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Protein components of fire ant venom (Solenopsis invicta)
Toxreon, Vol . 17, pp . 3y7-M)S . Pergsmon Prag Ltd . 1979 . Printed in Grcat Britain .
PROTEIN COMPONENTS OF FIRE ANT VENOM (SOLENOPSIS INVICTA) HARULI) i3AER, * T.-Y. LIU, * MARTHA C. ANDERSON, * MURRAY BLUM,t WILLIAM H . SCHMID$ and FRANK J . JAMES§ *Bureau of Biologics, Food and Drug Administration, Bethesda, MD 20014, U.S .A., j'University oFGeorgia, Athens, GA 30602, U.S .A ., $St. Petersburg, FL 33710, U.S .A. (Private Practice] and §San Antonio, TX 78234, U.S.A. (Private Practice) (Accepted jor publication 15 January 1979)
H. BAER, T .-Y . Ltu, M. C. ANDERSON, M. GLUM, W . H . ScrlMln and F. J. JaMar. Protein components of fire ant venom (Solenopsis invicta), Toxlcon 17, 39705, 1979 .-Venom of the fire ant (Solenopsis invicta), long thought to tmntain only alkaloids, is shown to contain proteins, which undoubtedly accounts for the induction of anaphylactic reactions from stings . Failure of previous investigators to identify proteins in this venom is probably due to the luw protein content of approximately 0~ 1 ~ of the venom weight . In common with other hymenoptera venoms, it contains phospholipase and hyaluronidase activity . Sephadex chromatography showed the presence of at least 3 proteins which were allergenically active, as determined by RAST using sera of individuals allergic to fire ant sting. The RAST also indicated that S. invicta venom was different from the venoms of S. richteri, S. xyloni and S. genrirrata, but probably shows extensive cross-reactivity . Commercially prepared extracts were shown to contain venom by BAST . INTRODUCTION IT Is now well established that fire ants of the genus Solenopsis that have invaded the south-
eastern and south-central states can induce an allergic reaction similar to that of other stinging insects, such as the bees, wasps and hornets . Clinical and other aspects of fire ants and their envenomation have been reviewed (LOCKEY, 1974 ; RHOADES et al., 1975 ; JAMES et al., 1976 ; RHOADES, 1977 ; MACCONNELL et al ., 1971). Immediate allergic reactions are usually associated with serum IgE directed against the specific allergens, which are almost always proteins (or large polypeptides) . The extensive studies of Solenopsis venom, however, have indicated that they only contain alkaloids (MACCONNELL et al., 1971 ; BRAND et al., 1972). This study describes the isolation of three proteins from the venom of the ant Solenopsis invicta (S. invicta), and indicates their role in the allergic reaction resulting from the sting of this insect . MATERIALS AND METHODS Vrnom Venom was obtained from venom sacs of S. invicto by expression into capillary tubes. The liquid venom was maintained at -30°C until used . S. invicta averages 5~2 mm in length with an average weight of 2~3 mg . Approximately 35 nl of venom can be obtained by milking an ant. All venom cannot be removed from the venom sac this way; the total volume is approximately 40 nl . Commercial samples of whole body extracts of S. invicta and S. richteri were purchased from Greer Laboratories, Lenoir, NC 28645 (lots GB47-1IAXIM and GB14-17-IAXiM) . Extracts of this type are prepared by grinding up and extracting whole ants with a buffer solution followed by filtration . Reprint requests to : Harold Baer, Ph.D ., Director, Allergenic Products Branch Division of Bacterial Products, Bureau of Biologics, 8800 Rockville Pike, Bethasch, Maryland 20014, U.S .A . 397
398
HAROLD BAER et ul .
Chromatography
Approximately 120 mg of liquid venom was mixed with 1 ml of 005 M ammonium bicarbonate and extracted three times with 1 ml of hexane to remove the alkaloids. The aqueous phase was freeze-dried, redissolved in 1 ml ammonium bicarbonate (005 M) and placed on a 0~6 x 60 cm column of Sephadex G50-(Pharmacia Fine Chemicals, Inc., Piscataway, NJ 08854). After collecting 50 one ml fractions, the column was eluted with 10 ~ acetic acid to remove anything left on the column that was not eluted by the ammonium bicarbonate, and a further 50 fractions collected. A 150111 aliquot of each fraction was analyzed for protein after alkaline hydrolysis (OLIVEIRA et al., 1977), using the ortho-phthalaldehyde procedure (RorH, 1971). Aliquots of each fraction (10-25111) were dried in a ventilated oven heated at 100°C for about 20 min. The peptides were then hydrolyzed with 50 ul of 4 N NaOH in the autoclave at 120°C for 20 min at 15 psi. The fluorímetric reaction was carried out at room temperature by the addition of 0~9 ml of the o-phthalaldehyde reagent. The tubes were individually stirred immediately after the addition of the reagent. The reagent was prepared just before use by adding 15 mg of o-phthalaldehyde dissolved in 1~0 ml of ethanol to 100 ml of 0~5 M boric acid (309 g/1) containing IS mg of dithiothreitol . The final pH of the reaction mixture was between 8~5 and 9~5. Under these conditions, the fluorescence was readily formed and decayed slowly with time . The readings were taken in an Aminco Fluorocolorimeter equipped with a primary filter with transmission peak at 360 nm and a secondary filter with cut off at 415 nm and transmission above that wavelength . An alanine standard curve was obtained using various amounts of the amino acid . A linear relationship was observed between relative fluorescence and quantity of alanine used within the range of 2-200 nmole. The presence of ammonia, N-ethylmorpholine, or pyridine in the buffers used for chromatography did not interfere with this ftuorimetric analysis . Pools were prepared from each peak (Fig. 1) and freeze-dried. The amino acid composition of each pool was obtained following hydrolysis with 4M methanesulfonic acid (LIU and CHANO, 1971), using a Beckman 121 M analyzer, according to the procedure of SPACKMAN It al ., 1958 . CHROMATOGRAPHY ON SEPHADEX G-50 LIiXßOcml OF THE VENOM PROTEINS FROM SOLENOPSIS INVICTA
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EFFLUENT Imll FIG. I . HEXANE EXTRACTED S. IliVICta VENOM (12O mg) WAS CHROMATOORAPHED ON A SEPHAUEX G-50 coLVMN (0~6 x 60 cm).
Each ml of eüiuent was sampled and tested by the phthalaldehyde procedure for fluorescence at 360 nm .
RAST Sera from individuals who had suffered allergic reactions to fire ant stings were used to determine the allergenic reactivity of whole venom, venom fractions and commercial products . Cyaaogen bromide activated paper discs (Schleicher and Scheull, Inc., Keene, NH, Type 595, }" diameter) were prepared by standard methods (WIDE et al ., 1967 ; AXEN et al., 1967 ; WIDE et al., 1967). Briefly, lOt>D discs were soaked in water for 30 min and the water removed on a sintered glass funnel . The remainder of the procedure was carried
Proteins of S.
lnvleta
Venom
399
out at 4°C. Tlae discs were resuspended in water, 2"5 g CNBr added and the pH maintained at 10"5 for 10 min with 3 N NaOH . The fluid was removed using a sintered glass funnel and the discs washed successively with 0"1 M NaHCO,, water and acetone. The dry discs wen stored in a desia:ator at 4°C. Sensitized discs were incubated for 1 hr with the test materials (incubation steps were at room temperature on a rotary shaker at low speed). Each disc was sensitized with approximately 1-2 pg of protein in 20 ul of water. Because of the limited amounts of material available, it was not possible to establish with certainty that the discs were maximally sensitized . Following complete washing with RAST B buffer (0~1 M phosphate buffer pH 7"5, containing 1 ~ Tween 20), the discs were allowed to stand overnight in 2 ml of RAST A buffer (0"1 M phosphate buffer, pH 7"5, containing 0"2 ~ BSA, 1 % Tween 20, and 0"01 ~ Sodium azide). After washing again with BAST B, each disc was incubated with 50 lIl of serum for 3 hr. The discs were washed with RAST B, followed by the addition of approximately 2 ng'ul labeled anti-IgE in 100 pl of RAST C buffer (0 "1 M phosphate buffer, pH 7"5 containing 4 ~ Fetal Calf Serum, 1 ~ Tween 20 and 0"1 ~ Sodium azide) . The unlabled anti-IgE was obtained from Atlantic Antibodies, Westbrook, Maine, and was prepared in goats against IgE myeloma PS (OanwA et al., 1969 ; KOCHWA et al., 1971) and chromatographically purified ; its preparation and purity have been described (MIYAWAKI and RITCHIE, 1974). After overnight incubation with the labeled antibody, the discs were again washed with RAST H and counted in a gamma counter. Enryme analyses Phospholipase was detxted using egg yolk agar (HAHERMANN Snd HARDT, 1972). Egg yolk is diluted with 085 ~ saline (1 :3), centrifuged to remove large particles, and 0'S mg of CaCI, (001 M) added to 50 ml of melted agarose in Tris buffer (0"03 M) . The mixture is poured onto a microscope slide and, after hardening. wells 3 mm in diameter punched into the agar . The wells are filled with venom and incubated at 30°-32°C for 48 hr and examined for clearing around the venom wells. Hyaluronidase was detected by the turbidometric procedure (Worthington Manual, Worthington Biochemical Corp . Freehold . NJ 07728) : proteinase with gelatin agar, and acid and alkaline phosphatase using p-nitrophenylphosphate as substrate (Sigma Chemical Co ., P .O . Box 14508, St . Louis, MO 63178). RESULTS Extraction of the venom with hexane removed most of the alkaloids. Chromatography of the hexane-extracted venom yielded four major peaks (Fig . 1). The amino acid analysis of fractions I-III is given in Table 1 and indicates that these three materials are different. Peak IV had only traces of amino acids. TABLE I . AMINO ACID COMPOSITION OF THE VENOM PROTEINS OF SOICnOpSIS l/1VlCtlf
Residues per 100 residues Amino acid ~ Fraction 1 Fraction 1I Fraction III Lysine 4.9 17 "4 11 "0 Histidine 1"7 7"4 17 "0 Arginine 2"8 4"2 7"5 Tryptophan 2"7 Aspartic acid 13 "7 5"9 11 "I Threonine 8"1 4~4 4"5 Serine 5"7 13 "2 4"S Glutamic acid 8"3 10 "0 7"5 Proline 8"4 3"6 3'7 Glycine 7"7 12 "4 6"7 Alanine 8"4 6"3 7.5 Valine 2"4 2"0 6"1 Methionine 4"6 1"5 l "3 Isoleucine S"6 2"3 3 "6 Leucine 5"9 3"2 4"8 Tryosine 5"5 3"4 2"0 Phenylalanine 3"2 2~7 1~4 Glucosemine -+Indicates the presence of glucosemine (quantitative data was not obtained), -indicates absence of the amino acid . The protein content of the venom put on the column was 104 Ng, as determined from its amino acid analysis, or 98 ltg as determined by the phthalaldehyde procedure. The
40 0
HAROLD BAER
et al.
amino acid analysis of the individual fractions resulted in an estimated protein content of 64 Itg for fraction I, 37 Itg for fraction Ii and 29 Itg for fraction III for a total of l31 llg of protein . The actual protein content of the venom was estimated from the mean of the three individual protein estimations to be 1 I 1 llg/ 120 mg of venom, or about 0~1 ~ of venom by weight. From Table 2 it is evident that whole venom was a good allergen, as determined by RAST using sera of seven allergic individuals . Fractions I, II and III were allergens, although all of the sera did not appear to react equally well with each fraction. Thus, serum 2 was more reactive to fraction III than fraction I, while the reverse is true for serum 5. Peak TV had no allergenic activity by RAST : no other work was done with this fraction. TABLE 2.
RAST REACTIVITY OF S.
Wholevenom Fraction I Serum Cpmt 1 8916 2 8061 3 6603 4 6510 5 1275 6 1185 7 1117 NHS$ 377 1 NHS
lnYlCla VENOM, VENOM FRACTIONS AND WHOLE BODY EXTRACTS
Fraction 2 Fraction 3 WBE§ Test l' Test 2 S. invicta XNHS Cpm X NHS Cpm X NHS Cpm X NHS Cpm X NHS Cpm XNHS 23'7 13,257 440 628 2~1 5153 171 214 677 2~3 407 1 ~4 425 1 ~4 5331 177 2192 5~5 175 3855 128 1135 3~8 408 1~4 3276 109 2470 6~2 173 8399 279 363 1~2 3704 123 2284 5~7 3~4 2197 7~3 277 0~9 336 1~1 396 1~0 3~1 824 2'7 272 0~9 476 1~6 543 1~4 3~0 444 1~5 206 0~7 276 0~9 297 0~7 301 301 301 400 3823 159 237 1~0 241
WBE
S. rlchteri
Cpm X NHS 675 1 ~7 305 0~8 2340 5~9 267 0~7 624 1~6 453 1~1
Average total activity = 26,888 . 'Time interval that Fraction 2 was stored at 4°C before test 2 was 10 days ; tCpm (counts per minute) values are the average of 2~4 experiments, each experiment carried out with duplicate samples; $NHS = Normal Human Serum; $ WBE = Whole Body Extract. The venom or venom fraction is coupled covalently to a filter paper disc by reaction with a cyanogen bromide and then reacted with IgE antibody to venom from the serum of an allergic individual and finally with 1~1 labeled anti-1gE. The radioactivity of the disc is a relative measure of the quantity of igE antibody against venom or its fractions. NHS is used as a control.
Fraction II presented some unusual problems. When a fresh solution was prepared and tested, it appeared to react with serum 3 ; a retest l0 days later showed very little activity for this fraction . The same apparent instability was obtained using a different lot of Fraction II ; the result ofthe analysis ofa fresh solution is in the lower portion ofTable 2, using serum 1 as a source of antibody . Table 2 demonstrates that commercially prepared extracts ofwhole bodies of S. invicta and S. richteri react with some of the same sera that react with S. invicta venom ; it also indicates that sera 2 and 3 which react with S. invicta whole body extract either do not react with S. richteri whole body extract, or do so only slightly, while serum 4 reacts equally well with both extracts. Table 3 gives the reactivity of 3 sera with venom from S. invicta worker ants, queen ants and S. geminata and S. xvloni venoms . Sera 1 and 4 appear to demonstrate a difference in reactivity between venom from worker and queen ants. These sera also demonstrate a difference in reactivity between S. invicta, S. geminata and S. .ryloni venoms . Tests for the various enryme activities showed the presence of phospholipase in all Solenopsis venoms and hyaluronidase in all venoms except S. x}Toni. However, the amount of x}Toni venom was insuflïcient to be certain of the hyaluronidase result . Although phos pholipase A_ from honeybee venom gives a completely clear circle in egg yolk agar, S. invicta venom gives a circle with a clear outer edge but cloudy interior (Fig. 2). Since it has
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Proteins of S. invicta Venom TABLE
Serum" 1 4 S NHSt
3. RAST
REACTIVITY OF VENOM FROM DIFFERENT SPSCIP3 OF SOLENOPSIs
S. invicta Worker cpm X NHS 48~0 7147 5537 37~2 8~6 1280 149
Queen cpm X NHS 3865 25~9 2036 13~7 210 1~4
S. geminates cpm 1346 738 218
X NHS 9~0 5~0 1~5
S. xytoni cpm 2697 1355 179
X NHS 18~1 9~1 1~2
Total activity = 19,757 cpm, the total number cpm of the anti-IgE added to each tube. "The serum numbers correspond to Table 2. tNHS = Normal Human Serum.
been demonstrated (HABERMANN and HA1tnT, 1972) that phospholipase A gives a clear circle, but that other phospholipases give either partially cleared areas or partially cleared areas with a clear outer edge in egg yolk agar, the venom of the fire ant is not pure phospholipase A. It was possible to localize the phospholipase to fraction I. DISCUSSION
The Solenopsis venoms are known to be composed largely of alkaloids, but it is not certain that the immediate systemic reactions in humans resulting from a sting are due to these components . Although all individuals allergic to sting are skin reactive to venom or whole body extract, only a few appear to be skin reactive to the alkaloids (JAMES et al., 1976). The alkaloid fraction was not reactive by RAST in our tests, but this may be due to a failure of the alkaloids to couple to the cyanogen bromide activated discs. Because they have been claimed to be skin-reactive and to release histamine (REnD et al., 1978), further work should be done with these substances. However, we did confirm the previous findings (JAMES, 197 that sera from all allergic individuals are positive by RAST to venom and whole body extracts . Since immediate allergic reactions and skin reactivity are usually due to proteins, it seemed reasonable to assume that these venoms contained proteins in addition to the alkaloids. Extraction of the liquid venom to remove most of the alkaloids followed by chromatography of the remaining material demonstrated at least three protein components. The single chromatographic separation procedure used in this study and yielding 4 peaks is not adequate to establish homogeneity of these components . The very small quantity of each component precluded the use of many additional procedures, such as further chromatography or other physical-chemical analyses . Since proteins were not heretofore known to be present in this venom, it was essential to determine that there were proteins and not merely nitrogen containing substances in the peaks, consequently they were analyzed for amino acid content. The quantity and number of amino acids in peaks I-III established that these were protein (or peptide) in nature. Peak N (Fig. 1) had no amino acids and therefore was not protein, although it gave fluorescence with the o-phthalaldehyde reagent. The substantial quantitative differences in amino acid content suggested that these were different proteins and their difference in allergenic activity established that they were different, although not necessarily pure . If sufficient venom becomes available, it may be possible to carry out further tests to determine homogeneity and then the physical-chemical properties of these fractions. Two ofthe proteins were very reactive in RAST with sera that react with whole venom, while a third component is probably reactive but appears to be unstable, solutions losing their reactivity when stored for more than a week . Thus, the allergenic activity of fire ant
HAROLD BAER et al.
venom is mainly due to proteins rather than the alkaloid components. This would make it similar to other Hymenoptera venoms (HABIItMANN, 1972) that are also allergenic . Due to the small quantity of pure S. invicta venom available and the very low protein content (approximately 0~1 ~), it was difficult to obtain many of its properties . It is undoubtedly this very low protein content that caused earlier investigators to assume that this venom contained only alkaloids. Because of the small quantity of protein and the need to use most of it for the amino acid analyses and to determine allergenic and enzymatic activities, it was not possible to carry out extensive studies to establish homogeneity. The chromatographic separation as well as the amino acid composition of each indicates that the three peaks probably represent different substances. 1n common with other Hymenoptera venoms, it contains phospholipase, possibly B or a mixture of A and B (HABERMANN et al., 1972), as well as hyaluronidase. Although it was possible to show that fraction I has phospholipase activity, the quantities available were insufficient to localize the hyaluronidase. When coupled to cyanogen bromide activated paper discs, venom and the three fractions reacted well with the sera of allergic individuals, although fraction Ii appears to be unstable in dilute solution. Since the sera of all individuals were not equally reactive to each fraction (Table 2), i .e. patient 5 was very reactive to whole venom and fraction I, but was unreactive to fraction III, each fraction is allergenically distinct and the sera of allergic individuals have antibodies to each venom component in different concentrations . The very small quantities (approximately 10 lt1) of S. invlcta worker and queen ant venoms did not permit extensive studies. From Table 3 it appears that these venoms may be different, i.e. patient 5 reacted very strongly with worker venom, but very slightly with queen venom, while sera from the other 2 patients showed small differences between venom from the two sources. Since it is unlikely that the allergic individuals received stings from multiple species of Solenopsis, and therefore sensitized to S. invicta, S. geminata and S. xyloni, these venoms appear to cross-react extensively (Table 3). The high reactivity of the two samples of whole body extract examined indicates that this type of preparation can contain venom . Acknowledgements-We thank Mr. Josarx Snr.EwsRr for certain enzyme analyses, and Dr . Hervan Fntps for analyzing the hexane extract of S. invicta venom for alkaloids. REFERENCES
Axex, R., PORATfi, J. and JoaAtvssort, S. G. O. (1967) Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214, 1302 . BRArro, J. M., Bt tnK, M. S., FAr.FS, H. M. and MACConttvet,L, J. G. (1972) Fire ant venoms : comparative analyses of alkaloidal components . Toxicon 10, 259. HASeRtKArrx, E. and HnxvT, K. L. (1972) A sensitive and specific test for the quantitation of phospholipases . Analyt. Btochem. S0, 163. HASeRMAtvrr, E. (1972) Bee and wasp venoms. Science 177, 314. JAt~s, F. K., PervCe, H. L., DRioceRS, D. P., JACOes, R. L. and HoRmrv, D. E. (1976) Imported fire ant sensitivity. J. Allergy Clin . Immunol. 58, 110. JAS, F. J. (1976) Fire ant sensitivity. J. Asthma Res. 13, 179. KocawA, S., TeRRY, W. D., CADRA, J. D. and YAtvc, N. L. (1971) Structural studies of immunoglobulin E : I. Physico-chemical studies of the IgE molecule . Ann. N.Y. Acad. Sci. 190, 49 . Ltv, T. Y. and Cxaxa, Y. H. (1971) Hydrolysis of proteins with p-toluene sulfonic acid . J. biol. Chun. 246, 2842. Locxev, R. F. (1974) Systemic reactions to stinging ants . 1. Allergy Clin. Inununol. 54, 132. MACCotvtvS.t, J. G., Brunt, M. S. and FALES, H. M. (1971) The chemistry of fire ant venom. Titrahedrnn 26, 1129 . MIYAWAICI, S, and RrrcHre, R. E. (1974) Heterogeneity of anti-nuclear antibody and IgE antinuclear antibody in patients with systemic rheumatic diseases . J. Immunol. 113, 1346.
Proteins of S. lnvlcta Venom
405
OGAWA, M., Kocxwn, S., Snnrx, C., IsHtzaxt, K. and Mcirv~rvaE, O. R. (1969) Clinical aspects of IgE myeloma. New F_rrgl . J. Med. 281, 1217. Ottvetn.~, E. B., GozscHLrcx, E. C. and Lru T. Y. (1977) Primary structure of human C-reactive protein. Proc . Nat. Arad. Sci. 74, 3148 . Re.~n, G. W., LtND N. K. and One, C. S. (1978) Histamine release by fire ant (Solenopsls) venom. Toxicon 16, 361 . RHOADFS, R. B., $CHAFFEIt., W. L., ScttrKtn, W. H., WUBHENA, P. F., Doztsa, R. M., TOWNFB, A. W. and Wrrrtc, J. (1975) Hypersensitivity to the imported fire ant. J. Allergy Clin . Immunol. 56, 84 . RHOnnFS, R. B. (1977) Medical Aspects of the Imported Fire Ant. Univ. of Florida Press, Gainesville, Florida RorH, M. (1971) Fluorescence reaction for amino acids. Analyt . Chenr . 43, 880. SPACxMAN, D. H., Srettv, W. H. and Mootee, S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt . Cáem . 30, 1190. WroE, L., BENNICH, H. and JOt-uNSSON, S. G.O . (1967) Diagnosis of allergy by an In vitro test for allergen antibodies . Lancet ü, 1105 . Wroe, L., Axent, R. and PORATH, J . (1967) Radioimmunosorbent assay for proteins chemical couplings of antibodies to insoluble dextran. Immunochem. 4, 381 .
PROTEIN COMPONENTS OF FIRE ANT VENOM (SOLENOPSIS INVICTA) HARULI) i3AER, * T.-Y. LIU, * MARTHA C. ANDERSON, * MURRAY BLUM,t WILLIAM H . SCHMID$ and FRANK J . JAMES§ *Bureau of Biologics, Food and Drug Administration, Bethesda, MD 20014, U.S .A., j'University oFGeorgia, Athens, GA 30602, U.S .A ., $St. Petersburg, FL 33710, U.S .A. (Private Practice] and §San Antonio, TX 78234, U.S.A. (Private Practice) (Accepted jor publication 15 January 1979)
H. BAER, T .-Y . Ltu, M. C. ANDERSON, M. GLUM, W . H . ScrlMln and F. J. JaMar. Protein components of fire ant venom (Solenopsis invicta), Toxlcon 17, 39705, 1979 .-Venom of the fire ant (Solenopsis invicta), long thought to tmntain only alkaloids, is shown to contain proteins, which undoubtedly accounts for the induction of anaphylactic reactions from stings . Failure of previous investigators to identify proteins in this venom is probably due to the luw protein content of approximately 0~ 1 ~ of the venom weight . In common with other hymenoptera venoms, it contains phospholipase and hyaluronidase activity . Sephadex chromatography showed the presence of at least 3 proteins which were allergenically active, as determined by RAST using sera of individuals allergic to fire ant sting. The RAST also indicated that S. invicta venom was different from the venoms of S. richteri, S. xyloni and S. genrirrata, but probably shows extensive cross-reactivity . Commercially prepared extracts were shown to contain venom by BAST . INTRODUCTION IT Is now well established that fire ants of the genus Solenopsis that have invaded the south-
eastern and south-central states can induce an allergic reaction similar to that of other stinging insects, such as the bees, wasps and hornets . Clinical and other aspects of fire ants and their envenomation have been reviewed (LOCKEY, 1974 ; RHOADES et al., 1975 ; JAMES et al., 1976 ; RHOADES, 1977 ; MACCONNELL et al ., 1971). Immediate allergic reactions are usually associated with serum IgE directed against the specific allergens, which are almost always proteins (or large polypeptides) . The extensive studies of Solenopsis venom, however, have indicated that they only contain alkaloids (MACCONNELL et al., 1971 ; BRAND et al., 1972). This study describes the isolation of three proteins from the venom of the ant Solenopsis invicta (S. invicta), and indicates their role in the allergic reaction resulting from the sting of this insect . MATERIALS AND METHODS Vrnom Venom was obtained from venom sacs of S. invicto by expression into capillary tubes. The liquid venom was maintained at -30°C until used . S. invicta averages 5~2 mm in length with an average weight of 2~3 mg . Approximately 35 nl of venom can be obtained by milking an ant. All venom cannot be removed from the venom sac this way; the total volume is approximately 40 nl . Commercial samples of whole body extracts of S. invicta and S. richteri were purchased from Greer Laboratories, Lenoir, NC 28645 (lots GB47-1IAXIM and GB14-17-IAXiM) . Extracts of this type are prepared by grinding up and extracting whole ants with a buffer solution followed by filtration . Reprint requests to : Harold Baer, Ph.D ., Director, Allergenic Products Branch Division of Bacterial Products, Bureau of Biologics, 8800 Rockville Pike, Bethasch, Maryland 20014, U.S .A . 397
398
HAROLD BAER et ul .
Chromatography
Approximately 120 mg of liquid venom was mixed with 1 ml of 005 M ammonium bicarbonate and extracted three times with 1 ml of hexane to remove the alkaloids. The aqueous phase was freeze-dried, redissolved in 1 ml ammonium bicarbonate (005 M) and placed on a 0~6 x 60 cm column of Sephadex G50-(Pharmacia Fine Chemicals, Inc., Piscataway, NJ 08854). After collecting 50 one ml fractions, the column was eluted with 10 ~ acetic acid to remove anything left on the column that was not eluted by the ammonium bicarbonate, and a further 50 fractions collected. A 150111 aliquot of each fraction was analyzed for protein after alkaline hydrolysis (OLIVEIRA et al., 1977), using the ortho-phthalaldehyde procedure (RorH, 1971). Aliquots of each fraction (10-25111) were dried in a ventilated oven heated at 100°C for about 20 min. The peptides were then hydrolyzed with 50 ul of 4 N NaOH in the autoclave at 120°C for 20 min at 15 psi. The fluorímetric reaction was carried out at room temperature by the addition of 0~9 ml of the o-phthalaldehyde reagent. The tubes were individually stirred immediately after the addition of the reagent. The reagent was prepared just before use by adding 15 mg of o-phthalaldehyde dissolved in 1~0 ml of ethanol to 100 ml of 0~5 M boric acid (309 g/1) containing IS mg of dithiothreitol . The final pH of the reaction mixture was between 8~5 and 9~5. Under these conditions, the fluorescence was readily formed and decayed slowly with time . The readings were taken in an Aminco Fluorocolorimeter equipped with a primary filter with transmission peak at 360 nm and a secondary filter with cut off at 415 nm and transmission above that wavelength . An alanine standard curve was obtained using various amounts of the amino acid . A linear relationship was observed between relative fluorescence and quantity of alanine used within the range of 2-200 nmole. The presence of ammonia, N-ethylmorpholine, or pyridine in the buffers used for chromatography did not interfere with this ftuorimetric analysis . Pools were prepared from each peak (Fig. 1) and freeze-dried. The amino acid composition of each pool was obtained following hydrolysis with 4M methanesulfonic acid (LIU and CHANO, 1971), using a Beckman 121 M analyzer, according to the procedure of SPACKMAN It al ., 1958 . CHROMATOGRAPHY ON SEPHADEX G-50 LIiXßOcml OF THE VENOM PROTEINS FROM SOLENOPSIS INVICTA
IV z
á
W Q
500
Û
3~
Ó
200
2 w U y W
H
400
III H
W
~.._- ..~~1 5o too 0.05M NH,HCO~ _ ~ 10% ACETIC ACID ~
o ~J
EFFLUENT Imll FIG. I . HEXANE EXTRACTED S. IliVICta VENOM (12O mg) WAS CHROMATOORAPHED ON A SEPHAUEX G-50 coLVMN (0~6 x 60 cm).
Each ml of eüiuent was sampled and tested by the phthalaldehyde procedure for fluorescence at 360 nm .
RAST Sera from individuals who had suffered allergic reactions to fire ant stings were used to determine the allergenic reactivity of whole venom, venom fractions and commercial products . Cyaaogen bromide activated paper discs (Schleicher and Scheull, Inc., Keene, NH, Type 595, }" diameter) were prepared by standard methods (WIDE et al ., 1967 ; AXEN et al., 1967 ; WIDE et al., 1967). Briefly, lOt>D discs were soaked in water for 30 min and the water removed on a sintered glass funnel . The remainder of the procedure was carried
Proteins of S.
lnvleta
Venom
399
out at 4°C. Tlae discs were resuspended in water, 2"5 g CNBr added and the pH maintained at 10"5 for 10 min with 3 N NaOH . The fluid was removed using a sintered glass funnel and the discs washed successively with 0"1 M NaHCO,, water and acetone. The dry discs wen stored in a desia:ator at 4°C. Sensitized discs were incubated for 1 hr with the test materials (incubation steps were at room temperature on a rotary shaker at low speed). Each disc was sensitized with approximately 1-2 pg of protein in 20 ul of water. Because of the limited amounts of material available, it was not possible to establish with certainty that the discs were maximally sensitized . Following complete washing with RAST B buffer (0~1 M phosphate buffer pH 7"5, containing 1 ~ Tween 20), the discs were allowed to stand overnight in 2 ml of RAST A buffer (0"1 M phosphate buffer, pH 7"5, containing 0"2 ~ BSA, 1 % Tween 20, and 0"01 ~ Sodium azide). After washing again with BAST B, each disc was incubated with 50 lIl of serum for 3 hr. The discs were washed with RAST B, followed by the addition of approximately 2 ng'ul labeled anti-IgE in 100 pl of RAST C buffer (0 "1 M phosphate buffer, pH 7"5 containing 4 ~ Fetal Calf Serum, 1 ~ Tween 20 and 0"1 ~ Sodium azide) . The unlabled anti-IgE was obtained from Atlantic Antibodies, Westbrook, Maine, and was prepared in goats against IgE myeloma PS (OanwA et al., 1969 ; KOCHWA et al., 1971) and chromatographically purified ; its preparation and purity have been described (MIYAWAKI and RITCHIE, 1974). After overnight incubation with the labeled antibody, the discs were again washed with RAST H and counted in a gamma counter. Enryme analyses Phospholipase was detxted using egg yolk agar (HAHERMANN Snd HARDT, 1972). Egg yolk is diluted with 085 ~ saline (1 :3), centrifuged to remove large particles, and 0'S mg of CaCI, (001 M) added to 50 ml of melted agarose in Tris buffer (0"03 M) . The mixture is poured onto a microscope slide and, after hardening. wells 3 mm in diameter punched into the agar . The wells are filled with venom and incubated at 30°-32°C for 48 hr and examined for clearing around the venom wells. Hyaluronidase was detected by the turbidometric procedure (Worthington Manual, Worthington Biochemical Corp . Freehold . NJ 07728) : proteinase with gelatin agar, and acid and alkaline phosphatase using p-nitrophenylphosphate as substrate (Sigma Chemical Co ., P .O . Box 14508, St . Louis, MO 63178). RESULTS Extraction of the venom with hexane removed most of the alkaloids. Chromatography of the hexane-extracted venom yielded four major peaks (Fig . 1). The amino acid analysis of fractions I-III is given in Table 1 and indicates that these three materials are different. Peak IV had only traces of amino acids. TABLE I . AMINO ACID COMPOSITION OF THE VENOM PROTEINS OF SOICnOpSIS l/1VlCtlf
Residues per 100 residues Amino acid ~ Fraction 1 Fraction 1I Fraction III Lysine 4.9 17 "4 11 "0 Histidine 1"7 7"4 17 "0 Arginine 2"8 4"2 7"5 Tryptophan 2"7 Aspartic acid 13 "7 5"9 11 "I Threonine 8"1 4~4 4"5 Serine 5"7 13 "2 4"S Glutamic acid 8"3 10 "0 7"5 Proline 8"4 3"6 3'7 Glycine 7"7 12 "4 6"7 Alanine 8"4 6"3 7.5 Valine 2"4 2"0 6"1 Methionine 4"6 1"5 l "3 Isoleucine S"6 2"3 3 "6 Leucine 5"9 3"2 4"8 Tryosine 5"5 3"4 2"0 Phenylalanine 3"2 2~7 1~4 Glucosemine -+Indicates the presence of glucosemine (quantitative data was not obtained), -indicates absence of the amino acid . The protein content of the venom put on the column was 104 Ng, as determined from its amino acid analysis, or 98 ltg as determined by the phthalaldehyde procedure. The
40 0
HAROLD BAER
et al.
amino acid analysis of the individual fractions resulted in an estimated protein content of 64 Itg for fraction I, 37 Itg for fraction Ii and 29 Itg for fraction III for a total of l31 llg of protein . The actual protein content of the venom was estimated from the mean of the three individual protein estimations to be 1 I 1 llg/ 120 mg of venom, or about 0~1 ~ of venom by weight. From Table 2 it is evident that whole venom was a good allergen, as determined by RAST using sera of seven allergic individuals . Fractions I, II and III were allergens, although all of the sera did not appear to react equally well with each fraction. Thus, serum 2 was more reactive to fraction III than fraction I, while the reverse is true for serum 5. Peak TV had no allergenic activity by RAST : no other work was done with this fraction. TABLE 2.
RAST REACTIVITY OF S.
Wholevenom Fraction I Serum Cpmt 1 8916 2 8061 3 6603 4 6510 5 1275 6 1185 7 1117 NHS$ 377 1 NHS
lnYlCla VENOM, VENOM FRACTIONS AND WHOLE BODY EXTRACTS
Fraction 2 Fraction 3 WBE§ Test l' Test 2 S. invicta XNHS Cpm X NHS Cpm X NHS Cpm X NHS Cpm X NHS Cpm XNHS 23'7 13,257 440 628 2~1 5153 171 214 677 2~3 407 1 ~4 425 1 ~4 5331 177 2192 5~5 175 3855 128 1135 3~8 408 1~4 3276 109 2470 6~2 173 8399 279 363 1~2 3704 123 2284 5~7 3~4 2197 7~3 277 0~9 336 1~1 396 1~0 3~1 824 2'7 272 0~9 476 1~6 543 1~4 3~0 444 1~5 206 0~7 276 0~9 297 0~7 301 301 301 400 3823 159 237 1~0 241
WBE
S. rlchteri
Cpm X NHS 675 1 ~7 305 0~8 2340 5~9 267 0~7 624 1~6 453 1~1
Average total activity = 26,888 . 'Time interval that Fraction 2 was stored at 4°C before test 2 was 10 days ; tCpm (counts per minute) values are the average of 2~4 experiments, each experiment carried out with duplicate samples; $NHS = Normal Human Serum; $ WBE = Whole Body Extract. The venom or venom fraction is coupled covalently to a filter paper disc by reaction with a cyanogen bromide and then reacted with IgE antibody to venom from the serum of an allergic individual and finally with 1~1 labeled anti-1gE. The radioactivity of the disc is a relative measure of the quantity of igE antibody against venom or its fractions. NHS is used as a control.
Fraction II presented some unusual problems. When a fresh solution was prepared and tested, it appeared to react with serum 3 ; a retest l0 days later showed very little activity for this fraction . The same apparent instability was obtained using a different lot of Fraction II ; the result ofthe analysis ofa fresh solution is in the lower portion ofTable 2, using serum 1 as a source of antibody . Table 2 demonstrates that commercially prepared extracts ofwhole bodies of S. invicta and S. richteri react with some of the same sera that react with S. invicta venom ; it also indicates that sera 2 and 3 which react with S. invicta whole body extract either do not react with S. richteri whole body extract, or do so only slightly, while serum 4 reacts equally well with both extracts. Table 3 gives the reactivity of 3 sera with venom from S. invicta worker ants, queen ants and S. geminata and S. xvloni venoms . Sera 1 and 4 appear to demonstrate a difference in reactivity between venom from worker and queen ants. These sera also demonstrate a difference in reactivity between S. invicta, S. geminata and S. .ryloni venoms . Tests for the various enryme activities showed the presence of phospholipase in all Solenopsis venoms and hyaluronidase in all venoms except S. x}Toni. However, the amount of x}Toni venom was insuflïcient to be certain of the hyaluronidase result . Although phos pholipase A_ from honeybee venom gives a completely clear circle in egg yolk agar, S. invicta venom gives a circle with a clear outer edge but cloudy interior (Fig. 2). Since it has
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Proteins of S. invicta Venom TABLE
Serum" 1 4 S NHSt
3. RAST
REACTIVITY OF VENOM FROM DIFFERENT SPSCIP3 OF SOLENOPSIs
S. invicta Worker cpm X NHS 48~0 7147 5537 37~2 8~6 1280 149
Queen cpm X NHS 3865 25~9 2036 13~7 210 1~4
S. geminates cpm 1346 738 218
X NHS 9~0 5~0 1~5
S. xytoni cpm 2697 1355 179
X NHS 18~1 9~1 1~2
Total activity = 19,757 cpm, the total number cpm of the anti-IgE added to each tube. "The serum numbers correspond to Table 2. tNHS = Normal Human Serum.
been demonstrated (HABERMANN and HA1tnT, 1972) that phospholipase A gives a clear circle, but that other phospholipases give either partially cleared areas or partially cleared areas with a clear outer edge in egg yolk agar, the venom of the fire ant is not pure phospholipase A. It was possible to localize the phospholipase to fraction I. DISCUSSION
The Solenopsis venoms are known to be composed largely of alkaloids, but it is not certain that the immediate systemic reactions in humans resulting from a sting are due to these components . Although all individuals allergic to sting are skin reactive to venom or whole body extract, only a few appear to be skin reactive to the alkaloids (JAMES et al., 1976). The alkaloid fraction was not reactive by RAST in our tests, but this may be due to a failure of the alkaloids to couple to the cyanogen bromide activated discs. Because they have been claimed to be skin-reactive and to release histamine (REnD et al., 1978), further work should be done with these substances. However, we did confirm the previous findings (JAMES, 197 that sera from all allergic individuals are positive by RAST to venom and whole body extracts . Since immediate allergic reactions and skin reactivity are usually due to proteins, it seemed reasonable to assume that these venoms contained proteins in addition to the alkaloids. Extraction of the liquid venom to remove most of the alkaloids followed by chromatography of the remaining material demonstrated at least three protein components. The single chromatographic separation procedure used in this study and yielding 4 peaks is not adequate to establish homogeneity of these components . The very small quantity of each component precluded the use of many additional procedures, such as further chromatography or other physical-chemical analyses . Since proteins were not heretofore known to be present in this venom, it was essential to determine that there were proteins and not merely nitrogen containing substances in the peaks, consequently they were analyzed for amino acid content. The quantity and number of amino acids in peaks I-III established that these were protein (or peptide) in nature. Peak N (Fig. 1) had no amino acids and therefore was not protein, although it gave fluorescence with the o-phthalaldehyde reagent. The substantial quantitative differences in amino acid content suggested that these were different proteins and their difference in allergenic activity established that they were different, although not necessarily pure . If sufficient venom becomes available, it may be possible to carry out further tests to determine homogeneity and then the physical-chemical properties of these fractions. Two ofthe proteins were very reactive in RAST with sera that react with whole venom, while a third component is probably reactive but appears to be unstable, solutions losing their reactivity when stored for more than a week . Thus, the allergenic activity of fire ant
HAROLD BAER et al.
venom is mainly due to proteins rather than the alkaloid components. This would make it similar to other Hymenoptera venoms (HABIItMANN, 1972) that are also allergenic . Due to the small quantity of pure S. invicta venom available and the very low protein content (approximately 0~1 ~), it was difficult to obtain many of its properties . It is undoubtedly this very low protein content that caused earlier investigators to assume that this venom contained only alkaloids. Because of the small quantity of protein and the need to use most of it for the amino acid analyses and to determine allergenic and enzymatic activities, it was not possible to carry out extensive studies to establish homogeneity. The chromatographic separation as well as the amino acid composition of each indicates that the three peaks probably represent different substances. 1n common with other Hymenoptera venoms, it contains phospholipase, possibly B or a mixture of A and B (HABERMANN et al., 1972), as well as hyaluronidase. Although it was possible to show that fraction I has phospholipase activity, the quantities available were insufficient to localize the hyaluronidase. When coupled to cyanogen bromide activated paper discs, venom and the three fractions reacted well with the sera of allergic individuals, although fraction Ii appears to be unstable in dilute solution. Since the sera of all individuals were not equally reactive to each fraction (Table 2), i .e. patient 5 was very reactive to whole venom and fraction I, but was unreactive to fraction III, each fraction is allergenically distinct and the sera of allergic individuals have antibodies to each venom component in different concentrations . The very small quantities (approximately 10 lt1) of S. invlcta worker and queen ant venoms did not permit extensive studies. From Table 3 it appears that these venoms may be different, i.e. patient 5 reacted very strongly with worker venom, but very slightly with queen venom, while sera from the other 2 patients showed small differences between venom from the two sources. Since it is unlikely that the allergic individuals received stings from multiple species of Solenopsis, and therefore sensitized to S. invicta, S. geminata and S. xyloni, these venoms appear to cross-react extensively (Table 3). The high reactivity of the two samples of whole body extract examined indicates that this type of preparation can contain venom . Acknowledgements-We thank Mr. Josarx Snr.EwsRr for certain enzyme analyses, and Dr . Hervan Fntps for analyzing the hexane extract of S. invicta venom for alkaloids. REFERENCES
Axex, R., PORATfi, J. and JoaAtvssort, S. G. O. (1967) Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214, 1302 . BRArro, J. M., Bt tnK, M. S., FAr.FS, H. M. and MACConttvet,L, J. G. (1972) Fire ant venoms : comparative analyses of alkaloidal components . Toxicon 10, 259. HASeRtKArrx, E. and HnxvT, K. L. (1972) A sensitive and specific test for the quantitation of phospholipases . Analyt. Btochem. S0, 163. HASeRMAtvrr, E. (1972) Bee and wasp venoms. Science 177, 314. JAt~s, F. K., PervCe, H. L., DRioceRS, D. P., JACOes, R. L. and HoRmrv, D. E. (1976) Imported fire ant sensitivity. J. Allergy Clin . Immunol. 58, 110. JAS, F. J. (1976) Fire ant sensitivity. J. Asthma Res. 13, 179. KocawA, S., TeRRY, W. D., CADRA, J. D. and YAtvc, N. L. (1971) Structural studies of immunoglobulin E : I. Physico-chemical studies of the IgE molecule . Ann. N.Y. Acad. Sci. 190, 49 . Ltv, T. Y. and Cxaxa, Y. H. (1971) Hydrolysis of proteins with p-toluene sulfonic acid . J. biol. Chun. 246, 2842. Locxev, R. F. (1974) Systemic reactions to stinging ants . 1. Allergy Clin. Inununol. 54, 132. MACCotvtvS.t, J. G., Brunt, M. S. and FALES, H. M. (1971) The chemistry of fire ant venom. Titrahedrnn 26, 1129 . MIYAWAICI, S, and RrrcHre, R. E. (1974) Heterogeneity of anti-nuclear antibody and IgE antinuclear antibody in patients with systemic rheumatic diseases . J. Immunol. 113, 1346.
Proteins of S. lnvlcta Venom
405
OGAWA, M., Kocxwn, S., Snnrx, C., IsHtzaxt, K. and Mcirv~rvaE, O. R. (1969) Clinical aspects of IgE myeloma. New F_rrgl . J. Med. 281, 1217. Ottvetn.~, E. B., GozscHLrcx, E. C. and Lru T. Y. (1977) Primary structure of human C-reactive protein. Proc . Nat. Arad. Sci. 74, 3148 . Re.~n, G. W., LtND N. K. and One, C. S. (1978) Histamine release by fire ant (Solenopsls) venom. Toxicon 16, 361 . RHOADFS, R. B., $CHAFFEIt., W. L., ScttrKtn, W. H., WUBHENA, P. F., Doztsa, R. M., TOWNFB, A. W. and Wrrrtc, J. (1975) Hypersensitivity to the imported fire ant. J. Allergy Clin . Immunol. 56, 84 . RHOnnFS, R. B. (1977) Medical Aspects of the Imported Fire Ant. Univ. of Florida Press, Gainesville, Florida RorH, M. (1971) Fluorescence reaction for amino acids. Analyt . Chenr . 43, 880. SPACxMAN, D. H., Srettv, W. H. and Mootee, S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt . Cáem . 30, 1190. WroE, L., BENNICH, H. and JOt-uNSSON, S. G.O . (1967) Diagnosis of allergy by an In vitro test for allergen antibodies . Lancet ü, 1105 . Wroe, L., Axent, R. and PORATH, J . (1967) Radioimmunosorbent assay for proteins chemical couplings of antibodies to insoluble dextran. Immunochem. 4, 381 .