Purification and characterization of three storage proteins in the common cutworm, Spodoptera litura

Pergamon 0965-1748(93)E0021-T

Insect Biochem. Molec. Biol. Vol. 24, No. 7, pp. 729 738, 1994 Copyright © 1994 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0965-1748/94 $7.00 + 0.00

Purification and Characterization of Three Storage Proteins in the Common Cutworm, Spodoptera litura SUMIO TOJO,*'~ TOYOSHI YOSHIGA*

Received 30 July 1993; revised and accepted 3 December 1993

Three storage proteins named SL-1, SL-2 and SL-3, the former two being synthesized only in the last larval instar, were purified from haemolymph of the common cutworm, Spodoptera litura. All three storage proteins have molecular sizes between 400 and 450 kDa, and are composed of subunit(s) which range in size from 70 to 80 kDa. Chemical cross-linking confirmed that these storage proteins are hexamers. SL-1 and -2 are basic proteins showing homogeneous amino acid compositions with c. 10% aromatic amino acids, the former being rich in methionine. Both are cross-reactive to antiserum against SP-1 (methionine-rich storage protein) of Bombyx mori at the native molecular level, but only SL-I is cross-reaction to it at the polypeptide level. SL-3 is a neutral protein with an amino acid composition that differs considerably from those of SL-1 and -2, having 20% aromatic amino acids. It is cross-reactive only to antiserum against SP-2 (arylphorin) of B. mori, both at native and subnnit molecular levels. From these results, it was concluded that SL-1 and -2 are 'methionine-rich' storage proteins with similar conformations but with different epitopes in subunit molecules, while SL-3 is an aryiphorin.

Storage protein Arylphorin Methionine-rich storage protein Storage hexamer Haemolymph Spodoptera litura

INTRODUCTION

In holometabolous insects, storage proteins are synthesized by the fat bodies and secreted into the haemolymph. Their concentrations increase markedly in the final larval instar, and then they are partially or wholly sequestered by the fat bodies (reviews: Riddiford and Law, 1983; Levenbook, 1985; Kanost et al., 1990; Teller and Kunkel, 1991).

In Lepidoptera, there are at least two kinds of storage proteins, i.e., arylphorin, which is rich in aromatic amino acids, and storage protein with high methionine (Riddi- ford and Law, 1983; Kanost et al., 1990; Telfer and Kunkel, 1991). All have molecular sizes of nearly 500 kDa and are composed of six subunits of c. 80 kDa each. Accordingly the name storage hexamer was pro- posed (Telfer and Kunkel, 1991).

In a previous report (Tojo et al., 1985), it was shown that in the common cutworm, Spodoptera litura, three storage proteins named SL-I, -2 and -3 prominently increase their concentration in the haemolymph early in the last larval instar and are detected in the fat body

*Laboratory of Entomology, Department of Applied Biological Sciences, Saga University, Saga 840, Japan.

tAu tho r for correspondence.

during pharate pupal development. SL-1 and -2 first appear in this instar and their synthesis is inhibited by juvenile hormone. While SL-3 is also present in the penultimate larval instar, its synthesis is not inhibited by juvenile hormone (Tojo et al., 1985).

In this article, we describe the purification procedure and characteristics of these three storage proteins from the haemolymph of last instar larvae of S. litura, and compare them with storage proteins of Bombyx mori (Tojo et al., 1980).

MATERIALS AND METHODS

Insect

Egg masses of S. litura were supplied from Sumitomo Chemical Co. Ltd, where these animals have been suc- cessively reared for more than 10 yr. The larvae were reared on an artificial diet composed mostly of kidney bean, yeast powder and wheat germ (Okada, 1977).

Chemicals

All chemicals used were of reagent grade. The follow- ing chemicals and products were purchased from the companies mentioned below:

729

730 SUMIO TOJO and TOYOSHI YOSHIGA

phenylmethylsulfonyl fluoride (PMSF) from Sigma; Bio-Gel Hx, SDS-PAGE molecular weight standard from Bio-Rad Laboratories; Butyl-Toyopearl 650 M, DEAE-Toyopearl , G-3000 S.W. column from Tosoh Co. Ltd; PBE 94, PBE 118, Pharmalite pH 8-10.5, gel filtration calibration kit for molecular weight determination of proteins from Pharma- cia Fine Chemicals; acrylamide, N,N'-methylenebisacrylamide, dimethyl suberimidate dihydrochloride (DMS) from Nacalai Tesque Inc.; N,N'-diallyltartardiamide from Aldrich Chemical Co. Inc.; agarose-I from Dojindo Co. Inc.; goat antirabbit IgG labelled with alkaline phosphatase from E. Y. Labs Inc.; nitrocellulose membrane from Sartorius GmbH.

Purification of storage proteins The experiments were performed at 0-4°C, unless

otherwise stated. All solutions used for extraction of proteins, ammonium sulphate precipitation and column chromatography were added with 0.5 mM PMSF to prevent proteolysis of proteins.

Prolegs of 3 day old sixth (last) instar female larvae were cut with scissors and haemolymph (8ml) was squeezed out into a tube containing 8 ml of phosphate buffered saline (20 mM potassium phosphate buffer - 200 mM NaCi - 2 mM sodium EDTA - 5 mM p h e n y l t h i o u r e a - 0 . 5 m M PMSF, pH 7.0) in which (NH4)2SO4 was dissolved in 80% saturation. SL-1 and -2 precipitated in the 40% (NHD2 SO4 saturated fraction, while SL-3 precipitated between the 40 and 55% (NH4)2SO 4 saturated fraction. Procedures for purification of SL-1 and -2 and that of SL-3 were carried out using different groups of larvae in order to improve the recovery. The (NH4)2SO 4 fractions were neutralized to pH 7.0 by adding HCI and kept in a refrigerator until the next procedure.

In subsequent column chromatography, fractions containing storage proteins had been previously checked immunologically, and the proteins were precipitated by adding (NH4)2SO 4 for SL-1 and/or SL-2 by 45% saturation, while for SL-3 60% saturation was used. Shortly before chromatography, precipitates were collected by centrifugation of the mixture at 12,000g for 20min, dissolved in the starting buffer for the next step, and passed through a Sephadex G25 column (1 x 45 cm), using the same buffer as the eluting buffer. The fraction eluted in the area of void volume was poured onto a column for the next chromatography.

The two fractions obtained by (NH4)2SO 4 salting-out were chromatographed on the columns (2 x 14 cm) with Bio-Gel "T, using 0.1 M potassium phosphate buffer, pH 7.0 (0.1 M K-P buffer), as the starting buffer. The column applied with the 40% (NH4)2SO a saturated fraction was then washed with 75 ml of 0.15 M K - P buffer at a flow rate of 30 ml per h, and finally with 0.25 M K- P buffer to elute out SL-I and -2. Another column which was applied with the 40-55% (NH4)2SO 4 saturated fraction, was washed with 100ml of 0.3 M

K -P buffer, and finally with 0.4 M K-P buffer to elute out SL-3. Effluents were fractionated at 5.5 ml intervals and those containing the corresponding storage protein(s) were collected for further purification. Also in subsequent chromatography, a flow rate of 30 ml per h and a fraction volume of 5.5 ml were adopted, except for HPLC.

The two fractions obtained by chromatography on Bio-Gel "v were then fractionated on columns (2 x 10cm) with Butyl-Toyopearl 650M, using 1 M (NH4)2SO4~.1 M Tris-HC1, pH 7.0, as the starting buffer. The (NH4)2SO 4 solution used in this procedure contained 0.1 M Tris and the pH was adjusted to 7.0 by adding HCI. The column applied with the fraction containing SL-1 and -2 was then washed with 75 ml of 0 .8M (NH4)2SO 4 solution, with 75ml of 0 .6M (NH4)2SO 4 solution to elute out SL-1, and finally with 0.4 M (NH4)2 SO 4 solution to elute out SL-2. The column applied with the SL-3 fraction was washed with 75 ml of 1 M (NH4)2 SO4 solution, and then with 0.8 M (NH4)2SO 4 solution to elute out SL-3.

The fraction containing SL-3 was further chromatographed on the column (2 x 18cm) with D E A E - Toyopearl, using 0.1 M Tris-HC1, pH 8.5 as the starting buffer. SL-3 was eluted out from the column by 0.1 M NaC1 - 0.1 M Tris-HC1, pH 8.5. We found that column chromatography on neither D EA E- nor CM type-Sephadex, including DEAE-Toyopearl , could be used for purification of either SL-I or -2, because of low recovery due to absorption to these resins.

The two fractions containing SL-1 and -2 from Butyl-Toyopearl chromatography and the one fraction containing SL-3 from DEAE-Toyopear l chromatography were further fractionated by chromatofocusing on Polybuffer exchangers, according to Pharmacia's man- ual, as follows. For SL-1 and -2 fractionation, two columns (1 x 2 5 c m ) with PBE 118 were run, with 0.025 M triethylamine-HC1, pH 10.3 being used as the starting buffer, and 1/45 Pharmalyte pH 8-10.5-HC1, pH 7.0 as the eluent. For SL-3 fractionation, one column (1 x 25 cm) with PBE 94 was run, with 0.025 M imida- zole HC1, pH 7.4, as the starting buffer, and 1/8 Poly- buffer 74-HC1, pH 4.0 as the eluent.

The final step of purification was gel filtration through G3000 S.W. column (0.75 x 60cm) by HPLC (Gilson Model 302), using 0.1 M Na2SO4-0 .1 M potassium phosphate buffer, at a flow rate of 1 ml per min at 25°C. The protein fractionation procedures previously described were used, as they consistently gave the best results.

SDS-polyacrylamide gel electrophoresis (PAGE) SDS-PAGE was performed as described by Laemmli

(1970) on a slab gel polymerized with acrylamide and N,N'-methylenebisacryl amide. In some cases, N,N'-diallyltartardiamide was used instead of N,N'- methylenebisacrylamide.

The protein samples for subunit analysis and immunoblotting were denatured for 2 min in the sample buffer

STORAGE PROTEINS OF SPODOPTERA LITURA 731

containing 1.25% mercaptoethanol-0.5% SDS-Tris- HC1 buffer, pH 6.8 in boiling water. The samples for chemical cross-linking were first incubated for 2h at 28°C in 0.2 M triethanolamine-HCl, pH 8.5, with dimethyl suberimidate dihydrochloride of different concentrations, and then heated for 2min in the sample buffer in boiling water, according to Davis and Stark (1970). After electrophoresis, proteins were stained with 0.1% Coomassie brilliant blue R250 dissolved in 10% acetic acid-40% methanol aqueous solution.

For immunoblotting, the proteins in the slab gel were transferred by electrophoresis to a nitrocellulose membrane, which was first treated with antiserum against storage protein prepared from rabbit, and then with goat anti-rabbit IgG labelled with alkaline phosphate to detect the antigen.

Immunological analysis of proteins

Antisera were prepared by injecting soluble proteins from pupal fat body of S. litura, as described by Tojo et al. (1985), which were reactive to three storage proteins. Antisera were also prepared by injecting storage proteins purified by the procedures described in this report. Rabbit antisera against SP-1 (methionine- rich storage protein) and SP-2 (arylphorin) of Bombyx mori were supplied by Dr M. Nagata, University of Tokyo.

Immunoelectrophoresis was performed on glass plates coated with 1.2% agarose 1 containing 0.01% NaN3 prepared in 0.06 M veronal buffer, pH 8.6. Double diffusion immunological tests of Ouchterlony were conducted in the agarose gel in the same buffer system.

Recovery of storage proteins was determined by the quantitative immunodiffusion technique of Oudin (Tojo et al., 1985).

Determination of components in storage proteins

Protein content in samples were determined by a protein-dye binding assay with Coomassie brilliant blue G250 (Bearden, 1978).

The neutral sugar composition was analysed by gas-liquid chromatography (Shimadzu, GC-4CM) using a column of 1.5% OV-1 on Chromosorb W (2 m × 3 mm), after methanolysis followed by acetyl- ation and trimethylsilylation of samples, as described by Chino and Yazawa (1986). For quantitative determination, ribitol was used as an internal standard.

Fatty acids were extracted from samples by chloroform-methanol (2:1), followed by methylation with BF3-methanol. The methylated fatty acids were analysed by gas chromatography (Shimadzu GC-14A) using a fused silica capillary column, CP-Sil 88 (50 m × 0.25 mm). For quantitative determination, pentadecanoic acid was used as an internal standard. Lipid contents were calculated by totalling the fatty acids detected by gas chromatography.

RESULTS

Purification of three storage proteins

Either seven or eight steps were undertaken for purification of three storage proteins from haemolymph of 3 day old last instar larvae of S. litura, as described in Materials and Methods. The most effective separation was done by chromatofocusing on a Polybuffer exchanger (Fig. 1). SL-I and -2 were eluted out from a PBE 118 column in the pH range of 9.4-9.7 and 8.3-8.8, respectively, and SL-3 was eluted out from a PBE 94 column in the pH range of 6.1~5.5. It should be noted that SL-1 and -2 are basic proteins with isoelectric points near 9.3 and 8.6, respectively, while SL-3 is a neutral protein with an isoelectric point near 6.3.

As shown in Fig. 2, the final step of purification was gel filtration through a G3000 S.W. column in HPLC, indicating SL-2 and -3 to be of a larger molecular size than SL-I. Immunoelectrophoresis demonstrated that the respective final fractions thus obtained contained only one antigen for which purification was intended (Fig. 3). SDS-PAGE indicates that the three storage proteins were highly purified by the procedures applied with SL-1 being composed of one subunit, while SL-2 and -3 were composed of two subunits (Fig. 4).

As shown in Table 1, the final recoveries of SL-1, -2 and -3 were 6.9, 6.5 and 9.4% of the starting materials, respectively. As SL-1 and -2 were found to be somewhat fragile in ammonium sulphate precipitation and also in a frozen condition, all steps of purification were per-

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FIGURE 1. Elution profiles of SL-1 ( ), SL-2 ( - - - ) and SL-3 ( ) containing fractions from Polybuffer exchanger columns (1 × 25 cm) by chromatofocusing. Polybuffer exchanger and chromatofocusing buffer: PBE 118 and 1/45 diluted Pharmalyte (pH 8-10.5~HC1, pH 7.0 for SL-1 and -2 fractionation (upper figure); PBE 94 and 1/8 diluted Polybuffer 74-HC1, pH 4.0 for SL-3 fractionation (lower figure): Flow rate: 30ml per h; fraction volume: 5.5ml. See

Materials and Methods for sample preparation•


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FIGURE 2. Elution profiles of SL-I ( ), SL-2 (-- --) and SL-3 ( . . . . ) containing fractions from G3000 S.W. column (7.5 mm × 60cm) on HPLC, using 0.1 M Na2SO 4 -0.1 M potassium phosphate buffer, pH 7.0~.02% NaN 3 as eluent. Flow rate: 1 ml per

h. See Materials and Methods for sample preparation.

formed within 1 week, and purified proteins were kept in solution at 4°C, for further analysis.

Physico-chemical characteristics o f the three storage proteins

As shown in Table 2, molecular weights of native proteins of SL-I, -2 and -3 were estimated to be 400, 450 and 450 kDa, respectively, by gel filtration

(-)

through a G3000 S.W. column on HPLC, comparing their retention times with those of standard proteins. The sizes of their subunit molecules were calculated to be 70kDa for SL-I, 72 and 7 4 k D a for SL-2, and 77 and 78 kDa for SL-3 (Table 2), when comparing their relative mobilities on SDS-PAGE with those of marker proteins. These results indicate that the three storage proteins are hexamers. This was confirmed by cross-linking of subunit molecules by DMS. Figure 5 shows that an increase in DMS concentration enhanced the production of oligometric proteins resulting in six differently migrating protein bands on SDS-PAGE for SL-3. Similar results were obtained for SL-1 and -2 by the chemical cross-linking (data not shown).

Lipid levels were as follows: 1.7% for SL-1, 2.6% for SL-2 and 2.0% for SL-3 (Table 2). As for carbohydrates, only mannose and N-acetylglucosamine were detected in all three storage proteins, and their total amounts in SL-I, -2 and -3 were 0.9, 2.8 and 3.3%, respectively (Table 2).

As shown in Table 3, the amino acid composition was nearly the same for SL-1 and -2, with both being rich in aspartate, leucine and lysine and having c. 10% aromatic amino acids (phenylalanine and tyrosine). Amino acid composition differed markedly for only methionine and glycine, the former being richer in SL-1, and the latter being richer in SL-2. On the other hand, SL-3 differed greatly from SL-I and -2 in amino acid composition, containing c. 20% aromatic amino acids, and being far richer in glutamate, but less in methionine.

A comparison of amino acid composition was carried out for storage proteins of three lepidopteran species (Fig. 6). It is clear that SL-1 of S. litura is similar to SP-1 of B. mori and SP-I of H. cecropia both being rich in methionine, while SL-3 is homologous to SP-2 (arylphorin) of B. mori and arylphorin of H. cecropia, in composition.

(+) H L S L - 1 S L - 2 S L - 3

FIGURE 3. lmmunoelectrophoresis of haemolymph proteins and purified storage proteins. HL, haemolymph (20 ~g) from 3 day old last instar larva of female S. litura; SL-I, -2 and -3 (each 5 #g) obtained by the final step of purification as shown in Fig. 2. For each channel, antisera against fat body proteins of female pupa of S. litura were

added.

Immunological characteristics o f the three storage proteins

Antisera against SL-I, -2 and -3 purified by the procedures described in this article were prepared from rabbits and used for the double diffusion immunological test of Ouchterlony. As shown in Fig. 7(A), SL-1 and -2 formed spurs of precipitation arcs, when reacted with antisera against fat body proteins of S. litura. However, when reacted with antiserum against SP-1 (methionine-rich) of B. mori, SL-1 and -2 produced fused arcs of precipitation [Fig. 7(B)]. While SL-I showed weak cross-reactivity against SL-2 antiserum, the arc of precipitation was not fused with that of SL-2 [Fig. 7(D)]. Further, SL-2 also showed weak cross-reactivity against SL-1 antiserum, with the arc of precipitation not being fused with that of SL-! [Fig. 7(C)].

Neither SL-I nor -2 showed cross-reactivity to antisera against SL-3 of S. litura [Fig. 7(E)], or SP-2

STORAGE PROTEINS OF SPODOPTERA LITURA 733

a b a b a b H L M

S L - I S L - 2 S L - 3

FIGURE 4. SDS-PAGE of haemolymph proteins and purified storage proteins. Proteins after treatment with SDS-mercaptoethanol were subjected to electrophoresis in gel with 12% acrylamide and 0.3% N,N'-diallyltartardiamide. SL-I: a, 0.5 pg; b, 2/~g. SL-2: a, 1 pg; b, 4/~g. SL-3: a, 0.8 pg; b, 3.2 #g. HL: haemolymph from 3 day old last instar larva of female S. litura, 15 pg. M: marker proteins, myosin (200 kDa), fl-galactosidase (I 16 kDa), phosphorylase b (97 kDa), phosphorylase

b (66 kDa) and ovalbumin (45 kDa).

(arylphorin) of B. mori [Fig. 7(F)]; only SL-3 was reactive against both antisera.

Fur ther immunologica l compar i son was carried out on their respective denatured molecules which were electrotransferred to nitrocellulose membranes . As shown in Fig. 8, a subuni t of SL-1 cross-reacted with antisera against SL-1 of S. litura and also SP-1 (methionine-rich) of B. mori, on the other hand, a subuni t of SL-2 only cross-reacted with ant iserum against itself, while SL-3 reacted both with ant isera against itself and against SP-2 (arylphorin) of B. mori.

DISCUSSION

Three storage proteins SL-1, -2 and -3 purified from haemolymph of S. litura last instar larvae, were shown to be similar in native molecular sizes ranging from 400 to 450 kDa, and in subuni t molecular size from 70 to 80 kDa. Chemical cross-l inking experiments indicate that their native molecules are hexamers, as in other storage proteins isolated from various insects, to which Telfer and Kunke l (1991) have given the name storage hexamers.

TABLE 1. Recovery of three storage proteins during the course of purification from S. litura haemolymph a

Total protein SL-I b Total protein SL-2 b Total protein SL-3 b Step (mg) (mg) (mg) (mg) (mg) (mg)

Haemolymph 122.23 (NH4)2 SO 4 precipitation 45.58 Bio-Gel Hr chromatography 25.57 Butyl-Toyopearl chromatography 4.05 DEAE-Toyopearl chromatography PBE chromatofocusing 2.40 Gel filtration on G3000 S.W. 1.31

18.92 (100.0) 122.23 24.42 (100.0) 142.50 50.32 (100.0) 11.42 (60.3) 45.58 13.62 (55.7) 88.53 30.39 (60.4) 5.63 (29.7) 25.57 8.52 (34.9) 23.45 18. I0 (36.0) 3.04 (16.1) 4.87 4.03 (16.5) 16.63 12.32 (24.5)

- - - - - - 12.70 9.52 (18.9) 2.03 (10.7) 2.23 1.92 (7.9) 8.95 7.90 (15.7) 1.31 (6.9) 1.61 1.61 (6.5) 4.84 4.84 (9.6)

~Haemolymph (8.0 ml) from 3 day old last instar female larvae were processed. bValues in parentheses are percentages of recovery, calculated on the assumption that the final protein was pure, from immunological activities

determined by the Oudin test (see Materials and Methods).


TABLE 2. Properties of three storage proteins purified from S. litura haemolymph

SL-1 SL-2 SL-3

Molecular weight Native molecule Subunit

400 kDa 450 kDa 450 kDa 70kDa a72kDa a77kDa

b74kDa b78kDa Carbohydrate content (%, w/w)

Mannose 0.5 1.8 2.4 N -acetylglucosamine 0.4 1.0 0.9

Lipid content (%, w/w) 1.7 2.6 2.0 Aromatic amino acid content (%)a 9.5 l l.8 19.6 Methionine content (%)a 7.6 3.5 2.0 pI 9.3 8.6 6.3

aMolar percentages in determined amino acids.

As expected from the mobilit ies on agarose gel at pH 8.3 (Fig. 3), SL-1 and -2 were found to be basic proteins

with isoelectric points of 9.3 and 8.6, respectively, estimated from elut ion profiles on electrofocusing chromatography, while SL-3 was found to be a neutral protein with an isoelectric point of 6.3. Similar findings have been reported by Jones et al. (1993) for Tricoplusia ni, where sequences of three hexamers, i.e. a ry lphor in and basic proteins, were also inferred from encoded D N A sequences. The two basic proteins with low aromatic acid contents (8.1 and 11.1%), one being rich in methionine (8.4%) and the other not so high in meth ionine (5.3%), are fundamenta l ly similar to SL-I and -2 of S. litura, based on amino acid composit ion. The

TABLE 3. Amino acid composition of three storage proteins a

Moles/1000 mol determined amino acids

SL-1 SL-2 SL-3

Cysteine ND ND ND Aspartate 139 141 121 Threonine 61 55 20 Serine 31 23 57 Glutamate 50 51 122 Proline 35 31 50 Glycine 85 140 33 Alanine 31 22 48 Valine 83 75 67 Methionine 76 35 20 Isoleucine 52 54 48 Leucine 89 97 74 Tyrosine 47 65 111 Phenylalanine 48 53 85 Lysine 80 82 82 Histidine 22 18 28 Arginine 71 60 35

aProteins were hydrolyzed in 6 N HC1 at 1 amino acid values thus obtained were hydrolysis.

lO°C for 24, 48 and 92 h, and corrected for the loss during

methionine-r ich SP-1 of B. mori is not a basic protein (Tojo et al., 1981, 1985), but was shown to be similar to SL-1 of S. litura in amino acid composi t ion and immunological character. Thus, it will be interesting to elucidate the specific part of the structure in the native molecule that causes the difference in electrical property between these two homologous proteins.

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1 2 3 4 5 6 7 FIGURE 5. SDS PAGE of chemically cross-linked SL-3. SL-3 was first incubated at 28c'C for 2 h in 0.2 M triethanolamine-HCl, pH 8.5 containing DMS of different concentrations as follows: from lanes 1 to 7~0, 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0 #g//~l, respectively. The proteins thus cross-linked were then treated with SDS-mercaptoethanol. Electrophoresis

was run on a 3 10% linear gradient acrylamide gel polymerized with N,N'-methylenebisacrylamide.

STORAGE PROTEINS OF SPODOPTERA L1TURA 735

SL-I and -2 of S. litura first appear in haemolymph of 1 day old last instar larvae and their appearance is largely retarded by external application of juvenile hormone or methoprene, while SL-3 is not specific to the last larval instar and its appearance is not influenced by juvenile hormone (Tojo et al., 1985). The patterns of appearance in haemolymph of the two basic proteins of T. ni (Jones et al., 1987) are quite similar to those of SL-1 and -2 of S. l i tura (Tojo et al., 1985) and female specific/methionine rich SP-1 of B. mor i (Tojo et al., 1980). Further, their appearance in the haemolymph and/or expression is largely suppressed by juvenile hormone treatment (Tojo et al., 1982; Jones et al., 1993). Accordingly, we believe these proteins are included in the same category. Immunological analyses and comparisons of amino acid composition clearly demonstrated that SL-1 and -2 differ from SL-3: the former two belong to the same group as the methionine-rich SP-1 of B. mori . Telfer and Kunkel (1991) grouped the storage hexamers with lower aromatic amino acids (9-13%) and higher methionine (4-11%), compared to arylphorins with 16-21% aromatic amino acids and 1-2% methionine.

In the lower aromatic amino acid with high methionine group, LSP-1 and -2 o fH . cecropia (Tojo et al., 1980) and female specific storage hexamer (LSP) of M a n d u c a sex ta (Ryan et al., 1985) were included Telfer and Kunkel (1991). The antiserum of LSP of M . sex ta is cross-reactive to the methionine-rich SP-I of B. mor i (Ryan et al., 1985), and the antiserum to SP-I of B. mor i is cross-reactive to SL-I and -2 of S. l i tura [Fig. 7(B)]. These facts indicate that SL-1 and -2 can be categorized in the same group as the high methionine storage hexamers, although SL-2 is not as rich in methionine as the others. Thus, we put quotation marks on methionine- rich storage hexamers to tentatively include SL-2 in this group. SP- 1 and -2 of H. cecropia first appear in haemo-

lymph of the last larval instar (Tojo et al., 1978), and SP-1 of B. mor i (Tojo et al., 1980) and LSP of M . sex ta

(Ryan et al., 1985), all of them being rich in methionine, show their first prominent increase in haemolymph of the last larval instar. Other proteins categorized as high methionine storage hexamers by Telfer and Kunkel (1991) are also specific to the last larval instar. There- fore, it may be more appropriate to name these storage hexamers, pupal storage proteins or juvenile hormone- sensitive storage proteins.

In spite of strong cross-reactivity of SL-1 and -2 to antiserum against methionine-rich SP-1 of B. mor i [Fig. 7(B)] and their partial cross-reactivity to antiserum against the partner protein at the native molecular level [Fig. 7(C), (D)], SL-1 cross-reacted only with antisera against SL-1 and -1, while SL-2 reacted only with antiserum against SL-2, in a denatured condition (Fig. 8). These results suggest that the polyclonal anti- bodies against SL-1 and -2 used in this experiment include at least two antisera: one being cross-reactive to surface epitope of native molecules bridging subunits, which can be destroyed by SDS-mercaptoethanol treatment, and the other being cross-reactive to an epitope of polypeptide molecules. Thus, it may be that SL-1 and -2 of S. litura have homologous epitopes on their surface conformation to that of methionine-rich SP- 1 of B. mori , but they have different epitopes in polypeptide molecules, the epitope of SL-1 being similar to that of methionine-rich SP-1, but different from that of SL-2. Tojo et al. (1985) reported that neither SL-1 nor -2 showed cross-reactivity to anti-SP-1 of B. mor i at the native molecular level, differing from the results of this study. This discrepancy was found to have resulted from the anti-SP-1 used in the previous study, which had almost lost its immunogenic reactivities to SL-1 and -2. The authors would like to revise the immunoreactivities of SL-1 and -2, as described above.

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Val Ala

S L - 3 ( S . l i t u r a ) . . . . . . . S P - 2 ( B . mor l ) . . . . A r y l p h o r ; n ( H . c e c r o p i a )

FIGURE 6. Comparisons of amino acid compositions of storage proteins isolated from three lepidopteran species. Data for SL-I and -3 are from Table 3, for SP-I and -2 (arylphorin) of B. mori from Tojo et al. (1980), and for SP-1 and arylphorin of H. cecropia from Tojo et al. (1978) and Telfer et al. (1983), respectively. Relative contents of respective components are

plotted on lines which equally divide a circle, based on their ratios. O, Centre of circle.


~ 4 ¸

FIGURE 7. Double diffusion immunological tests of storage proteins purified from S. litura. ASLF, antisera against fat body proteins of S. litura pupae; ASLI, ASL2 and ASL3, antiserum against SL-1, -2 and -3, respectively; ASPI and ASP2, antiserum

against SP-1 (methionine-rich) and SP-2 (arylphorin) of B. mori, respectively.

SL-3 showed cross-react iv i ty to an t i se rum agains t SP-2 (a ry lphor in) o f B. mori, on both the nat ive and subuni t molecu la r levels, and was quite s imilar to SP-2

(a ry lphor in) o f B. mori and a ry lphor in o f H. cecropia in amino acid compos i t i on (Fig. 6). The an t i -H, cecropia a ry lphor in an t i se rum is cross-react ive to a ry lphor in o f

STORAGE PROTEINS OF SPODOPTERA L1TURA 737

M. sexta (Telfer et al., 1983), and the antiserum against the latter arylphorin is cross-reactive to B. mori SP-2 (Ryan et al., 1985). These facts indicate that SL-3 of S. litura should be categorized as an arylphorin, like the arylphorins of H. cecropia and M. sexta.

Although SP-1 (methionine-rich) and SP-2 (arylphorin) of B. mori do not show any immunological similarity (Tojo et al., 1980), the deduced primary structures of SP-1 and -2 exhibit nearly 30% homology (Izumi et al., 1988; Fujii et al., 1989). We are now sequencing cDNAs of SL-1, -2 and -3 of S. litura, which should contribute significantly to a better under- standing of the immunological and evolutionary re- lationships of the two different groups of storage hexamers.

The lipid content in storage hexamers is generally in the range of 1-2% of the mass of the proteins (a review: Kanost et al., 1990), which coincides with the values in SL-I, -2 and -3 in the present report (Table 2). In many cases, arylphorins contain cova

(a)

(b)

SL-1 SL-2 SL-3

i i Anti-SL-1

Anti-SL-2

Anti-SL-3

Anti-SP-1

Anti-SP-2

FIGURE 8. Immunoblotting of subunit molecules of storage proteins purified from S. litura. Proteins denatured by SDS-mercaptoethanol were subjected to electrophoresis in a gel with 7.5% acrylamide-0.2% N,N'-methylenebisacrylamide, transferred onto nitrocellulose membranes, and then reacted with rabbit antiserum against one of the storage proteins of S. litura (SL-1, -2 and -3), SP-1 (methionine-rich), or SP-2 (arylphorin) of B. mori. The gels at the moving area of three storage proteins after SDS-PAGE were stained with Coomassie brilliant blue R250 (a), or immunostained with an antiserum as

described above (b).

lently bound carbohydrates. The arylphorin of H. cecropia contains 3.5% mannose and 0.7% glucosamine, and that of M. sexta contains 1.6% mannose and 0.4% glucosamine. Both carbohydrate moieties were identified not only in SL-3, but also in SL-1 and -2 of S. litura (Table 2). It had apparently been accepted that the lack of carbohydrate in the protein molecule seen in M. sexta and B. rnori (Ryan et al., 1985) was characteristic of 'methionine-rich storage hexamers' (Kanost et al., 1990; Telfer and Kunkel, 1991). How- ever, the present analyses for SL-I and -2 do not support the criterion of distinguishing these proteins from arylphorins based on the lack of covalently bound carbohydrates. More extensive studies are required to elucidate the characteristics of this group of proteins, especially their carbohydrates.

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Acknowledgements--The authors wish to express their sincere thanks to Mrs L. Tsukamoto and Mr E. O. Ayoade for proof-reading the text, to Miss N. Hoashi for typewriting the manuscript, to Dr Makoto Hatakoshi for supplying S. litura, to Dr Masao Nagata for supplying antisera against storage proteins of B. mori, to Dr Haruo Chino for performing carbohydrate analysis, and to Dr Teruyoshi Yanagita for lipid analysis. This research was supported by a Grant-in-Aid for the Bio-Media Program from the Ministry of Agriculture, Forestry and Fishery (BMP 93-I-2-3).

Documents

Purification and characterization of three storage proteins in the common cutworm, Spodoptera litura