Isolation of Three Main Sericin Components from the Cocoon of the ...

Biosci. Biotechnol. Biochem., 66 (12), 2715–2718, 2002

Note

Isolation of Three Main Sericin Components from the Cocoon of the Silkworm, Bombyx mori Yoko TAKASU,† Hiromi YAMADA, and Kozo TSUBOUCHI National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba, Ibaraki 305-8634, Japan Received June 7, 2002; Accepted July 31, 2002

To characterize the sericin components of the cocoon of silkworm Bombyx mori, fresh cocoon shells were dissolved in saturated aqueous lithium thiocyanate containing 2-mercaptoethanol, and fractionated by ethanol precipitation. Cocoon sericin was found to mainly consist of three polypeptides having molecular masses of the 400, 250, and 150 kDa estimated by SDS-PAGE, which corresponds to the sericin present in the middle, anterior, and posterior part of the middle silk gland. The amino acid compositions of the 400 and 150 kDa components were similar to each other, but that of the 250 kDa component was diŠerent. This suggests diŠerences in the coding gene and properties of the 250 kDa sericin from the other two. Key words:

sericin; Bombyx mori; SDS-PAGE; cocoon; middle silk gland

Sericin is a group of glue proteins produced in the middle silk gland (MSG) of the silkworm, Bombyx mori. It surrounds ˆbroin ˆbers and ˆxes them to each other in the cocoons. The molecular heterogeneity of sericin had been controversial for a long time, and a lot of researchers have tried molecular separation of sericin. Mosher1) extracted sericin from the cocoon by boiling water and divided it into the insoluble and soluble fractions at pH 4.1. Simizu2) and Komatsu3) tried to fractionate cocoon sericin by the diŠerence of the dissolution rate in boiling water. These studies, however, could neither prove the heterogeneity of sericin nor achieve molecular separation, because Gamo's report showed that sericin was degraded by boiling water.4) The molecular heterogeneity of sericin was ˆrst demonstrated by analyzing native molecular proteins in the silk gland; Tashiro and Otsuki showed three kinds of sericin by ultracentrifugation,5) and Gamo separated at least six sericins by PAGE.4) Gamo et al. estimated the molecular masses of sericins to be 309, 177, 145, 80, and 134 kDa,6) while Sprague reported at least 15 diŠerent sericins ranging from about 20 to 220 kDa in the anterior portion of the middle silk gland.7) Recent studies on sericin genes provided further information

about the constituents of sericin. The Ser1 gene was reported to produce mRNAs with sizes of 10.5, 9.0, 4.0, and 2.8 kb by alternative splicing,8) and the Ser2 gene was found to produce mRNAs with sizes of 6.4 or 5.0 and 3.1 kb.9) In spite of these studies, we have not reached a consensus about the constitution of sericin, and especially, there has been no attempt to estimate sericin components quantitatively. Additionally, the properties of individual sericin polypeptides were not studied, since no eŠective separation of sericins was established, although the diŠerences of the properties among sericins are expected to be closely related to the diversity of sericin molecules. In this study we intended to clarify the predominant polypeptides of sericin and to establish the isolation procedures of them. Fresh cocoons of silkworms ( Bombyx mori ) (C145 ×J140) reared on an artiˆcial diet (Nihon Nosan Kogyo) were used throughout the experiments. To obtain the whole cocoon protein solution, a 50-mg piece of the cocoon shell was peeled into several ‰akes and dissolved at room temperature in 2 ml of sat. LiSCN, which was prepared by adding 10 ml of water to 25 g of LiSCN hydrate (Kanto Chemicals). After centrifugation at 3000 rpm for 15 min, about 2.5z of the silk protein solution was obtained as the supernatant. Silk gland protein solution containing sericin was prepared according to the method of Simizu et al.10) One of the MSG was removed from a mature larva, washed in water, and cut into three parts at the windings. The gland cells were taken away from each part in water with forceps, and the contents of the silk gland were put into beakers with water, which were shaken gently for 30 min. The supernatant was used as the silk protein solution from the MSG. Sample solutions for SDS-PAGE were prepared by adding the same volume of sample buŠer (0.1 M sodium phosphate buŠer of pH 7, containing 6 M urea, 1z SDS, 2z 2-mercaptoethanol, and 0.025z bromophenol blue) to each of the protein solutions. When the protein solution contained excess electro-

To whom correspondence should be addressed. Tel W Fax: +81-298-38-6265; E-mail: takasu＠aŠrc.go.jp Abbreviations : MSG, middle silk gland; sat. LiSCN, saturated aqueous lithium thiocyanate solution

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Fig. 1. SDS-PAGE of Cocoon Proteins. Total cocoon protein (lane t) was prepared by the method described in the text. Fibroin solution was prepared by dissolving the degummed cocoon in sat. LiSCN. The degummed cocoon was obtained by removing sericin, in 8 M urea containing 2z 2mercaptoethanol at 809C for 2 h, from the cocoon shell (lane f ). Silk gland proteins of anterior (lane a), middle (lane m), and posterior (lane p) parts of the MSG were extracted in the method described in the text. M L and MH indicate low and high molecular mass markers, respectively. Fibroin heavy chains and light chain are indicated by f-H and f-L, sericins A, M, and P are by s-A, s-M, and s-P, respectively, and molecular masses are expressed in kDa.

lytes such as LiSCN, the solvent was changed to 40 mM Tris-sulfate buŠer (pH 8) containing 8 M urea using Sephadex G-25 (PD-10, Amersham Pharmacia Biotech) before adding sample buŠer. Each sample solution was incubated at 609C for 15 min and put on a 2-15z gradient gel purchased from Daiichi Pure Chemicals. Electrophoresis was done in 0.1 M sodium phosphate buŠer of pH 7 with 0.1z SDS at the current density of 40 mA W cm2, and then polypeptides were stained with Coomassie Brilliant Blue R. When the silk protein solution obtained by dissolving fresh cocoons in aqueous LiSCN was analyzed by SDS-PAGE, seven polypeptide bands, the molecular masses of which were from about 20 to over 400 kDa, were observed (Fig. 1, lane t). Among them the two dense bands above 300 kDa and another around 20 kDa are derived from ˆbroin, since the cocoon protein after removal of sericin showed only the three bands corresponding to them (Fig. 1, lane f ). The bands other than those derived from ˆbroin were considered to correspond to the main components of sericin. The largest polypeptide, which moved a little more slowly than ˆbroin heavy chain, was estimated to have a molecular mass of around 400 kDa. As it was considered to be the same component as seen mainly in the middle part of the MSG (Fig. 1, lane m), it was named sericin M. The two close bands around 250 kDa, which were considered to be the products of alleles, were named sericin A, because their sizes agreed with the polypeptide which was speciˆcally found in the anterior part of the

Fig. 2. Fractionation of Silk Proteins by Ethanol Precipitation in Sat. LiSCN. A cocoon shell (0.5 g) was dissolved in 10 ml of LiSCN, into which ethanol was added stepwise, and the generated precipitate was analyzed by SDS-PAGE. The volumes of added ethanol were 18, 20, 22, 24, 26.5, 35, 40, 42, 52, 62, and 82 ml (lane 1–11), respectively. The dry weights of the obtained fractions 1–11 were 14, 2, 95, 8, 6, 4, 281, 1, 1, º1, and º1 mg, respectively. Lane M, molecular marker. Abbreviations are to be referred to Fig. 1.

MSG (Fig. 1, lane a). The clear band at about 150 kDa was observed abundantly in the posterior part of the MSG (Fig. 1, lane p) and thus named sericin P. The trace near the top of lane t was considered to be some aggregation formed in the cocoon secondarily, because the corresponding band could not be found in the polypeptides in the MSG. Fractionation of the whole cocoon protein was done by adding ethanol, portion by portion, into an about 5z protein solution in sat. LiSCN containing 2z 2-mercaptoethanol, while stirring. The precipitates generated were collected at intervals by centrifugation. The total amount of added ethanol was 82 ml to 10 ml of the protein solution, and eleven fractions were obtained as precipitates. A small part of each fraction was analyzed by SDS-PAGE after it was dissolved in sat. LiSCN (Fig. 2). The result showed that each cocoon protein precipitates at a particular concentration of ethanol. The main components of the cocoon protein were fractionated as precipitates in the following order: sericin A in fractions 1 and 2, sericin M in fractions 3 and 4, sericin P in fractions 5 and 6, ˆbroin heavy chain in fractions 7 and 8, and ˆbroin light chain in fractions 9–11. The sum of fractions 3 and 4 was 103 mg, but the sum of fractions 1 and 2 was 16 mg, and that of fractions 5 and 6 was 10 mg, which implied that sericin M exists much more abundantly than sericins A and P. To purify the three main components of sericin, a whole sericin solution free from ˆbroin was prepared by extracting the ˆnely peeled cocoon pieces in 8 M urea containing 2z 2-mercaptoethanol for 5 min at 809 C.11) After residual ˆbers were removed, the extract was centrifuged, and then an about 0.7z sericin solution was obtained as the supernatant. The extracted sericin was once collected as a precipitate by

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Isolation of Sericins from the Cocoon of Bombyx mori

Table 1. Amino Acid Compositions of Sericins (in molez) a.a.

Fig. 3. SDS-PAGE of the Puriêd Sericin Samples. Three main components of sericin were puriêd by the method described in the text. Puriêd sericins A, M, and P were analyzed by SDS-PAGE in, respectively, lanes A, M, and P. Lane MH, molecular marker.

Gly Ala Val Leu Ile Ser Thr Asp+Asn Glu+Gln Lys Arg His Tyr Phe Pro Trp Met Cys —: nd:

adding 3 times the solution volume of ethanol and then dissolved in sat. LiSCN again for further puriˆcation. Sericin A was collected as the precipitate at the ethanol concentration below 65z and puriêd by a subsequent repetition of dissolving and precipitation. In the same way, sericin M was obtained from the fraction at the ethanol content between 67z and 71z, and sericin P, from the fraction between 72z and 75z (Fig. 3). To analyze the amino acid composition, each puriêd sample was hydrolyzed in 6 N HCl at 1109 C for 24 h, and put through a Hitachi L-8800 apparatus. Alternatively, it was hydrolyzed in 6 N HCl at 1509C for 1 h and then analyzed by the PTC method.12) Table 1 shows amino acid compositions of sericins A, M, and P. All three sericin components showed high contents of serine, glycine, and aspartic acid (containing asparagine), which has been known as a common feature of sericin. Besides, sericin A showed a characteristic proˆle in which the contents of glutamic acid (containing glutamine) and lysine were rich but those of threonine and tyrosine were less, compared to sericins M and P. These diŠerences in amino acid compositions between sericin A and the others suggest two important aspects of the sericins. One relates to the correspondences of the three polypeptides to sericin genes: sericins1 deduced from the Ser1 gene8) showed the same characteristics as sericins M and P, and S-2 polypeptide produced by the Src-2 gene13) was very similar to sericin A (Table 1, Ser1C is one of sericins1), when attention was focused on the contents of glutamic acid containing glutamine, threonine, lysine, and tyrosine. This suggests the possibilities that sericins M and P are the products of the Ser1 gene, and that sericin A is identical to the S-2 polypeptide produced by the Src-2 gene. The correspondences cannot be

Sericin A Sericin M Sericin P Ser1C8) S-213) 250 kDa 400 kDa 150 kDa 331 kDa 227 kDa 14.3 5.5 0.7 0.5 0.2 39.0 3.3 13.3 12.8 5.4 2.9 1.0 0.7 0.4 nd — nd 0.1

16.0 4.1 3.2 0.9 0.5 35.4 9.7 15.7 3.1 1.8 3.4 1.3 4.0 0.2 0.6 — nd 0.0

14.1 8.1 3.9 1.6 0.8 33.2 12.2 11.3 3.1 1.0 4.0 nd 4.6 0.7 1.3 — nd nd

13.4 3.8 3.4 0.6 0.5 38.0 9.9 14.6 2.5 2.1 4.7 1.0 4.4 0.3 0.3 0.3 0.1 0.1

14.9 4.4 1.2 1.4 0.6 36.8 4.0 14.9 11.1 6.0 3.2 1.0 0.1 0.4 nd — nd nd

not determined not detected

proved only by the present data in Table 1 regarding the discrepancies in some amino acids, but the possibilities are thought to be high, considering the experimental errors and the polymorphism among the silkworm races and individuals. Another aspect is noted by reference to the conformational preferences of amino acids in globular proteins:14) tyrosine and threonine are classiêd into the b-sheet favoring residues, but glutamic acid, glutamine, and lysine are put into the b-sheet breaking group. This leads to the prediction that sericin A is a less b-forming protein than sericins M and P, which agrees with our data that the solubility of sericins M and P is decreased by steaming, but not that of sericin A (unpublished data), because the b-structure formation decreases the solubility of sericin15) and bstructure of sericin was proved to be formed through humidity.16) This study clariêd that sericin is mainly composed of three polypeptides, sericins A, M, and P, together with presenting a practical method to isolate them. Hereafter, this method can be eŠectively used to characterize each sericin molecule, especially in its properties as a structural protein.

Acknowledgments We would like to thank Dr. M. Kiuchi for oŠering the mature silkworms used in this study, and Ms. Y. Igarashi for complementing amino acid analysis data. This work was supported by the Center of Excellence, Special Coordination Funds for Promoting Science and Technology, Science and Technology Agency, Japan.

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References 1) 2) 3)

4)

5)

6)

7)

8)

Mosher, H., The sericin fractions of silk. Am. Silk Rayon J., 53, 43–44 (1934). Simizu, M., X-ray studies on silkworm sericin. Bull. Sericult. Exp. Sta. (in Japanese), 10, 441–474 (1941). Komatsu, K., Studies on dissolution behaviors and structural characteristics of silk sericin. Bull. Sericult. Exp. Sta. (in Japanese), 26, 135–256 (1975). Gamo, T., Electrophoretic analyses of the protein extracted with disulphide cleavage from cocoons of the silkworm, Bombyx mori. J. Sericult. Sci. Jpn. (in Japanese), 42, 17–23 (1973). Tashiro, Y., and Otsuki, E., Studies on the posterior silk gland of the silkworm Bombyx mori IV. Ultracentrifugal analyses of native silk proteins, especially ˆbroin extracted from the middle silk gland of the mature silkworm. J. Cell Biol., 46, 1–16 (1970). Gamo, T., Inokuchi, T., and Laufer, H., Polypeptides of ˆbroin and sericin secreted from the diŠerent sections of the silk gland in Bombyx mori. Insect Biochem., 7, 285–295 (1977). Sprague, K., The Bombyx mori silk proteins: characterization of large polypeptides. Biochemistry, 14, 925–931 (1975). Garel, A., Deleage, G., and Prudhomme, J., Structure and organization of the Bombyx mori sericin 1 gene and of the sericins 1 deduced from the sequence

9)

10)

11)

12)

13)

14)

15) 16)

of the Ser 1B cDNA. Insect Biochem. Molec. Biol., 27, 469–477 (1997). Michaille, J., Garel, A., and Prudhomme, J., Cloning and characterization of the highly polymorphic Ser2 gene of Bombyx mori. Gene, 86, 177–184 (1990). Simizu, M., Fukuda, T., and Kirimura, J., Chemistry of Proteins 5. Kyoritsu Shuppan Co., Tokyo (in Japanese), pp. 317–377 (1957). Takasu, Y., Yamada, H., and Tsubouchi, K., Extraction and chromatographic analysis of cocoon sericin of the silkworm Bombyx mori. J. Insect Biotech. Sericology, 71, 151–156 (2002). Kobayashi, R., and Tarr, G. E., Protein sequencing analysis by manual Edman degradation. Protein Nucleic Acid and Enzyme (in Japanese), 31, 991–1002 (1986). Gamo, T., Genetic variants of the Bombyx mori silkworm encoding sericin proteins of diŠerent lengths. Biochem. Genet., 20, 165–177 (1982). Levitt, M., Conformational preferences of amino acids in globular proteins. Biochemistry, 17, 4277– 4285 (1978). Kataoka, K., On the solubility of sericin in water. J. Sericult. Sci. Jpn. (in Japanese), 46, 227–230 (1977). Kataoka, K., On the amorphousª b transition of silk sericin. Kobunshi Ronbunshu (in Japanese), 34, 1–6 (1977).