequence of the H-chain gene; E1, exon1 of the H-chain gene; Intron, intron 1 of the H-chain gene; 50 -E2, 50 -terminal sequence of exon2 of the H-chain gene; EGFP, enhanced green fluorescence protein gene (green box); 30 -E2, 30 -terminal sequence of exon2 of the H-chain gene; poly(A), polyadenylation signal sequence of the H-chain gene.
cocoons. In addition, paraffin sections of silk from the cocoons of TSL and TSP silkworms were viewed under a laser scanning confocal microscope (Fig. 3B). The results showed that EGFP was located in the fibroin layer, but not the sericin layer, of cocoon silk from TSL and TSP silkworms (Fig. 3B, b,d). Analysis of EGFP/H-chain fusion transcripts in the PSGs of transgenic silkworms To determine the different constructs of EGFP/Hchain fusion genes in TSL and TSP individuals, PCR was done on genomic DNAs from TSL, TSP and wild-type strain 871 adults with the primer pair EGFP-MF/poly(A)-MR (Fig. 4A). As shown in Fig. 4B, the PCR products were a 306 bp DNA fragment for the TSL individuals, a 134 bp DNA fragment for TSP-1–TSP-4 individuals and no amplified frag-
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
ment for wild-type strain 871 silkworm control, which is consistent with the putative pattern. RT-PCR was performed as described in Materials and methods to further confirm the expression of these EGFP/H-chain fusion genes in the PSGs of TSL and TSP silkworms. As shown in Fig. 4C, the expected 1418 bp EGFP/H-chain fusion gene and 717 bp EGFP gene PCR products were observed for cDNA from transcripts in the PSG of TSL individuals, and the expected 1246 bp EGFP/H-chain fusion gene and 717 bp EGFP gene PCR products were observed for cDNA from transcripts in the PSG of TSP-1–TSP-4 individuals by using the primer pair FibH-50 UTR-F/poly(A)-MR. The cDNA from transcripts in the PSG of wild-type 871 strain and fat body of TSP-1 were used as controls. All PCR products were sequenced and, as expected, no structural change was detected (data not shown). These results
91
D. Long et al.
The mechanism of fibroin secretion in silkworm
A a
b
c
d
e
f
a
b
c
d
e
f
B
Fig. 2. Distribution of EGFP in the silk glands and cocoons from transgenic silkworms. (A) Silk glands were dissected from one individual of wild-type strain 871 (a, d), TSL (b, e) and TSP-1 (c, f) of 2-day-old fifth instar larvae, and were viewed under white light (a–c) and GFP fluorescence (d–f). (B) Cocoons produced by one individual of wild-type 871 (a, d), TSL (b, e) and TSP-1 (c, f) were observed under white light (a–c) and GFP fluorescence (d–f). White scale bar represents 5 mm.
confirmed that the different EGFP/H-chain fusion transcripts of EGFP/H-chain fusion genes were synthesized specifically in the cells from PSGs of TSL and TSP silkworms. Analysis of EGFP/H-chain fusion proteins in the cocoon silk from transgenic silkworms The cocoon silk proteins from TSL, TSP-1 and wild-type 871 silkworms were dissolved in 60% (w/v) 92
lithium thiocyanate without or with b-mercaptoethanol (2-ME) and separated by SDS/PAGE to further investigate whether the different EGFP/H-chain fusion proteins were linked with the L-chain via a disulfide bond in the cocoon silk from TSL and TSP silkworms. The same diluted samples were subjected to western blotting with anti-GFP antibody (Fig. 5). The results of SDS/PAGE suggested that the EGFP/H-chain fusion proteins derived from TSL silkworms (TSL-FP) were produced as a TSL-FP FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
The mechanism of fibroin secretion in silkworm
A a
b
c
d
e
f
g
h
b
c
d
B a
Fig. 3. Distribution of EGFP in PSGs, MSGs and cocoon silks of transgenic silkworms. (A) Paraffin sections were prepared from PSGs (a, b, e, f) and MSGs (c, d, g, h) of the TSL (a, c, e, g) and TSP-1 (b, d, f, h) silkworms, and observed under white light (a–d) and GFP fluorescence (e–h) using a fluorescence microscope. White scale bar represents 25 lm. (B) Paraffin sections were prepared from cocoon silks of the TSL (a, b) and TSP-1 (c, d), and observed under white light (a, c) and GFP fluorescence (b, d) using a laser scanning confocal microscope. Scale bar represents 10 lm.
and L-chain disulfide-linked (TSL-FP-L) heterodimer of 82 kDa without treatment with 2-ME, and a single TSL-FP of 57 kDa with 2-ME treatment, which could break the disulfide bond between TSL-FP and the L-chain (Fig. 5A). The EGFP/H-chain fusion proteins derived from TSP-1 silkworms (named TSPFP) were produced as a single TSP-FP of 43 kDa whether without or with 2-ME treatment (Fig. 5A). Western blotting with an anti-GFP antibody showed these bands at the same position and size as SDS/ PAGE in the TSL and TSP-1 samples (Fig. 5B). The results confirmed that TSL-FP was linked with the L-chainvia a disulfide bond in the fibroin of cocoon silks from TSL silkworms, but TSP-FP was
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
secreted directly into the fibroin of cocoon silk from TSP silkworms without forming an H-L heterodimer.
Discussion The essential H-chain domain for the secretion of fibroin Silk has long attracted attention as a biodegradable fiber of considerable strength, elasticity and durability. The properties of B. mori silk fiber depend on the amino acid repeats that interact during fibroin H-chain polymerization [18]; however, the true mechanism
93
D. Long et al.
The mechanism of fibroin secretion in silkworm
A
B
C
Fig. 4. Analysis of EGFP/H-chain fusion transcripts in the PSGs of transgenic silkworms. (A) Schematic maps of the transgene constructs in genomes of TSL (top) and TSP (bottom) individuals. The EGFP-MF/poly(A)-MR primer pair was used for PCR analysis of genomic DNAs from TSL and TSP individuals. FibH-50 UTR-F/poly(A)-MR and EGFP-F/EGFP-R primer pairs were used for RT-PCR analysis of cDNA from EGFP/H-chain fusion transcripts in the PSGs of TSL and TSP individuals. (B) PCR analysis of genomic DNA using primer pair EGFP-MF/poly (A)-MR. The expected 306 bp PCR products were observed for TSL, and the expected 134 bp PCR products were observed for TSP-1–TSP4. WT, wild-type 871 strain was used as a control; lane M, trans2K Plus DNA marker. (C) RT-PCR confirmation of EGFP/H-chain fusion gene expression in the PSGs. The expected 1418 bp PCR products were observed for TSL, and the expected 1246 bp PCR products were observed for TSP-1–TSP-4 by using primer pair FibH-50 UTR-F/poly(A)-MR (top). The expected 717 bp EGFP gene PCR products were observed for TSL and TSP-1–TSP-4 by using primer pair EGFP-F/EGFP-R (bottom). The cDNA from transcripts in the PSG of wild-type 871 strain (WT) and fat body (FB) of TSP-1 was used as control. The amplification of BmActin A3 using primer pair BmActin3-f/BmActin3-r served as an internal control.
underlying the secretion of silk fibroin is not entirely clear. In an earlier study, we demonstrated that the NTD of the H-chain was important in the secretion of fibroin, and the signal peptide from amino acid residues 1–21 of the NTD, but not the repeated region of the H-chain, was essential for the secretion of the H-chain into the PSG lumen [15]. In this experiment, the EGFP/H-chain fusion protein TSL-FP (named R2R3-FP in our earlier work 94
[15]) lacks the repeated region of the H-chain, and fusion protein TSP-FP lacks the repeated region and CTD of the H-chain (Fig. 6), but TSL-FP and TSPFP were both secreted into the PSG lumen, aggregated in the MSG and spun into cocoons. By analyzing the secretion of TSL-FP and TSP-FP in the PSGs of transgenic silkworms, the present study demonstrated the repeated region and CTD of the H-chain were not necessary for secretion of the H-chain into the PSG FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
A
The mechanism of fibroin secretion in silkworm
B
Fig. 5. SDS/PAGE and western blotting analysis of transgenic silkworm cocoon silk proteins using anti-GFP antibody. (A) Cocoon silk protein samples from wild-type 871, TSL and TSP-1 silkworms were subjected to SDS/10% PAGE without or with b-galactosidase (2-ME) treatment. (B) Western blotting of proteins in the gel as in (A) with the anti-GFP antibody. M, molecular mass markers. Sizes are indicated on the left of the panels. Triangles indicate TSL-FP-L heterodimer, asterisks indicate TSL-FP, and arrowheads indicate TSP-FP. and + indicate the protein samples without or with 2-ME treatment, respectively.
Fig. 6. Domain structure of the normal Hchain and three different EGFP/H-chain fusion proteins, TSL-FP, TSP-FP and R1FP. The repeated region of the normal Hchain comprises 12 hydrophobic crystalline domains separated by 11 hydrophilic noncrystalline domains [6,7]. The TSL-FP is also named R2R3-FP in our earlier work [15].
lumen. In accord with our earlier studies, we confirmed that only the NTD of the H-chain was essential for the secretion of the H-chain into the PSG lumen. Different functions for the H-chain domains in the assembly and secretion of fibroin Some earlier studies found the repeated region of the normal H-chain comprises 12 low-complexity hydrophobic crystalline domains separated by 11 hydrophilic noncrystalline domains (Fig. 6) [6,7,9]. Twelve hydrophobic crystalline domains composed of Gly-X repeats account for 94% of the H-chain; X is Ala in 65%, Ser in 23% and Tyr in 10% of the repeats [6,7]. The main function of the repeated region was suggested to be a
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
contribution to maintenance of the physical properties of fibroin [9,10], but not for the secretion of fibroin, which was confirmed by the results of this study. On the basis of what was discussed above, we suggest the most important function of the NTD of the H-chain is that it contains a signal peptide sequence (21 amino acids long) essential for the secretion of fibroin (Fig. S1). The CTD of the H-chain has a cysteine residue (Cys-c20) that links with the L-chain (Cys172 in the mature L-chain) via a disulfide bond and the other two cysteine residues (Cys-c4 and Cysc1) form an intramolecular disulfide bond [11,19]. The H-L heterodimers and N-glycosylated fhx/P25 assemble into an elementary unit (6 : 6 : 1 molar ratio of H-chain/L-chain/fhx/P25) through hydrophobic inter-
95
D. Long et al.
The mechanism of fibroin secretion in silkworm
actions between fhx/P25 and H-chains and by hydrogen bonding between N-linked oligosaccharide chains of fhx/P25 and H-chains [4]. Some earlier studies suggested that the H-L disulfide bond was critical for secretion of the fibroin molecule into the cell lumen on the basis of the evidence that L-chain-deficient mutant silkworms (Nd-s and Nd-sD mutants) are unable to secrete silk fiber [12,13], and it has been reported that the mutant chimeric Nd-sD L-chain lacks Cys172, which was encoded by the original sixth exon and consequently could not form a disulfide bond with the H-chain [14]. In this study, the TSP-FP lacks the CTD (and Cys-c20) in the PSG cells of TSP silkworms, so the TSP-FP could not link with the L-chain via a disulfide bond forming an H-L heterodimer in the PSG cells of TSP silkworms; however, we observed secretion of the TSP-FP into the lumen of the PSG cells of TSP silkworms. This result revealed that formation of the H-L heterodimer was not essential for secretion of the H-chain into the PSG lumen. We suggested that the CTD of the H-chain was necessary only for forming the H-L heterodimer in the PSG cells. In this study, the molecular mass of the TSL-FP and TSL-FP-L heterodimers was estimated by SDS/ PAGE to be ~ 57 kDa and ~ 82 kDa, respectively, which is 6.6 kDa larger than the predicted molecular mass of the TSL-FP (49.4 kDa) and TSL-FP-L heterodimers (75.4 kDa). This might be owing to the abnormal mobility of the H-chain fusion proteins in SDS/ PAGE or the presence of post-translational modifications (PTMs), such as glycosylation or phosphorylation, as reported in our earlier study and elsewhere [15,17,20,21]. In general, the presence of a cluster of basic amino acid residues causes a delay in protein migration during SDS/PAGE [22]. An earlier report suggested there were basic amino acids (R, K and H) in the cluster at the CTD of the H-chain fusion protein (Fig. S1), and this might have been the cause of the molecular mass difference determined by SDS/ PAGE [17]. However, the possibility of PTMs (phosphorylation or glycosylation) cannot be excluded. Phosphorylation of fiber proteins has been reported in spider silk [23] and in the L-chain and fhx/P25 of silkworms [24] and glycosylation of fibroin proteins has been reported in fhx/P25 [11,19], and we reported the presence of potential PTMs on the H-chain [15]. The identity and function of these PTMs on the H-chain are unclear but there is the possibility of PTMs on the normal H-chain, TSL-FP and TSP-FP, since the NTD and CTD of the normal H-chain, TSL-FP and TSPFP, contain many amino acid residue sites that could be phosphorylated and glycosylated (Fig. S1). Actu96
ally, we observed that the molecular mass of TSP-FP without the CTD of the H-chain was estimated to be about 43 kDa by SDS/PAGE, which was consistent with the predicted molecular mass of TSP-FP (43.6 kDa), so we speculated that the likeliest cause of the abnormal migration is the presence of a C-terminal basic amino acid cluster and the most likely PTM of the H-chain is also at the CTD. New insights into the fibroin secretion mechanism in the PSG cells of B. mori Inoue et al. reported the use of the piggyBac transposon for germline transformation of the Nd-sD mutant with a normal L-chain-GFP fusion gene to construct a transgenic mutant [25]. Normal development of the PSG in the transgenic mutant was restored and it formed a normal cocoon. Inoue et al. had confirmed that the molar ratio of the H-chain/L-chain-GFP fusion protein/fhx/P25 in the secreted fibroin of the transgenic mutant was strictly 6 : 6 : 1 [25]. Combined with the present study, we propose two new insights about the secretion of normal H-chain and H-chain fusion protein in the PSG cells of the silkworm: (a) H-L linkage could contribute to the maintenance of the elementary unit but the formation of the H-L heterodimer is not essential for secretion of the H-chain into the PSG lumen; (b) the H-chain and the H-chain fusion protein could be secreted directly into the PSG lumen and spun into cocoons without forming the HL heterodimer, especially for the small molecules and hydrophilic H-chain fusion proteins. Most of the H-chains were secreted into the silkworm PSG in the form of the elementary unit of fibroin [4]. Because the normal H-chain has a high molecular mass (~ 350 kDa) [26] and contains 12 hydrophobic crystalline domains of high molecular mass which account for 94% of the H-chain [6,7], the form of the elementary unit could contribute to secretion of the H-chains. When the normal H-chain or H-chain fusion protein (such as the TSL-FP) containing the CTD of the H-chain is first linked with the normal L-chain (containing Cys172) to form the H-L heterodimer, six H-L heterodimers are linked with one molecule of fhx/P25 to form the elementary unit for the secretion of fibroin [4]. It has been reported that fhx/P25 interacts with the H-chain in the absence of H-L linkage but its content of oligosaccharides was reduced when the H-L linkage was not formed [19], so H-L linkage could contribute to maintenance of the elementary unit. The disulfide bond between the L-chain and Cysc20 of the H-chain does not appear to be essential for secretion of the H-chains, however; actually, a FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
small amount of the L-chain-free H-chain (< 1% compared to the normal level fibroin-producing silkworms) could be secreted from the naked pupa mutants [4]. In addition, the present study demonstrated that TSP-FP from TSP silkworms not containing the CTD of the H-chain could be secreted into the PSG lumen and spun into cocoons. These results strongly suggested that direct secretion of the H-chain or H-chain fusion proteins without forming the H-L heterodimer into the PSG lumen of the silkworms was a pathway for secretion of the H-chain into PSG cells. In this study, we used a small molecule and the hydrophilic EGFP domain to replace these hydrophobic crystalline domains of the normal H-chain in two EGFP/H-chain fusion proteins (Fig. 6). Because TSL-FP could be linked preferentially with the L-chain through a disulfide bond, the TSL-FP-L heterodimer further assembled with fhx/ P25 to form the elementary unit for secretion of large amounts of the TSL-FP, the same as secretion of the normal H-chain. On the other hand, TSP-FP could not be linked to the L-chain to form the H-L heterodimer and it could be secreted into the PSG lumen directly; understanding the real reasons requires further study. Our results showed that TSLFP and TSP-FP were expressed highly efficiently in the silk glands and cocoons of TSL and TSP silkworms using fluorescence detection; further, there was no obvious difference in fluorescence intensity between the two types of cocoons from TSL and TSP silkworms, respectively. Advantages and prospects of the new fibroin H-chain expression system Since Kurihara et al. [27] first reported using the Hchain gene for the production of an active feline interferon in the cocoon of transgenic silkworms, the fibroin H-chain expression system has been used widely to produce exogenous proteins in the silk gland and cocoon of transgenic silkworms [15,17,21,28–30]. Earlier studies suggested production of the recombinant proteins mediated by the fibroin H-chain expression system required both the NTD and CTD of the H-chain [31]. However, this study revealed that production of the TSP-FP using the new fibroin H-chain expression system required only the NTD or a signal peptide sequence of the H-chain, which would make the construction of this system simpler compared to using the earlier system containing both the NTD and CTD of the H-chain. In addition, the expression efficiency of the H-chain fusion protein mediated by this new system was comparable to the earlier system. FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
The mechanism of fibroin secretion in silkworm
More importantly, PTMs of recombinant proteins often have a critical effect on the function of the exogenous target proteins [32]. Because both the NTD and CTD of the H-chain contained potential N-glycosylation and generic phosphorylation sites (Fig. S1), and the H-chain fusion proteins (such as the TSL-FP) containing the CTD of the H-chain could be linked to the normal L-chain, the activity of the target proteins might be affected by the possible PTMs of the NTD and CTD of the H-chain fusion proteins and the formation of the H-L heterodimer. Kurihara et al. [27] reported that the NTD and CTD of the H-chain in the fusion protein reduced the biological activity of the target protein from transgenic silkworms. Our new fibroin H-chain expression system has only a short NTD with an essential signal peptide for the encoded fusion proteins. Therefore, this new system would give a greater chance to express the targeted protein in an active form, which would enable production of protein drugs or novel biomaterials on the basis of transgenic silk. In this study, we confirmed that only the non-repetitive N terminus with a signal peptide of the H-chain is essential for the secretion of the H-chain into the PSG lumen, through observation and analysis of the secretion of different EGFP/H-chain fusion proteins in the PSG and cocoon of transgenic silkworms. Our results suggested that PSG cells of transgenic silkworms could secrete the elementary unit of fibroin into the PSG lumen and could direct secretion of the H-chain fusion protein into the PSG lumen and spinning into cocoons without forming the H-L heterodimer. A new fibroin Hchain expression system with only a shorter N-terminal sequence of the H-chain for fusion with the exogenous genes could give a greater chance to express the targeted protein in an active form. In addition, exploitation of this new system in commercial silkworm strains would contribute to the development of silkworm resources and the utilization of silkworm bioreactors and show great potential to increase the value of the commercial production of exogenous proteins.
Materials and methods Silkworm strain used for germline transformation The Chinese lineage diapause B. mori strain 871 (white cocoon, commercial strain) was maintained at the Gene Resource Bank of Domesticated Silkworms (Southwest University, Chongqing, China). The 15 °C-IMES germline transformation strategy [33] was used to change the diapause character of strain 871 eggs for DNA pre-blastoderm
97
D. Long et al.
The mechanism of fibroin secretion in silkworm
microinjection. Microinjection with non-diapause strain 871 embryos and screening of transgenic silkworms were done as described elsewhere [34,35].
Construction of vectors and generation of transgenic silkworms The piggyBac-derived vector pBac{3 9 P3-DsRed}-R3 [15] (Fig. 1A) was maintained in our laboratory. pBac{3 9 P3DsRed}-R3 contains an EGFP/H-chain fusion gene with the LBS, which consists of the fibroin H-chain gene 30 -terminal sequence including the deduced Cys-c20 of the H-chain and the polyadenylation signal sequence of the H-chain gene (Fig. 1A). To construct the vector pBac {3 9 P3-DsRed; Fib-H P3-EGFP-poly(A)} (Fig. 1B), the poly(A) sequence was amplified by PCR using the primer pair poly(A)-F-BamHI/LBS-R-SalI (Table S1) with the pBac{3 9 P3-DsRedaf}-R3 plasmid as a template. The 182 bp PCR product was inserted into the BamHI/SalI site of the plasmid pSL-LBS-EGFP+Fib-H P3 [15] to generate plasmid pSL{Fib-H P3-EGFP-poly(A)}. pBac{3 9 P3DsRed; Fib-H P3-EGFP-poly(A)} was constructed by cloning a 3.2 kb AscI fragment from pSL{Fib-H P3-EGFPpoly(A)} into AscI-cut pBac{3 9 P3-DsRedaf} [36]. pBac {3 9 P3-DsRed; Fib-H P3-EGFP-poly(A)} contains an EGFP/H-chain fusion gene with the H-chain gene 30 -terminal sequence, which includes only the poly(A) (Fig. 1B). The piggyBac-derived vectors were injected with the helper plasmid pHA3PIG [16] into G0 non-diapause 871 eggs at the pre-blastoderm stage. G0 adults were backcrossed with wild-type strain 871. We obtained a total of five G1 transgenic strains, including one TSL strain from the pBac{3 9 P3-DsRed}-R3 vector insertion and four TSP strains from the pBac{3 9 P3-DsRed; Fib-H P3EGFP-poly(A)} vector insertion (Table S2).
Inverse PCR The chromosomal insertion sites of the piggyBac-derived vectors on the chromosomes were determined by inverse PCR. About 10 lg of genomic DNA was digested with HaeIII overnight at 37 °C and circularized by overnight ligation at 16 °C. The ligated product was PCR-amplified with the transposon-specific primer pairs PLF/PLR (for piggyBac left arm) and PRF/PRR (for piggyBac right arm) (Table S1). The PCR fragments were cloned and sequenced. Localization of the silkworm genomic insertion sites of the piggyBac-derived vectors were completed using SILKMAP software (www.silkdb.org/silksoft/silkmap.html). The results showed that the sequence of the two piggyBac arms was bordered by the characteristic TTAA site in all transgenic strains, and the piggyBac inserts in the genome of TSL and TSP strains were located on different chromosomes (Table S3).
98
EGFP fluorescence observation of the silk glands and cocoons of transgenic silkworms The silk glands of transgenic silkworms on day 2 of the fifth instar were dissected and placed onto glass slides to determine EGFP expression using a fluorescence stereomicroscope [Olympus MacroViewMVX10-AUTO (Olympus, Tokyo, Japan) equipped with a GFP filter]. Cocoons from transgenic silkworms were selected at random and photographed using the same microscope.
Histological fluorescence detection of expression of EGFP in transgenic silkworms The fresh silk glands from transgenic silkworms on day 6 of the fifth instar were washed and then fixed in Bouin fixative [0.9% (v/v) picric acid, 5% (v/v) acetic acid and 9% (v/v) formaldehyde] for 24 h at room temperature. After washing with PBS, the silk glands were dehydrated by passage through a graded series of ethanol, added to a 1 : 1 (v/v) mixture of dimethylbenzene and ethanol for 1 h and then into dimethylbenzene for 20 min before embedding in paraffin wax and were cut into 5-lm-thick slices, which were transformed to gelatin-coated slides and dried at 40 °C overnight. Subsequently, sections were observed under a light microscope (Olympus BX51TRF). Transgenic silk fibers were collected and twisted into a narrow bundle, and then embedded directly into paraffin wax and cut into 7lm-thick slices before observation under a laser scanning confocal microscope (Leica TCS-SP2; Leica, Mannheim, Germany).
Genomic PCR The primer pair EGFP-MF/poly(A)-MR (Table S1) was used to confirm transgenic individuals. Genomic DNA extracted from transgenic and wild-type strain 871 adults was used as a template for PCR. The purified PCR fragments were cloned and sequenced.
RT-PCR Total RNA was extracted from the PSG and the fat body of day 4 of the fifth instar using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as described previously [15]. cDNA was synthesized using the Perfect Real Time version of the PrimerScriptTM RT reagent kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China) according to the manufacturer’s instructions. PCR amplifications were done using the primer pairs FibH-50 UTR-F/poly(A)-MR, EGFP-F/EGFP-R and BmActin3-f/BmActin3-r (Table S1). Amplification of the BmActin A3 (AAC47446) served as an internal control.
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
SDS/PAGE and western blotting About 25 mg of each transgenic silkworm cocoon shell was dissolved in 1 mL of 60% (w/v) lithium thiocyanate by vortex-mixing for 10 min, incubation at room temperature for 1 h followed by centrifugation at 10 000 g in a microcentrifuge for 5 min. Then, 4-lL samples were subjected to SDS/PAGE [10% (w/v) polyacrylamide slab gel] by dissolving in an equal volume of sample loading buffer [5% (v/v) glycerol, 1% (w/v) SDS, 0.05% (w/v) bromophenol blue, 0.0625 M Tris/HCl, pH 6.8] without or with 2% (v/v) 2-ME and boiled for 5 min. After electrophoresis, the gel was stained with 0.1% (w/v) Coomassie Brilliant Blue R250, 10% (v/v) acetic acid, 50% (v/v) methanol. About 2 lL of each sample was diluted five-fold with 10 mM Tris/ HCl (pH 6.8) and then 2 lL of the diluted sample was dissolved in sample loading buffer without or with 2% 2-ME and subjected to SDS/PAGE. The gel was transferred directly onto a polyvinylidene difluoride membrane (Roche, Mannheim, Germany), rinsed briefly and blocked for 1 h at room temperature with TBST (0.136 M NaCl, 20 mM Tris/HCl pH 7.6, 0.1% (w/v) Tween 20) containing 5% (v/ v) skim milk. After rinsing twice and washing three times with TBST, the membrane was incubated at room temperature for 1 h in TBST containing 2500-fold diluted GFP antibody (Beyotime, Jiangsu, China). The membrane was rinsed twice and washed three times (1 9 15 min and 2 9 5 min) with TBST, and then incubated at room temperature for 1 h in TBST containing 10 000-fold diluted horseradish-peroxidase-labeled rabbit IgG secondary antibody (Beyotime). After rinsing and washing with TBST, the antibody was detected with ECL plus Western Blotting Detection Reagents according to the manufacturer’s instructions (Beyotime). The signals were obtained by a chemiluminescence imaging system (Clinx ChemiScope series, Shanghai, China).
Acknowledgements This work was supported by grants from the Hi-Tech Research and Development (863) Program of China (No. 2013AA102507) and the China Agriculture Research System (No. CARS-22-ZJ0102).
Author contributions A.Z. and Z.X. planned the experiments; D.L., W.L. and Y.Z. performed the experiments; D.L. and Q.G. analyzed the data; D.L. and A.Z. wrote the paper.
References 1 Vepari C & Kaplan DL (2007) Silk as a biomaterial. Prog Polym Sci 32, 991–1007.
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
The mechanism of fibroin secretion in silkworm
2 Omenetto FG & Kaplan DL (2010) New opportunities for an ancient material. Science 329, 528–531. 3 Grzelak K (1995) Control of expression of silk protein genes. Comp Biochem Physiol B Biochem Mol Biol 110, 671–681. 4 Inoue S, Tanaka K, Arisaka F, Kimura S, Ohtomo K & Mizuno S (2000) Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6: 6: 1 molar ratio. J Biol Chem 275, 40517–40528. 5 Inoue S, Tanaka K, Tanaka H, Ohtomo K, Kanda T, Imamura M, Quan GX, Kojima K, Yamashita T & Nakajima T (2004) Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L-chain for protection of a1, 2-mannose residues in Nlinked oligosaccharide chains of fibrohexamerin/P25. Eur J Biochem 271, 356–366. 6 Zhou C, Confalonieri F, Medina N, Zivanovic Y, Esnault C, Yang T, Jacquet M, Janin J, Duguet M & Perasso R (2000) Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res 28, 2413– 2419. 7 Zhou C, Confalonieri F, Jacquet M, Perasso R, Li ZG & Janin J (2001) Silk fibroin: structural implications of a remarkable amino acid sequence. Proteins 44, 119–122. 8 Wang SP, Guo TQ, Guo XY, Huang JT & Lu CD (2006) In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector. Biochem Biophys Res Commun 341, 1203–1210. 9 Jin HJ & Kaplan DL (2003) Mechanism of silk processing in insects and spiders. Nature 424, 1057– 1061. 10 Mita K, Ichimura S & James TC (1994) Highly repetitive structure and its organization of the silk fibroin gene. J Mol Evol 38, 583–592. 11 Tanaka K, Kajiyama N, Ishikura K, Waga S, Kikuchi A, Ohtomo K, Takagi T & Mizuno S (1999) Determination of the site of disulfide linkage between heavy and light chains of silk fibroin produced by Bombyx mori. Biochim Biophys Acta 1432, 92–103. 12 Takei F, Oyama F, Kimura K, Hyodo A, Mizuno S & Shimura K (1984) Reduced level of secretion and absence of subunit combination for the fibroin synthesized by a mutant silkworm, Nd (2). J Cell Biol 99, 2005–2010. 13 Takei F, Kikuchi Y, Kikuchi A, Mizuno S & Shimura K (1987) Further evidence for importance of the subunit combination of silk fibroin in its efficient secretion from the posterior silk gland cells. J Cell Biol 105, 175–180. 14 Mori K, Tanaka K, Kikuchi Y, Waga M, Waga S & Mizuno S (1995) Production of a chimeric fibroin lightchain polypeptide in a fibroin secretion-deficient naked
99
D. Long et al.
The mechanism of fibroin secretion in silkworm
15
16
17
18
19
20
21
22
23
24
25
100
pupa mutant of the silkworm Bombyx mori. J Mol Biol 251, 217–228. Zhao A, Zhao T, Zhang Y, Xia Q, Lu C, Zhou Z, Xiang Z & Nakagaki M (2010) New and highly efficient expression systems for expressing selectively foreign protein in the silk glands of transgenic silkworm. Transgenic Res 19, 29–44. Tamura T, Thibert C, Royer C, Kanda T, Eappen A, Kamba M, K^ omoto N, Thomas J-L, Mauchamp B & Chavancy G (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposonderived vector. Nat Biotechnol 18, 81–84. Kojima K, Kuwana Y, Sezutsu H, Kobayashi I, Uchino K, Tamura T & Tamada Y (2007) A new method for the modification of fibroin heavy chain protein in the transgenic silkworm. Biosci Biotechnol Biochem 71, 2943–2951. Fedic R, Zurovec M & Sehnal F (2003) Correlation between fibroin amino acid sequence and physical silk properties. J Biol Chem 278, 35255–35264. Tanaka K, Inoue S & Mizuno S (1999) Hydrophobic interaction of P25, containing Asn-linked oligosaccharide chains, with the H-L complex of silk fibroin produced by Bombyx mori. Insect Biochem Mol Biol 29, 269–276. Teule F, Miao Y, Sohn B, Kim Y, Hull JJ, Fraser MJ, Lewis RV & Jarvis DL (2012) Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties. Proc Natl Acad Sci USA 109, 923–928. Iizuka T, Sezutsu H, Tatematsu KI, Kobayashi I, Yonemura N, Uchino K, Nakajima K, Kojima K, Takabayashi C & Machii H (2013) Colored fluorescent silk made by transgenic silkworms. Adv Funct Mater 23, 5232–5239. Takano E, Maki M, Mori H, Hatanaka M, Marti T, Titani K, Kannagi R, Ooi T & Murachi T (1988) Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis. Biochemistry 27, 1964–1972. Michal CA, Simmons AH, Chew B, Zax DB & Jelinski LW (1996) Presence of phosphorus in Nephila clavipes dragline silk. Biophys J 70, 489–493. Zhang P, Aso Y, Yamamoto K, Banno Y, Wang Y, Tsuchida K, Kawaguchi Y & Fujii H (2006) Proteome analysis of silk gland proteins from the silkworm, Bombyx mori. Proteomics 6, 2586–2599. Inoue S, Kanda T, Imamura M, Quan G, Kojima K, Tanaka H, Tomita M, Hino R, Yoshizato K & Mizuno S (2005) A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins. Insect Biochem Mol Biol 35, 51– 59.
26 Shimura K (1983) Chemical composition and biosynthesis of silk proteins. Cell Mol Life Sci 39, 455– 461. 27 Kurihara H, Sezutsu H, Tamura T & Yamada K (2007) Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system. Biochem Biophys Res Commun 355, 976–980. 28 Shimizu K, Ogawa S, Hino R, Adachi T, Tomita M & Yoshizato K (2007) Structure and function of 50 flanking regions of Bombyx mori fibroin heavy chain gene: Identification of a novel transcription enhancing element with a homeodomain protein-binding motif. Insect Biochem Mol Biol 37, 713–725. 29 Zhu Z, Kikuchi Y, Kojima K, Tamura T, Kuwabara N, Nakamura T & Asakura T (2010) Mechanical properties of regenerated Bombyx mori silk fibers and recombinant silk fibers produced by transgenic silkworms. J Biomater Sci Polym Ed 21, 395–411. 30 Nagano A, Tanioka Y, Sakurai N, Sezutsu H, Kuboyama N, Kiba H, Tanimoto Y, Nishiyama N & Asakura T (2011) Regeneration of the femoral epicondyle on calcium-binding silk scaffolds developed using transgenic silk fibroin produced by transgenic silkworm. Acta Biomater 7, 1192–1201. 31 Tatemastu K, Sezutsu H & Tamura T (2012) Utilization of transgenic silkworms for recombinant protein production. J Biotechnol Biomaterial S9, 1–8. 32 Walsh CT, Garneau-Tsodikova S & Gatto GJ Jr (2005) Protein posttranslational modifications: the chemistry of proteome diversifications. Angew Chem Int Ed Engl 44, 7342–7372. 33 Zhao A, Long D, Ma S, Xu L, Zhang M, Dai F, Xia Q, Lu C & Xiang Z (2012) Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains. Insect Sci 19, 172–182. 34 Long D, Zhao A, Chen X, Zhang Y, Lu W, Guo Q, Handler AM & Xiang Z (2012) FLP recombinasemediated site-specific recombination in silkworm, Bombyx mori. PLoS One 7, e40150. 35 Long D, Zhao A, Xu L, Lu W, Guo Q, Zhang Y & Xiang Z (2013) In vivo site-specific integration of transgene in silkworm via phiC31 integrase-mediated cassette exchange. Insect Biochem Mol Biol 43, 997– 1008. 36 Horn C & Wimmer EA (2000) A versatile vector set for animal transgenesis. Dev Genes Evol 210, 630–637.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site:
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
D. Long et al.
Fig. S1. Amino acid sequence alignment between three different EGFP/H-chain fusion proteins, TSL-FP, TSP-FP and R1-FP. Table S1. Summary of primers used in the present study.
FEBS Journal 282 (2015) 89–101 ª 2014 FEBS
The mechanism of fibroin secretion in silkworm
Table S2. Injection of piggyBac-derived vectors in G0 silkworm embryos of the strain 871. Table S3. Identification of genomic insertion sites of piggyBac vectors by inverse PCR.
101
