Journal of Insect Biotechnology and Sericology 86, 105-112 (2017) The physiological accumulation of mutant fibroin light chains induces an unfolded protein response in the posterior silk gland of the Sericin cocoon Nd-sD strain of silkworm Bombyx mori Tadashi Takahashi1, Masao Miyazaki1, Shin-ichiro Kidou2, Yoshiki Matsui1, Ying An1, Taku Ozaki3, Koichi Suzuki1＊＊ and Tetsuro Yamashita1＊ 1 Faculty of Agriculture, Iwate University, Ueda, Morioka, Iwate 020-8550, Japan 2 Graduate School of Natural Sciences, Nagoya City University, Mizuho-cho, Mizuho-ku, Nagoya 467-8501, Japan 3 Faculty of Science and Engineering, Iwate University, Ueda, Morioka, Iwate 020-8550, Japan (Received May 15, 2017; Accepted July 4, 2017) Fibroin, a major component of silk fiber, is composed of light (L) chains, heavy chains, and fhx/P25 in the silk- worm Bombyx mori. Sericin cocoon (Nd-sD) is a silkworm strain expressing a mutant fibroin L chain that is accu- mulated in the endoplasmic reticulum (ER) of posterior silk glands (PSGs). However, little is known about the effects of accumulation in PSGs in Nd-sD strain. We compared the PSG gene expression profiles of 5th-instar larvae of Nd-sD strain and the fibroin-producing normal strain. cDNA representational difference analysis identi- fied candidate genes whose expression levels were higher in Nd-sD strain than in normal strain. We focused on heat shock proteins and cathepsin B, a major lysosomal protease. We confirmed upregulation of BiP(GRP78), Hsp20.8, and cathepsin B at the transcriptional and/or translational levels. These results suggest that the accu- mulation of mutant L chain induces the unfolded protein response in PSGs of Nd-sD strain, which will be a valu- able tool for protein quality-control studies of silk grand. Key words: Bombyx mori, fibroin, unfolded protein response, molecular chaperone ponents (Inoue et al., 2000). The fibroin L chain, fibroin INTRODUCTION H chain, and fhx/P25 are assembled in the endoplasmic Fibroin is the major silk protein component synthesized reticulum (ER) in a 6:6:1 molecular ratio of L chain:H in the posterior silk gland (PSG) of the silkworm Bombyx chain:fhx/P25. A previous study demonstrated that the (H- mori. Fibroin is composed of three proteins: 30-kD light L)6 fhx/P25 complex structure is essential for the secre- (L) chain (Yamaguchi et al., 1989), 350-kD heavy (H) tion of fibroin into the PSG lumen; therefore, it is called chain (Zhou et al., 2000), and 25-kD fibrohexamerin (fhx)/ the “fibroin elementary unit” (Inoue et al., 2004). P25 (Chevillard et al., 1986). Intact fibroin L chain is The silkworm mutant, Sericin cocoon (Nd-sD), has fi- composed of 262 amino acid residues, the first 18 of broin secretion levels that are less than 0.3% of those of which are cleaved as a signal peptide and the subsequent fibroin-producing strains (Takei et al., 1987). Therefore, N-terminal serine is acetylated (Yamaguchi et al., 1989). this fibroin-secretion -deficient mutant produces a very thin, The deduced fibroin L-chain polypeptide contains three naked-pupa cocoon that consists mostly of sericin (Fig. 1A). Cys residues. An intramolecular disulfide bond is formed Genetic analyses revealed that Nd-sD strain has a deletion between Cys83 and Cys142 in the fibroin L chain. Cys172 of approximately 10 kbp in the Fib-L gene (Fig. 1B). Since forms an intermolecular disulfide linkage to Cys-c20 (20th the deleted region contains four exons (IV, V, VI, and VII) residues from the carboxyl terminus) of the fibroin H of the fibroin L chain, the resulting gene has two new ex- chain (Tanaka et al., 1999). The middle portion of the ons (IV′ and V′). The L-chain polypeptide of the Nd-sD amino acid sequence of the fibroin H chain contains re- mutant is a hybrid amino acid sequence in which the first peated Gly-Ala sequences that form a highly hydrophobic 105 residues are identical to the native fibroin L-chain crystalline domain. Fhx/P25 is a glycoprotein that pos- polypeptide, but the remaining sequence containing Cys172 sesses three high mannose-type Asn-linked oligosaccha- is completely different from the original (Mori et al., ride chains that interact with the H chain, which inhibits 1995). Therefore, the mutant fibroin L chain cannot form non-specific interactions and aggregations of fibroin com- a disulfide bond with the fibroin H chain. The lack of this disulfide bond causes the production of misfolded fibroin; *To whom correspondence should be addressed. i.e., a secretion-deficient phenotype. A previous study ob- D Fax &Tel: +81-19-621-6157. served the enlarged ER in the PSG of the Nd-s mutant Email: [email protected] using an electron microscope and found that fibroin com- ** Present address: Biococoon Laboratories Inc., Research and De- ponents accumulated in the ER (Gamo and Sato, 1985). velopment Center by Collaboration of Morioka city and Iwate Uni- Consequently, the deficiency in the fibroin elementary unit versity, Morioka, Iwate 020-8551, Japan because of mutant fibroin L-chain production results in 106 Takahashi et al. Fig. 1. (A) Fifth instar larvae and cocoons of DAIZO and Nd-sD strain. (B) Genetic map of normal fib-L and Nd-sD strain fib-L genes. the fibroin secretion-deficient phenotype in Nd-sD strain. strain. We compared the gene expression profiles in the Based on these findings, we hypothesized that the pro- PSGs of days-2-6 5th-instar larvae of normal strain, a fi- duction of mutant fibroin L chain is involved in quality broin-producing silkworm strain, and Nd-sD strain using control of proteins in the PSG. Molecular chaperones pro- cDNA representational difference analysis (RDA). RDA is vide the machinery for protein quality control in cells a PCR-based subtractive hybridization method to clarify (Buchberger et al., 2010). They discriminate between na- the differences between two DNA samples (Hubank and tive and misfolded proteins or protein complexes after Schatz, 1994). Because we found that genes including which they can induce the folding of unfolded proteins heat shock proteins were more highly expressed in Nd-sD into their appropriate folded states. If they find mis- or strain than in normal strain, we also compared protein ex- unfolded proteins in the ER, the intracellular trafficking pression levels between the strains. for such proteins is inhibited. Mis- and unfolded proteins that were already transported to the Golgi apparatus are MATERIALS AND METHODS returned to the ER for restoration. Further expression and accumulation of mis- and unfolded proteins leads to the Silkworms unfolded protein response (UPR) (Mori, 2000). The UPR The Sericin cocoon (Nd-sD) mutant strain was gifted by consists of four stages: suppression of translation, induc- the Insect Genetics Laboratory of the National Institute of tion of chaperone genes, activation of ER-associated deg- Agrobiological Sciences, Japan. The silkworm Bombyx radation (ERAD), and finally induction of apoptosis. We mori DAIZO strain, whose secretion level of fibroin is therefore suggest that the accumulation of mutant fibroin normal, was supplied by the Laboratory of Applied Ento- components in the PSG of the Nd-sD mutant strain leads mological Science of Iwate University, Japan. These strains to the UPR in the ER. were maintained by feeding artificial foods under a 12-h The aim of the present study was to examine the effect light/dark photoperiod at 25°C. Fifth-instar larvae of both of the expression of the mutant fibroin L chain on the strains were used in this study. quality control of proteins in the PSG of the Nd-sD mutant Unfolded protein response of silk gland 107 Table 1. Primer pairs used for RT-PCR Target gene Forward primer Reverse primer Fibroin L chain (normal) 5’-GGATCCGTCATTAACTCTTACACAG-3’ 5’-GAATTCTTAGACGTGAACCTGGCTGG-3’ Fibroin L chain (Nd-sD) 5’-GGATCCGT ATGGGTTCGGTTATTTCG-3’ 5’-GAATTCCTATTCACACATT CGTTTAT-3’ fhx/P25 5’-GTCTAGCTGTAGCCGCTGTG-3’ 5’-C TGCGCTGTGCGTTGGTCAT-3’ Cathepsin B 5’-GGATCCATGTTTATTTCGCG TGCGGCGTAT-3’ 5’-GTCGACCTAGTGTTCATCTAAGAACG GTTC-3’ Hsc 70-4 5’-GAATTCATGGCAAAAGCACCCGCAGT-3’ 5’-GTCGACAAGTGTGGTGTGGAATGTTG-3’ BiP 5’-GTCGACATGGT CAAGATGCGGTGGAG-3’ 5’-GTCGACCATCTTCAAGCGTCTTCTTCT -3’ Hsp 20.8 5’-GGATCCATGTCTCTTCTACCATTC GTG-3’ 5’-GAATTCACCCTACTTATTTTCGGCAG-3’ Representational difference analysis (RDA) of for 8 h with probes for BiP, Hsp20.8, Hsc70-4, or cathep- cDNA sin B, which were obtained using RT-PCR and labeled us- A modified method established by Hubank and Schatz ing an AlkPhos Direct Labeling Module (GE Healthcare). (Hubank and Schatz, 1994) was used for RDA of cDNA. The primers for these genes are summarized in Table 1. Total RNAs were extracted from PSGs of the DAIZO and The membranes were incubated with CDP-Star detection Nd-sD strains at appropriate stages using the TRIzol re- reagent (GE Healthcare). agent (Invitrogen, Carlsbad, CA, USA). cDNA of each sample was synthesized from 1 μg of total RNA using the Western blotting cDNA Synthesis Kit (M-MLV version, Takara). After di- PSGs were prepared from days-2-6 5th-instar larvae of gesting the synthesized cDNA with Sau3AI, two linker DAIZO and Nd-sD strains, and lysed in RIPA buffer [50 mM primers, RBA1 (5’-AGCACTCTCCAGCCTCTCACCG Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 1% DOC, 1% AG-3’) and RBA2 (5’-GATCCTCGGTGA-3’), were ligat- SDS, 1 mM EDTA, 1× Complete Mini Protease Inhibitor ed on both ends of the cDNA. RBA-linked cDNA frag- Cocktail (Roche, Mannheim, Germany)]. The fibroin aggre- ments were amplified by PCR using Ex Taq (Takara) and gates were precipitated by the centrifugation at 15,000 × g, RBA1. PCR products were digested with Sau3AI and ex- and the supernatant (20-μg aliquot) was separated by cess primers were removed. The same protocol was ap- SDS-PAGE using a 10% polyacrylamide gel with under plied to substitute the RBA linkers for the NBA linkers, reducing conditions.