Structural properties of Musca domestica storage protein

Insect Biochem. Vol. 16, No. 4, pp. 709-716, 1986 Printed in Great Britain

0020-1790/86 $3.00+0.00 Pergamon Journals Ltd

STRUCTURAL PROPERTIES OF MUSCA DOMESTICA STORAGE PROTEIN OSVALDO MARINOTTI and ANTONIO G. DE BIANCHI* Departamento de Bioquimica, Instituto de Quimica, Universidade de Silo Paulo, CP 20780, CEP 01498-S~0 Paulo, Brasil (Received 3 April 1985; revised and accepted 24 September 1985)

Abstract--The storage protein of Musca domestica is a hexameric protein with an apparent molecular weight of 500,000. The hexamers are assembled by at least three types of polypeptides: pl and p2 with apparent molecular weights of 83,000 each, and p3 with an apparent molecular weight of 89,000. The storage protein contains 26.1% of tyrosine plus phenylalanine and 0.68% of carbohydrates, which were identified as mannose and/or glucose and possibly N-acetylglucosamine by means of their interaction with lectins. About 50% of the storage protein molecules bind to a Concanavalin A affinity column. The storage protein hexamers begin to dissociate into monomers near pH 7.0 and become totally dissociated at pH 8.5. The hexamers of Musca storage protein are partially dissociated by iodination and are not affected by treatment with Triton X-100. Key Word Index: Musca domestica, housefly, storage protein, haemolymph protein

INTRODUCTION The storage proteins are the major proteins of holometabolous insect larval haemolymph (for a review see Roberts and Brock, 1981). These proteins are used as an amino acid store for adult development ( M u n n and Greville, 1969; Levenbook and Bauer, 1984). The storage proteins contain high amounts of tyrosine and phenylalanine and in general are hexamers with molecular weights of about 5 x 105 (Roberts and Brock, 1981). The first storage protein to be described was calliphorin ( M u n n et al., 1967) which is the best studied storage protein so far. The calliphorin molecules are hexamers, composed of at least nine types of subunits that dissociate when the pH is raised from 6.5 to 9.5 (Levenbook, 1983). Musca domestica storage protein was described by Bianchi et al. (1983). It is a hexameric protein with an apparent molecular weight of 500,000 constituted of subunits with apparent molecular weights 83,000 and 89,000. In this paper we present further data characterizing these subunits and their formation from the hexamer by pH variation. We also performed analyses of the amino acids and carbohydrate components of the storage protein and studied the effects of certain chemical treatments on the hexamerization of the storage protein subunits. MATERIALS AND METHODS Animals Musca domestica of a wild strain were reared at 25°C by the method of Targa and Peres (1979).

Musca storage protein purification The Musca storage protein was purified as previously described (Bianchi et al., 1983). The preparation homogeneity was tested by polyacrylamide-SDS gel electrophoresis. *To whom reprint requests should be addressed. 709

Protein radioiodination About 200/Jg of purified storage protein or 200/~1 of a 100-fold diluted haemolyrnph solution or 250/1g of Concanavalin A (Sigma Chemical Co.) were used for radioiodination (Fraker and Speck, 1978). Dilution was with a buffer of 50 mM KH2 PO4-NaOH, pH 6.5, containing 0.1 M NaC1. The samples were added to tubes containing 30 ttg of Iodogen (Pierce Chemical Co.) and 100/JCi of [13q]Na. After 10m i n at 0°C, the reaction mixture was passed through a Sephadex G-25 (or a Bio-Crel P-6) column (0.75 x 17era). Fractions of 0.5ml were collected and the radioactivity of each was determined by a gamma counter (Gamma 5500, Beckman). The fractions containing the iodinated proteins were pooled and used for the experiments. The specific activity obtained for purified storage protein was 3 x 105epm//zg protein and for Concanavalin A 4 x 105cpm//zg protein. Polyacrylamide gel electrophoresis Electrophoresis on polyacrylamide-SDS slab gel was carried out by the method of Laemmli (1970). Materials to be submitted to this electrophoretic procedure were solubilized in sample buffer [60mM Tris-HCl, pH 6.8; 2.5% (w/v) SDS; 2.5% (v/v) #-mercaptoethanol; 0.5 mM EDTA; 10% (v/v) glycerol and 0.005% (w/v) bromophenol blue], and heated at 50°C, for 1 hr. Alkaline pH polyacrylamide gel electrophoresis was carried out by the method of Davis (1964) omitting the stacking gel. Haemolymph to be submitted to alkaline pH polyacrylamide gel electrophoresis was obtained from larvae at the wandering stage, centrifuged at 12,000g, at 4°C, for 10rain after the addition of some crystals of phenylthiocarbamide (PTC). The supernatant was diluted 25-fold with 25raM Tris, 192raM glycine pH8.3 and dialyzed against 500 ml of this same buffer containing 0.2 mM PTC, for t 5 hr at 4°C. After dialysis the haemolymph was diluted with an equal volume of 40% (v/v) glycerol and 0.01% (w/v) bromophenol blue was added prior to electrophoresis. In some experiments haemolymph samples were subjected to alkaline pH polyacrylamide gel eleetrophoresis and regions of the gel containing the storage protein bands were cut out with a razor blade. The pieces of gel containing the protein were used as samples for a polyacrylamide--SDS slab gel electropboresis (see above). The molecular weights of

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OSVALDO MARINOTTI and ANTONIO G. DE BIANCH!

proteins analyzed by alkaline pH polyacrylamide gel electrophoresis were estimated by the method of Hedrick and Smith (1968).

Amino acids determination Purified storage protein (650 #g) was submitted to hydrolysis in 6 N HC1 for 25 hr, at 110°C. The HC1 was removed by lyophilization and the amino acids present in the hydrolysates were analyzed by the method of Spackman et al. (1958). Carbohydrate determinations The total amount of storage protein carbohydrates was determined by the method of Dubois et al. (1956), using glucose as standard. For qualitative determination of carbohydrates present in Musca storage protein, different lectins (about 50pg of each) were immobilized on nitrocellulose filters (HAWP, Millipore). The nitrocellulose filters were then incubated for 2 hr, at room temperature, with 3 0 bovine serum albumin [pretreated with periodic acid according to Glass et al. (1981)], in 10mM KH2PO4-NaOH, pH6.5, containing 128 mM NaCI ("incubation buffer"). After this the nitrocellulose filters were incubated with 12 ml of radioiodinated storage protein solution in incubation buffer (about 106 cpm/ml), for 2 hr. The filters were washed three times for 10min with incubation buffer, dried and exposed to a Kodak X-Omat X-ray film. After preliminary identification of the carbohydrates, the experiment was repeated once more adding 0.1 M ~-methyl-D-mannoside and 0.3 M Nacetyl-D-glucosamine to the incubation buffer, and to the storage protein solution. Protein determination Protein determinations were made according to the method of Elmann (1962), using bovine serum albumin as standard. Affinity chromatography Haemolymph obtained from wandering larvae was dialyzed, overnight, against 10 mM KH2PO4-NaOH, pH 6.5, 128 mM NaC1, 0.2% PTC and 0.01% NAN3. The dialyzed haemolymph was applied to a Sepharose--Concanavalin A column (0.8 × 6.0cm). After standing for l hr, at room temperature, the unbound material was eluted with 15 ml of the buffer described above. The same buffer containing 0.1 M ~-methyl-D-mannoside or 0.1 M ~-methyl-omannoside plus 0.1 M ~-methyl-D-glucoside, was used to elute bound material. Western blotting Haemolymph proteins separated on polyacrylamide SDS slab gel were electrophoretically blotted onto a nitrocellulose filter (HAWP, Millipore) by the method of Towbin et al. (1977). After blotting, the filters were incubated for 2 hr at room temperature with incubation buffer (see above). The filters were then incubated with a solution of [13q]Concanavalin A in incubation buffer (6.7 x 105 cpm/ml) for 2 hr. The filters were washed three times with incubation buffer, dried, and exposed to a Kodak X-Omat X-ray film. Storage protein dissociation as a function of pH Haemolymph from wandering larvae was dialyzed against 10 mM Tris-HCl, 128 mM NaCI, 0.01% NaN 3 and 0.2 mM PTC or 10mM KH2PO4-NaOH, 128mM NaC1, 0.01% NaN 3 and 0.2 mM PTC adjusted for several pH values, during 24 hr at 4°C. The dialyzed haemolymph was centrifuged for 10 min, at 12,000g and diluted 10-fold with the same buffer used in dialysis. The different haemolymph samples so. obtained were applied to the top of 5-30% (w/v) glycerol gradients. The glycerol was diluted with the same buffer used in dialysis. The glycerol gradients were centrifuged for 15 hr at 32,000 rpm on a SW 50.1 rotor (Beck-

man), at 4°C. After fractionation the absorbance at 280 nm of each fraction was determined.

Photooxidation of storage protein Larval haemolymph was diluted 35-fold with 10mM KH:PO4-NaOH, pH 8.0 (or 6.5), 128 mM NaCI, 0.2 mM PTC and dialyzed against 1 litre of the same buffer for 12 hr, at 4°C. Aliquots of dialyzed haemolymph, containing 5 mg of protein, were added to 0.005% methylene blue and irradiated for 30min with two tungsten light sources of 100 W each, displaced at a distance of 15 cm at opposite sides of samples contained in test tubes. Control samples were run in which methylene blue or irradiation were omitted. During the irradiation period the tubes were shaken by hand at each 10min interval. After photooxidation the samples were dialyzed against 10mM KH2PO4-NaOH, pH6.5, 128mM NaCI, 0.2mM PTC. After dialysis the proteins were analyzed by glycerol gradient ultracentrifugation (see above) and by electrophoresis on 1% agarose in 50 mM Tris HCI, pH 7.2. RESULTS

Amino acid composition and carbohydrate contents o[ M u s c a storage protein The a m i n o acid c o m p o s i t i o n of Musca domestica storage protein (Table 1) shows a very high c o n t e n t o f a r o m a t i c a m i n o acids. T h e value o f A280 1% is 16.7 a n d the ratio A2s0/A250 is 3.1. This protein is therefore a typical representative o f the g r o u p defined by Teller et al. (1983) as arylphorins. The storage protein o f Musca is a glycoprotein (Bianchi et al., 1983) a n d its c a r b o h y d r a t e c o n t e n t is 0.68 + 0 . 0 8 % ( m e a n + S E M for three i n d e p e n d e n t determinations). Since this low level of c a r b o h y d r a t e m a k e s the use o f traditional m e t h o d s for carbohydrate identification impracticable, we have undert a k e n a study of binding o f 131I-storage protein to lectins immobilized o n nitrocellulose filters. F r o m the several lectins used, positive results were o b t a i n e d only for Canavallia ensiformis, Lens cullinaris and Triticum vulgaris lectins (Fig. IC). T h e binding of Musca storage protein to Triticum lectins was not prevented by 0.3 M N-acetyl-D-glucosamine. This result is unexpected a n d suggests t h a t the binding o f Table 1. Amino acid composition of Musca storage protein Mol Amino acid (%) Lys 9.4 His 1.6 Arg 2.9 Asp 13.1 Thr 4. I Ser 3.7 Glu 10.1 Pro 3.9 Gly 5.3 Ala 3. I Cys* Val 5.3 Met 3.5 lie 2.9 Leu 5.0 Tyr 14.1 Phe 12.0 Trp* *Tryptophan and cystein were not determined.

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