Purification and characterization of a biliverdin-binding protein from the molting fluid of the eri silkworm, Samia cynthia ricini (Lepidoptera: Saturniidae)

Comp. Biochem. PhysioL Vol. 105B,Nos 3/4, pp. 473-479, 1993 Printed in Great Britain

0305-0491/93$6.00+ 0.00 PergamonPress Ltd

PURIFICATION AND CHARACTERIZATION OF A BILIVERDIN-BINDING PROTEIN FROM THE MOLTING FLUID OF THE ERI SILKWORM, SAMIA CYNTHIA RICINI (LEPIDOPTERA: SATURNIIDAE) HITOSHISAITO* Department of Sericulture, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan (Received 4 January 1993; accepted 12 February 1993)

Abstract--1. The biliverdin-binding protein (BBP) was found in the molting fluid of the saturniid silkmoth, Samia cynthia ricini at the time of the larval-pupal ecdysis. 2. It was purified from the molting fluid using hydrophobic interaction chromatography, gel-filtration and high-performance liquid chromatography (HPLC). 3. The highly purified BBP showed a M~ of approximat*.ly 49,000 by gel permeation of HPLC analysis using a TSKgel G3000SW column. 4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band of Mr approximately 24,000, indicating that the native BBP is dimer. 5. The blue coloration of BBP from the molting fluid was due to the presence of biliverdin type pigments, as judged from its absorption spectra. 6. These results suggested that BBP from Samia molting fluid may be a new type ofinsecticyanin (INS).

Goodman et al., 1985; Holden et al., 1986, 1987). The larval epidermis of Manduca synthesizes I N S (Riddiford, 1982; Kiely and Riddiford, 1985), and the I N S are stored in granules within the larval epidermis and also secreted into both the cuticle and hemolymph (Riddiford et al., 1990). More recently, the two major isoelectric forms of Manduca insecticyanin (INS-a and INS-b) were found to be encoded by two different genes (Li and Riddiford, 1992). In the saturniid silkmoth, Samia cynthia ricini, blue pigment is seen only in the cuticle of fourth and fifth instar larvae, and this pigment is intimately associated with a protein. We have not found this pigment in the hemolymph, epidermis or any other organs during either the larval or pupal stages. However, a particularly rich source of a biliverdin-binding protein (BBP) is found in the molting fluid during the larval-pupal ecdysis. In this paper, I report the purification and characterization of BBP from the molting fluid of Samia.

INTRODUCTION

Blue-green coloration has been observed in the integument and hemolymph of several insects. It is generally known that the blue-green pigmentation is due to two components of the blue pigments: the bile pigments, most often biliverdins, and yellow pigments, usually carotenoids. These pigments are usually associated with protein (Kayser, 1985; Law and Wells, 1989). Bile pigments have been isolated and identified in the integument and hemolymph of several insect species (Fuzeau-Braesch, 1985; Kayser, 1985). The bilin binding protein of the butterfly, Pieris brassicae has been crystallized and analyzed for molecular structure (Huber et al., 1987a, b). Chino and his coworkers have also reported the purification and characterization of a biliverdin-binding cyanoprotein from the hemolymph of Locusta migratoria (Chino et al., 1983). In the tobacco hornworm, Manduca sexta, the blue biliprotein, insecticyanin (INS) has been purified and characterized from the hemolymph and epidermal cells of the larvae (Cherbas, 1973; Riley et al., 1984; *Present address: Department of Bioscience, Kitasato University School of Hygienic Sciences, Sagamihara, Kanagawa 228, Japan (Tel.: 0427-78-9487). Abbreviations--BBP, biliverdin-binding protein; INS, insecticyanin; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; STI, soybean trypsin inhibitor; HLPC, high-performance liquid chromatography.

MATERIALS AND METHODS

Animals

Larvae of the saturniid silkmoth, Samia cynthia ricini, were reared on fresh leaves of Ailanthus glandulosa at 25°C under a natural photoperiod. The larval stages took 21 days and pupal ecdysis occurred usually 4 days after spinning, when the pharate-pupae were maintained at 25°C under a photoregime of 16 hr light-8 hr dark.

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Materials PMSF, Coomassie Brilliant Blue R-250 and biliverdin (dihydrochloride form) were obtained from Sigma Chemical Co. (St Louis, MO). PhenylSepharose CL-4B, Sephacryl S-200 HR and Mono Q HR 5/5 were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). The HPLC column of TSKgel G3000SW Glass for gel-filtration was obtained from Tosoh (Tokyo, Japan). All other reagents used were of the highest grade available commercially.

Preparation of molting fluid

KCI in buffer A. The volumes of BBP fractions were reduced to 0.2 ml by an Ultrafree CL filter (Millipore Japan Ltd, Tokyo). (4) Gel-permeation HPLC on TSKgel G3OOOSW. The Mono Q fraction was further purified by HPLC using a column (0.8 cm i.d. × 30cm) of TSKgel G3000SW Glass. Purified BBP solution (100 #1) was injected, then the BBP was eluted with 0.2 M KCI containing 50mM phosphate buffer (pH 6.8) at a flow rate of 0.6 ml/min under a constant pressure of 10 kgf/cm z. The peak fraction obtained was used to determine the molecular weight. For polyacrylamide gel electrophoretic analysis, BBP was concentrated using an Ultrafree C3 filter (Millipore Japan Ltd, Tokyo).

Molting fluid appeared in the exuvial space between the new and old cuticles during the last part of the pupal molt. This fluid persisted for a short period of about 1-2 hr before pupal ecdysis. Molting fluid samples (5 ml) were obtained from pharate-pupae by cutting the dorsal region of their abdominal knobs and caudal legs. Samples were dropped into saturated ammonium sulfate solution and concentrated by centrifugation at 10,000g for 20min at 4°C. The precipitate dissolved in 10ml buffer A[200mM Tris-HCl buffer (pH 7.8)/0.1 mM PMSF] containing ammonium sulfate at 40% saturation. The precipitate was removed by centrifugation at 10,000g for 20 min at 4°C, then the supernatant was used as the crude extract for purification of a biliverdin-binding protein (BBP). In addition, molting fluid stored at - 30°C for up to 5 years showed no BBP degradation.

Subunit analysis was carried out on SDS-PAGE. SDS-PAGE was performed on 15% polyacrylamide gel containing 0.1% SDS (Laemmli, 1970). Proteins were stained with Coomassie Brilliant Blue R-250. Molecular weight was estimated using low molecular weight calibration kit from Pharmacia Fine Chemicals (Uppsala, Sweden).

Purification of a biliverdin-binding protein (BBP)

Amino acid analysis

All procedures were carried out at 4°C or in ice, unless otherwise stated. (1) Hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B. The crude extract was applied on a column (l.5cm i . d . × 8 c m ) of Phenyl-Sepharose CL-4B, previously equilibrated with buffer A containing 40% saturation of ammonium sulfate. The column was eluted with a linear gradient of 40-0% saturation of ammonium sulfate in buffer A. BBP fractions were pooled and dialyzed against three changes of buffer A containing 0.1 M KC1 overnight. The dialyzed solutions were concentrated to 0.5 ml by ultrafiltration with a Minimodule NM-3 fiber filter (Asahikasei, Tokyo). (2) Gel-filtration on Sephacryl S-200. The Phenylfraction applied on a gel-filtration column (1.2 cm i.d. x 105era) of Sephacryl S-200 HR, previously equilibrated with buffer A containing 0.1 M KC1 at a flow rate of 4.5ml/hr. These BBP fractions were pooled and concentrated to 1 ml using a Minimodule NM-3 fiber filter. (3) HPLC on Mono Q. The S-200 fraction was subjected to HPLC (Model Bio-LC system, Tosoh, Tokyo) using a column on Mono Q HR 5/5 (0.5 cm i.d. x 5 era), previously equilibrated with buffer A containing 0.05 M KC1. The column was separately eluted with a linear gradient between 0.05 and 0.4 M

The purified BBP (5 #g) was hydrolyzed with 6 M HCI for 21 hr at 110°C. After hydrolysis, the sample was analyzed on a Waters Pico-Tag amino acid analysis system.

Detection of BBP BBP in the indicated fractions was determined by measuring the absorbance at 668 nm with a spectrophotometer (Shimadzu UV-150, Kyoto). The absorption spectra of BBP was measured with a spectrophotometer (Shimadzu MPS-2000, Kyoto).

Polyacrylamide gel electrophoresis (PAGE)

Protein determination Protein content was determined by a Bio-Rad protein assay kit from Bio-Rad Laboratories (Richmond, CA) using bovine serum albumin (BSA) as a standard protein. RESULTS

Purification of BBP from the molting fluid The molting fluid collected from the exuvial space at the pharate-pupal stage contained about 3 mg BBP/ml. The first step of hydrophobic interaction chromatography on a Phenyl-Sepharose CL-4B column of the molting fluid extracts showed that most protein components bound to the column (Fig. 1). The blue peak of BBP was eluted with fractions (55-68) of the major peak at 20-10% saturation of ammonium sulfate. Thus, Phenyl-Sepharose column chromatography is a reliable first step in the largescale purification of BBP. The Phenyl-fraction was collected and dialyzed against buffer A containing 0.1 M KC1. The dialyzed sample was concentrated,

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Determination of the molecular weight

then put on a column of Sephacryl S-200 gel-filtration and duted with the same buffer (Fig. 2). Further purification was obtained by HPLC on Mono Q column. The blue peak of BBP eluted as a symmetric sharp peak at 0.2 M KC1 (Fig. 3). The BBP was greatly enriched at this step (Fig. 5A, lane 5). The purification procedure of BBP from the Samia molting fluid is summarized in Table 1, which shows the recovery and yield at each step for a typical preparation. Our standard purification scheme allowed us to obtain about 1.6 mg of the purified BBP from 137 mg of Samia molting fluid. During purification, the specific activity was increased 10.5-fold with 12% recovery.

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Amino acid composition o f BBP The amino acid composition of the biliverdin-binding protein from Samia molting fluid is shown in Table 2 and compared with amino acid composition of the biliverdin-binding proteins from two distinct insect species, the Locusta cyanoprotein and the Manduca insecticyanin. The BBP of Samia contains high amounts of aspartic acid and valine, and low amounts of methionine and cysteine. Except for the high valine, these characteristics are also found in the other two proteins.

Identification o f chromophore The absorption spectra for purified BBP are shown in Fig. 6 together with the spectrum of standard biliverdin for comparison. The purified protein dissolved in 50 mM phosphate buffer (pH 6.8) has three characteristic absorption peaks at 278, 382 and 668 nm, while under the same conditions a standard biliverdin exhibits peaks at 375 and 655nm. In addition, the absorption spectra of the extracted blue pigments from BBP was essentially similar to the absorbance spectrum of standard biliverdin dissolved with 80% methanol (data not shown). These obser-

vations suggest that the blue pigments of BBP are biliverdin, or at least a closely related compound. DISCUSSION The blue pile pigments, biliverdins, have been found with a wide distribution in the insect species (Kayser, 1985). Especially, biliverdin IX 7 is most common in the lepidopteran insects. For example, biliverdin has been detected in the wings of Lcpidoptera (Barbier, 1981, 1983; Kayser, 1985; Huber et al., 1987a,b), the bemolymph and cocoons of Antheraea yamamai (Kate et al., 1989; Yamada and Kate, 1989, 1991), the hemolymph of Locusta migratoria (Chino et al., 1983) and the hemolymph of Manduca sexta (Cherbas, 1973; Riley et al., 1984; Goodman et al., 1985; Law and Wells, 1989). These pigments are usually associated with proteins as pigment-protein complexes. However, the physiological and biological function of the insect bile proteins is unclear. We have observed that the blue pigments are confined to the cuticle and transported into the molting fluid during the pupal molt of Samia cynthia ricini. This pigment is not detected in the hemolymph and epidermal cells throughout the larval or pupal stage. In the present study, we purified a biliverdinbinding protein (BBP) from the molting fluid of Samia at pupal molt. This BBP was purified by a

Table 1. Purificationof BBP from Sam/a moltingfluid Total protein Total activity* Specificactivity Purificationstep (rag) (units) (units/mg) 1. Moltingfluid 137.0 24.3 0.18 2. 40% (NH4)2SO4 73.1 23.9 0.33 3. Phenyl-SepharoseCL-4B 26.4 12.3 0.47 4. SephaerylS-200 2.3 3.9 1.70 5. Mono Q 2.1 3.8 1.81 6. TSKgelG3000SW 1.6 3.0 1.89 *One unit represented 1.0 absorbanceat 668nm.

Yield (%) 100 98 51 16 16 12

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The Samia BBP shows a native M, approximately 49,000 as determined by gel-filtration of HPLC (Fig. 4B) and apparently is a dimer of identical subunits with a M, 24,000 (Fig. 5B). In contrast to Samia, the M, of cyanoprotein from Locusta hemolymph is considerably larger (M, approximately 350,000) and is composed of four identical M, 83,000 subunits (Chino et al., 1983). Insecticyanin (INS) from Manduca hemolymph has a Mr of 71,600 and appears to be a trimer of three subunits of Mr 23,000 (Goodman et al., 1985), yet its apoprotein is a tetramer with a

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Table 2. Aminoacid compositionof BBP Amino BBP from Samia Cyanoprotein from Insecticyaninfrom acid molting fluid Locusta hemolymph* Manduca hemolympht Asp 11.3 11.7 13.5 Glu 6.7 9.3 7.1 Ser 8.1 5.1 4.3 Gly 8.3 6.2 6.5 His 3.5 2.9 3.1 Arg 4.4 4.9 1.0 Thr 6.0 4.2 g.0 Ala 6.7 6.8 7.8 Pro 3.3 7.0 3.7 Tyr 4.7 6.0 7.0 Val 10.9 7.5 7.7 Met 0.5 2.0 0.8 Cys 1.3 -1.7 Ile 4.8 4.7 4.2 Leu 6.1 9.2 5.8 Ph¢ 6.2 6.5 4.4 Lys 6.7 7.8 9.4 Tryptophan was not determined. *Chino et al. (1983). tRiley et al. (1984).

single polypeptide chain of 189 amino acid residues of Mr 21,378 and two disulfide bridges (Riley et al., 1984). INS has been crystallized in a form suitable for high resolution X-ray analysis (Holden et al., 1986) and its tetrameric molecular structure determined at 2.6 A, resolution (Holden et al., 1987). The crystallographic asymmetric unit of the Pieris bilin binding protein contains a tetramer of identical subunits with a M, approximately 90,000 (Huber et aL, 1987a). Interestingly, a comparison of the amino acid composition of BBP with other insects indicates no homology (Table 2). Thus, the BBP of S a m i a is quite different from that of the biliverdin-binding proteins in other insect species. The larval epidermis of M a n d u c a synthesizes and stores two major isoelectric forms of INS (INS-a and INS-b), of identical Mr, but separated by charge. Both INS-a and INS-b are stored in the pigment granules, but INS-b is the only form secreted into the

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hemolymph (Riddiford, 1982; Kiely and Riddiford, 1985; Riddiford et al., 1990). Both forms are secreted into the cuticle (Riddiford et al., 1990). More recently, Li and Riddiford (1992) have reported that two distinct genes encode two major isoelectric forms (INS-a, pI 5.5 and INS-b, pI 5.7) of INS. In contrast, the BBP of S a m i a is synthesized in the larval epidermis and secreted into the cuticle, but it cannot be found in the hemolymph throughout the larval-pupal stage. In the preliminary isoelectric focusing experiments, the BBP from the molting fluid was found to have two isoelectric forms, which are more basic (pI 8.3 and pI 8.5) than INS-a and INS-b (data not shown). The functional significance of the different isoelectric forms of BBP is still unclear. In this study, we describe the purification and characterization of a biliverdin-binding protein (BBP) from the S a m i a molting fluid. The purification of BBP and identification of its chromophore should help to elucidate the physiological function of this protein during the larval-pupal molt. The physiological function of BBP remains unknown, although it seems likely that BBP is essential in the camouflage system during the larval feeding stage. Presently, one of the interesting aspects of BBP is its physiological role of absorption of the molting fluid during the larval-pupal ecdysis. It is generally believed that the ability to absorb the molting fluid is a generalized property of the integument and that absorption takes place through the general surface of the new cuticle. However, it is not known whether specific regions of the integument are specialized as sites of absorption (Lensky et al., 1970). This BBP will be an effective marker protein for the study of the mechanism of the concentration and absorption of the cuticle proteins in the molting fluid during the larval-pupal transformation. Thus, this insect may serve as a useful model for further research on molting fluid resorption. Finally, these results suggest that BBP from S a m i a

Biliverdin-binding protein in Sam/a molting fluid molting fluid may be a new type of INS. Further studies are needed on the determination of the subunit composition of BBP and comparison of the amino acid sequence of BBP with INS from Manduca hemolymph. It would be useful in the elucidation of the principles underlying the life strategies and regulation of color changes among the insects. Acknowledgements--The author thanks Professor L. M. Riddiford, University of Washington, for useful comments and critical reading of this manuscript. The author also thanks Dr K. Dohke, Kitasato University Sachool of Liberal Arts and Sciences for his valuable comments, and Dr K. Kiguchi, National Institute of Sericultural and Entomological Science, Japan, for his helpful advice. Thanks are also due to Emeritus Professor F. Mukaiyama, Tokyo University of Agriculture and Technology, for provision of certain facilities in his laboratory.

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crystal structure analysis and preliminary molecular model of the bilin binding protein from the insect Pieris brassicae. J. molec. Biol. 195, 423-434. Huber R., Schneider M., Mayr I., Muller R., Deutzmann R., Suter F., Zuber H., Falk H. and Kayser H. (1987b) Molecular structure of the bilin binding protein (BBP) from Pieris brassicae after refinement at 2.0 A resolution. J. molec. Biol. 198, 499-513. Kato Y., Onuma Y., Sakurai K. and Yamada H. (1989) Role of light in green pigmentation of cocoons of Anteraea yamamai (Lepidoptera: Saturniidae). Appl. Ent. Zool. 24, 398-406. Kayser H. (1985) Pigments. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol 10, pp. 367-415. Pergamon Press, Oxford. Kiely M. L. and Riddiford L. M. (1985) Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation of Manduca sexta. Roux's Arch. Dev. Biol. 194, 325-335. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Law J. H. and Wells M. A. (1989) Insects as biochemical models. J. biol. Chem. 264, 16335-16338. Lensky Y., Cohen C. and Schneiderman H. A. (1970) The origin, distribution and fate of the molting fluid proteins of the Cecropia silkworm. Biol. Bull. 139, 277-295. Li W.-c. and Riddiford L. M. (1992) Two distinct genes encode two major isoelectric forms of insecticyanin in the tobacco horuworm. Manduca sexta. Fur. J. Biochem. 205, 491-499. Riddiford L. M. (1982) Changes in translatable mRNAs during the larval-pupal transformation of the epidermis of the tobacco hornworm. Dev. Biol. 92, 330-342. Riddiford L. M., Palli S. R., Hiruma K., Li W.-C., Green J., Hice R. H., Wolfgang W. J. and Webb B. A. (1990) Developmental expression, synthesis, and secretion of insecticyanin by the epidermis of the tobacco hornworm, Manduca sexta. Arch. Insect Biochem. Physiol. 14, 171-190. Riley C. T., Barbeau B. K., Keim P. S., Kezdy F. J., Heinrikson R. L. and Law J. H. (1984) The covalent protein structure of insecticyanin, a blue biliprotein from the hemolymph of the tobacco hornworm, Manduca sexta L. J. biol. Chem. 259, 13159-13165. Yamada H. and Kato Y. (1989) Purification and properties of a blue chromoprotein in the haemolymph of the Japanese oak silkworm Antheraea yamamai. In Wild Silkmoths '88 (Edited by Akai H. and Wu Z. S.), pp. 41-48. International Society for Wild Silkmoths. Yamada H. and Kato Y. (1991) Characteristic properties of a blue chromoprotein in larval haemolymph of Antheraea yamamai. In Wild Silkmoths '89. '90 (Edited by Akai H. and Kiuchi M.), pp. 89-95. International Society for Wild Silkmoths.