Developmental regulation of silk protein P 25 in the silkworm Bombyx mori

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1 J. Biosci., Vol. 20, Number 2, March 1995, pp Printed in India. Developmental regulation of silk protein P 25 in the silkworm Bombyx mori 1. Introduction Κ MUTHUMANI, S MATHAVAN* and S MAYILVAHANAN Sericulture Research Unit, Department of Genetics, School of Biological Sciences, Madurai Kamaraj University. Madurai , India MS received 23 May 1994; revised 24 February 1995 Abstract. P 25 protein was extracted from cocoons of the silkworm Bombyx mori by alkali solubilization and purified by gel elution. The purity and authenticity of the protein were confirmed by SDS-PAGE, 2-dimensional gel electrophoresis and peptide mapping. Polyclonal anti-p 25 sera were raised in rabbit and mice. The relative abundance of P 25 protein present in the larva during different developmental stages was analysed by SDS-PAGE, and quantified by sandwich ELISA. The minimum level (0 2 μg/animal) of this protein was recorded at the beginning of the first instar and maximum (16 7mg/pair of silkgland) on the final day of the V instar. During each moult period, P 25 protein level was suppressed; the level increased with the initiation of feeding and reached maximum on the 3rd day of each instar except the final instar where the maximum was recorded prior to pupal moult. Western blot analysis also confirmed the developmental stage-specific accumulation of P 25 protein in the silkworm Bombyx mori. Keywords. Bombyx mori; P 25 protein purification and quantification; stage-specific regulation. Silk fibroin produced by the silkworm Bombyx mori is composed of one heavy chain ( 350 kda) and one light chain (25 kda) (Sasaki and Noda 1973). Shimura et al (1976) demonstrated the occurrence of minor fibroin (L-chain) in the cocoon of B. mori. Couble et al (1983) identified a new mrna species in the posterior silkgland encoding a silk protein of kda which was termed as P 25. Fibroin L-chain cdna was cloned and the amino acid sequence was determined by Yamaguchi et al (1989) and they also estimated the size of the L-chain as 24 8 kda which is different from the P 25 protein identified by Couble et al (1983). Subsequently, the P 25 gene has been cloned and sequenced, and the putative amino acid sequence of the protein has been deduced (Couble et al 1985; Chevillard et al 1986). Recently, Durand et al (1992) reported tissue- and developmental stage-specific binding factors of the P 25 gene and their involvement in gene regulation. Couble and Prudhomme (1993) identified cis-acting DNA sequences and trans-acting factors that regulate the expression of the P 25 gene. Synthesis of major fibroin protein is turned on during the feeding period and turned off during the moulting period of the larvae (Maekawa and Suzuki 1980). P 25 protein is produced in equimolar concentrations to major fibroin (Couble et al 1983). It is not known whether P 25 protein is also developmentally regulated as in * Corresponding author. 211

2 212 Κ Muthumani, S mathavan and S Mayilvahanan the case of major fibroin. Shimura (1983) quantified the amount of major fibroin protein in the silkgland during development but such quantification has never been made for P 25 protein. The present study attempts to demonstrate the regulation and quantitative changes in P 25 protein level during development in B. mori. 2. Materials and methods 2.1 Experimental animal Larvae and cocoons of the silkworm Β. mori (TW NB 4 D 2 ) were used in the experiments. The larvae were reared in the laboratory following the method recommended by Krishnaswamy (1978) and fed ad libitum on fresh mulberry leaves. 2.2 Purification of P 25 protein P 25 protein was purified from cocoons following the method of Shimura et al (1982) with slight modification. The cocoons were cut into small pieces, desericinated, dissolved in 60% Lithium thiocyanate (LiSCN) solution and incubated overnight at 4 C. The protein was fractionated with 0 20% ammonium sulphate solution and dialyzed against 20 mm Tris-HCI (ph 8 0), 5 Μ urea and 0 1% β-mercaptoethanol. Protein content was estimated according to Lowry et al (1951). Proteins were further purified using a preparative SDS-polyacrylamide gel. The P 25 protein band was excised from the gel and eluted as described by Hager and Durgess (1980). 2.3 Immunization schedule Three to five month-old male albino rabbits were injected subcutaneously with 5 mg/ml of P 25 protein in Freund's adjuvant. Immunization was repeated three times at fifteen days interval. Antibody titres in the sera were examined by double immunodiffusion. After the final booster, the animals were bled completely and the sera were decomplemented at 57 C for 30 min. Both test and control sera were subjected to ammonium sulphate precipitation (40% saturation at 4 C). The precipitated immunoglobulins were dissolved in 10 mm Na 2 PO 4 buffer (ph 7 0) and dialyzed extensively against the same buffer. Antibody for the P 25 protein was raised in the Balb/C mice following the same method. Pre-immune sera were also collected and used on requirement. 2.4 Analysis of P 25 protein 2.4a SDS-PAGE: Total larval proteins from I III instars were collected (from entire larvae) by homogenizing the larvae in phosphate buffer (ph 7 0) with 1 mm PMSF. It is not possible to remove the gut content in the I and II instar. However, in III instar the gut content was removed as far as possible. In IV and V instar larvae, total silkgland protein was collected by homogenizing the silkgland (both from MSG and PSG) in the above mentioned buffer. The homogenates were filtered through cheese cloth and the filtrates were centrifuged at 11,850 g for 10 min. The supernatant was collected and stored at -20 C. Proteins (150 µg) from each sample were electrophoresed in a 1 5 mm, 10% polyacrylamide gel containing 1% SDS

3 Development regulation of silk protein P (w/v) (Laemmli 1970). The gels were stained with Coomassie brilliant blue R250 (Sigma, USA). 2.4b Two-dimensional electrophoresis: Two-dimensional electrophoresis of total silkgland proteins and purified P 25 protein was carried out according to O'Farrel (1975) as modified by Dorel and Coulon (1988). 2.4c Peptide mapping: P 25 protein was mapped by limited proteolysis in the presence of SDS, with V8 protease from Staphylococcus aureus (Sigma, USA) following the method of Cleveland et al (1977). Briefly, purified P 25 protein (150 μg) was electrophoresed on a 1 mm thick 10% polyacrylamide slab gel. The 25 kda protein band was identified by staining as mentioned above, excised and incubated in 125 mm Tris-HCl (ph 6 8) and 0 1% SDS for 1 h at room temperature. The gel piece was placed on the well of 17% acrylamide gel with 3% stacking gel, and overlayered with sample buffer containing 20 μg of V8 proteases. After the migration of protease and silk proteins into the middle of the stacking gel, electrophoresis was interrupted for 40 min for digestion and then continued. The proteins were transferred to a nitrocellulose membrane and immunoblotted using anti P 25 sera. 2.4d Indirect ELISA : Indirect ELISA was performed following the method of Voller et al (1979). Briefly, 20 μg/ml of P 25 protein was coated on polystyrene plates in carbonate-bicarbonate buffer (ph 9 6). Blocking and washing were done with PBS-Tween 20 (0 05%) with and without bovine serum albumin (BSA), respectively. Subsequently, various dilutions (1:400 to 1:6400) of P 25 antisera were added to the wells. HRP-conjugated goat anti-rabbit IgG (Bangalore Genei, India; 1 : 2000 dilution) was used for detection. The o-phenylenediamine (Sigma, USA) and H 2 O 2 were used as substrates and the intensity of colour development was measured at 492 nm. Wells without primary antibody served as negative controls. 2.4e Western blot analysis: Western blot analysis was carried out as described by Towbin et al (1979). The total protein extracted from I to III instar and silkgland protein extracted from IV and V instar were subjected to immunoblot analysis using P 25 antibody raised in rabbit and mice. The total silkgland protein and purified P 25 protein were also immunoblotted and probed using fibroin L-chain antibody (obtained from Prof. Shigeki Mizuno, Tohoku University, Japan). 2.5 Quantification of P 25 protein To quantify the P 25 protein present in the larva/silkgland during the developmental stages, sandwich ELISA was performed. Purified IgG of P 25 antibody raised in mice (50 μg/well : coating antibody) and the antibody raised in rabbit were used (1 :2000 dilution). HRP-conjugated goat anti-rabbit IgG (1:2000 dilution) was used as secondary antibody. To construct the standard graph (Ternynck and Avrameas 1990) purified P 25 protein concentrations ranging from μg/well to 128 μg/well were used. Quantification of P 25 protein was done either from total larval protein (I to III instar) or from silkgland protein (IV and V instar).

4 Κ Muthumani, S mathavan and S Mayilvahanan Results and discussion 3.1 Purity and authenticity of P25 protein Total protein extracted from the silkgland (fifth day of V instar), cocoon and P25 protein purified from the cocoons were analysed by SDS-PAGE. The purified protein gave a single band corresponding to 25 kda, and a band with same molecular weight was observed in total silk gland proteins and cocoon proteins (figure 1). Figure 1. SDS-PAGE analysis of P25 protein: Total protein extracted from the entire silkgland and P25 protein purified from cocoons were separated in 10% SDS-PAGE and stained with Coomassie brilliant blue. (A), Molecular weight markers; (B), Total silkgland protein (15(µg); (C), Total cocoon protein (15 µg); (D), Purified P25 protein (50 µg).

5 Development regulation of silk protein P The P 25 protein has a P I value of (Couble et al 1983; Mounier et al 1991). To confirm the authenticity of the purified protein two-dimensional gel electrophoresis was performed using total proteins from silkgland as well as the P 25 protein from the cocoon. The isoelectric points and the molecular weights confirm the P 1 value of both in the total protein and purified protein indicating that the purified fraction is P 25 protein (figure 2). Figure 2. Two-dimensional gel electrophoresis of proteins from total silkgland (15µg) (A) and purified P 25 protein from cocoons (50µg) (B). The focusing gels were run from left ( ) and SDS gels from top to bottom. The isoelectric points and the molecular weights are indicated on the horizontal and vertical axis, respectively. The arrows indicate the P 25 protein. 3.2 Peptide mapping Peptide mapping analysis of P 25 protein was reported by Couble et al (1983) by subjecting the protein to limited digestion, with V8 protease from S. aureus. They showed the presence of two cleaved small peptides. The protein that has been purified in the present study was subjected to peptide mapping using V8 protease from S. aureus. Two cleaved polypeptides observed in the present study corresponds to the peptides reported by Couble et al (1983) confirming that the purified protein is the authentic P 25 protein (figure 3). 3.3 lmmunoblot with P 25 and L-chain antisera To determine the optimal dilution of P 25 antibody for further analysis, ELISA was carried out using the antibody raised in rabbit and mice and the optimal dilution was recorded as 1 : 2000 for both antibodies. Total silkgland (5th day of V instar) proteins and proteins purified from cocoons were separated by SDS-PAGE and transferred to nitrocellulose membrane. Three membranes containing these two proteins were prepared; one membrane was immunoblotted against P 25 sera raised in rabbit, the second one with P 25 antibody raised in mice and the third one against.

6 216 Κ Muthumani, S mathavan and S Mayilvahanan Figure 3. Peptide mapping of purified P25 protein from the cocoons: The purified P25 protein was initially separated by 10% SDS-PAGE (1D). The protein was digested with V8 protease from Staphylococcus aureus in a 3% stacking gel of second dimensional electrophoresis and separated in 17 5% acrylamide resolving gel (2D). The cleaved proteins were transferred to a nitrocellulose membrane and immunoprobed using P25 antisera raised in rabbit. The arrow indicates the uncleaved P25 protein. Lane A is uncleaved P25 protein. The two smaller peptides in lane Β are cleaved P 25 protein.

7 Development regulation of silk protein P the fibroin L-chain antibody (obtained from Prof. Shigeki Mizuno). Figure 4 shows that sera raised against P25 protein alone reacted with P25 antigen of total silkgland and purified fraction (lane Β and C; rabbit antibody, lane D and E; mice antibody). The L-chain antibody reacted only in the total silkgland protein with a lower molecular weight protein compared to P25 antibody because fibroin L-chain is smaller (mol. wt kda) than Ρ25 protein ( kda) (lane F). Further, L-chain antibody does not react with P25 protein purified from the cocoon (lane G). These results clearly indicate that the antibodies raised (both in rabbit and mice) are anti P25 and do not show cross reactivity Figure 4. Total silkgland protein (lanes B, D and F) and purified P25 protein (lanes C, Ε and G) were electrophoresed in in 10%SDS-PAGE and the proteins were electrotransferred to nitrocellulose membrane. After transfer the filter was cut into 3 parts containing lanes Β and C (I), lanes D and Ε (II) and lanes F and G (HI). The filters I, II were immunoprobed using P25 antibody raised in rabbit and in mice. The filter III was immunoprobed using fibroin L-chain antibody. After immunoblotting the membranes were kept together and photographed. Lane A, Molecular weight markers.

8 218 K Muthumani, S mathavan and S Mayilvahanan 3.4 SDS-PAGE and Western blot analysis Production of P 25 protein during the larval developmental period was analysed in 10% SDS-PAGE and stained with Coomassie blue (figure 5) and the same proteins from different developmental stages were transferred to a nitrocellulose filter and immunoblotted against P 25 antibody raised in rabbit (figure 6). A single positive band was observed in all five instars. The pattern obtained in the fifth instar shows increase in the concentration of P25 protein as a function of developmental period. Figure 5. Total larval protein From I. II und III instars and total silkgland proteins from IV and V instars were analysed in 10% SDS-PAGE. The panels A to Ε indicate the instars and the Arabic numerals indicate developmental period (day). The arrow indicates P 25 protein and the lane Ρ represents purified P25 protein (50µg). 150 µg protein was loaded in each lane. 3.5 Quantification of P25 protein Concentration of P25 protein was quantified using sandwich ELISA technique. Since it is extremely difficult to dissect out the silk gland for Ι, Π and III instars, total protein was extracted from the larvae and the concentration of P 25 protein was

9 Development regulation of silk protein P Figure 6. Total larval protein from I. II and III instars and total silkgland proteins from IV and V instar were extracted during the entire period of the larval development analysed in 10% SDS-PAGE and immunoblotted against P 25 ant i body raised in rabbits. The Roman numericals indicate the instar and the Arabic numericals indicate the developmental period (day). quantified. The concentration was represented as unit weight of protein/larva. In the IV and V instars, the P 25 protein was estimated from the total silkgland protein and represented as concentration of P 25 protein/pair of silkgland. P 25 protein level was 0 2 µg/animal on 1st day of I instar and it increased to 0 3 µg and 0 5 µg/animal on 2nd and 3rd day respectively. Subsequently the level decreased to 0 1 µg/animal on the 4th day of I instar (moulting period). Similarly, in the II instar the level of P 25 protein was 0 5, 0 8, 1 3, and 0 4 µg/animal on 1, 2, 3 and 4th day, respectively. Thus, there is a cyclic change in the protein level in each instar and the maximum level of the protein was on the third day of the instar (mid instar period). Almost the same pattern of cyclic change was observed in the III and IV instars, where the concentration of the protein increased from the 1st day to 3rd day of the instar and decreased on 4th day of the respective instars (figure 7A). The first three days of each instar represent the active feeding period and 4th day represents the premoulting and moulting period. Analysing the pattern of food consumption and utilization in an instar, Scriber and Slansky (1981) showed that the peak of relative rates of consumption, growth and metabolism occur prior to moulting (see also Waldbauer 1968: Mathavan and Pandian 1975; Muthukrishnan and Pandian 1987). The maximum level of P 25 protein observed in the present study at the pre moulting stages of each instar can be correlated to the maximum feeding period of the instar. Earlier workers have shown a correlation between feeding and major fibroin mrna synthesis and cyclic variation of fibroin and sericin mrna synthesis during development. With the initiation of feeding after each moult, the silk gene transcription was also initiated and continued till the larvae reduced the feeding and settled for moulting (Mackawa and Suzuki 1980: Prudhomme et al 1985). The transcription stopped after apolysis and pre-existing

10 220 Κ Muthumani, S mathavan and S Mayilvahanan Figure 7. A, B.

11 Development regulation of silk protein P molecules of mrna were degraded during the moulting process. The breakdown of mrna during apolysis has been recorded both for major fibroin and P 25 (Couble et al 1983). Maekawa and Suzuki (1980) showed that the production of fibroin mrna was mol gene -1 min -1 during the feeding period while it was mol gene -1 min -1 during the moulting period. Couble et al (1983) have shown the presence of residual P 25 mrna even during moulting process. The cyclical changes observed in the concentration of P 25 protein was similar to the pattern observed in mrna production. Most of the pre-existing P 25 protein was secreted out prior to each larval moulting and accumulation of the protein starts only with the initiation of feeding at subsequent instars. During moulting stages some P 25 residual protein was present in the silkgland. During the last instar period (V instar), the level of P 25 protein increased throughout the feeding period and the maximum was observed on the final day of the instar. It is contrary to the pattern observed in other instars where the maximum was observed in mid instar period. B. mori larvae consume more than 70% of the total food intake during the final instar and this period is characterized by active growth and specifically in silkgland weight (Hiratsuka 1920; Walbauer 1986). Maximum level of P 25 protein accumulation during the V instar can be correlated to the maximum feeding. Accumulation of the P 25 protein/pair of total silkgland/day was minimum-on first day (0 085 mg/silkgland/day) and steadily increased to mg/silkgland/day on 5th day and subsequently decreased (figure 7B). The cumulative accumulation of the stable P 25 protein is also shown in figure 7B. The level increased from 1st day to final day reaching maximum of 16 7 mg/pair of silkgland. The pattern of cumulative and daily accumulation of stable P 25 protein during V instar was compared with cumulative and daily accumulation of major fibroin protein (Shimura 1983). The cumulative accumulation of the P 25 protein was determined following the sandwich ELISA technique. The daily accumulation of the protein was deduced by subtracting the total P 25 at a particular day from the protein content in the previous day. For instance, the accumulation of P 25 protein on 5th day was deduced by subtracting the P 25 content at the end of fifth day from the P 25 protein content at the end of fourth day. Data reported by Shimura (1983) was used for comparison. It is known that P 25 protein is produced in equimolar concentration (1:1) like that of major fibroin (Couble et al 1983). The molecular weight of major fibroin is about 350,000 Dalton and P 25 is 25,000 Dalton. Assuming that they are expressed in equimolar concentration. P 25 protein should be 14 times less than the major fibroin. Shimura (1983) reported that the accumulation of stable major fibroin protein was about 240 mg/pair of silkgland on the final day of the V instar. In the present study we have estimated that the accumulation of P 25 protein was 16 7 mg/pair of silkgland on final day of the V instar, which is about 14 times less than the fibroin accumulation. On comparing the daily accumulation of P 25 and fibroin protein, the pattern is almost similar in both. Accumulation of P 25 protein during development is similar to that observed for fibroin proteins. Figure 7. Using the standard graph constructed by sandwich ELISA techniques the cumulative accumulation of stable P 25 protein was quantified per larva (A) in the I, II and III instars and per pair of silkgland (B) in IV and V instars. Daily accumulation of P 25 protein during V instar is also represented (B).

12 222 Κ Muthumani, S mathavan and S Mayilvahanan Acknowledgements Financial support received from the World Bank/Central Silk Board through a project (No. CSB: 46/107/ TS) to SM is gratefully acknowledged. SRF awarded to KM (CSIR/New Delhi) and SM (UGC) are acknowledged. We are thankful to Prof. Κ Dharmalingam, for providing facility for two-dimensional gel electrophoresis and to Prof. Shigeki Mizuno, Tohoku University, Sendai 981, Japan For providing fibroin L-chain antibody. References Chevillard M. Deleage G and Couble P (1986) Amino acid sequence and putative conformational characteristics of the 25 kd silk protein of Bombyx mori, Sericologia Cleveland D W. Fischer S G. Kirschner Μ W and Laemmli U Κ 1977 Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electruphoresis; J. Biol. Chem Couble P, Moine A. Garel A and Prudhomme J C 1983 Developmental variations of a nonfibroin mrna of Bombyx mori silk gland, encoding for a low molecular weight silk protein; Dev. Biol Couble P. Chevillard Μ, Moine A, Ravel-Chapuis Ρ und Prudhomme J C 1985 Structural organization of the P 25 gene of Bambyx mori and comparative analysis of its flanking DNA with that of the fibroin gene; Nucleic Acids Res Couble Ρ and Prudhomme J C 1993 The regulation of the silk P 25 gene in the silkgland of Bombyx mori; Indian J. Seric Durand Β. Drevet J and Couble J. Ρ 1992 P 25 Gene regulations in Bombyx mori silkgland 2: Promoter-binding factors have distinct tissue and developmental specificities; Mol. Cell. Biol Dorel C and Coulon Μ 1988 Regulation of gene expression in prediapausing embryos of the silkworm Bombyx mori: Pattern of protein synthesis; Cell Differ Hager D A and Durgess R R 1980 Elution of proteins from sodium sulfate poly acrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity. Results with Sigma subunit of E. coli RNA polymerase, wheat germ DNA topoisomerase and other enzymes: Anal. Biochem Hiratsuka Ε 1920 Researches on the nutrition of the silkworm; Bull. Serie. Exp. Station Tokyo Krishnaswami Κ 1978 New technology of silkworm rearing; CSR Tl Mysore (India) Bull Laemmli U Κ 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage Τ 4 : Nature (London) Lowry Ο Η, Rosebrough Ν J, Farr A L and Randall R J 1951 Protein measurement with the folin phenol reagent; J. Biol. Chem Maekawa Η and Suzuki Υ Repealed turn-off and turn-on of Fibroin gene transcription during silk gland development of Bombyx mori; Dev. Boil Mathavan S and Pandian Τ J 1975 Effect of temperature on food utilization in the monarch butterfly Danaus chrysippus; Oikos Mounier N, Coulon Μ and Prudhomme J C 1991 Expression of a cytoplasmic Actin gene in relation to the silk production cycle in the silk glands of Bombyx mori: insect Biochem Muthukrish J and Pandian T J 1987 Animal energetics(insecta) (eds) TJ Pandian and F John Vernberg (New York; Academic Press) vol 1, pp O'Farrel Ρ Η 1975 High resolution two dimensional electrophoresis of proteins; J. Biol. Chem Prudhomme J C. Couble P. Garel J P and Daillie J 1985 Silk synthesis, in Comprehensive insect physiology biochemistry and pharmacology (eds) G A Kerkut and L I Gilbert (New York: Pergamon Press) vol 10, pp Sasaki Τ and Noda Η 1973 Studies on silk fibroin of Bombyx mori directly extracted from the silk gland. 1, Molecular weight determination in guanidine hydrochloride or urea solutions; Biochim. Biophys. Acta Scriber J Μ and Slansky F Jr 1981 The Nutritional ecology of immature Insects; Annu. Rev. Entomol

13 Development regulation of silk protein P Shimura Κ 1983 Chemical composition and biosynthesis of silk proteins; Experientia Shimura K, Kikuchi A, Ohotomo K, Katagala Υ and Hyodo A 1976 Studies on the silk fibroin of Bombyx mori I. Fractionation of fibroin prepared from the posterior silk gland; J. Biochem. (Tokyo) Shimura Κ, Kikuchi A, Katagata Υ and Ohotomo Κ 1982 The occurrence of small component proteins in the cocoon fibroin of Bombyx mori; J. Sericult. Sci Jpn Temynck Τ Η and Avrameas S 1990 Immunoentymatic techniques (Amsterdam: Elsevier Publishing Company) vol 1, Towbin H, Staehellin Τ and Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedures and some application; Proc, Natl Acad. Sci USA Voller A, Bidwell D and Bartlett 1979 The Enzyme Linked Immuno Sorbant Assay (ELISA) (Alexandria, Virginia: Dyantech Laboratories) p 35 Yamaguchi K, Kikuchi Υ, Tokagi Τ, Kikuchi Α. Oyama F. Shimura Κ and Mizuno S 1989 Primary structure of the silk fibroin fight chain determined by cdna sequencing and peptide analysis; J Mol. Biol Waldbauer G P 1968 The consumption and utilization of food by insects: Adv. Insect Physiol