A neutral DNase in venom sac extract of the oriental hornet Vespa orient alis: Characterization, specificity and mode of action

0041-0101/811020211-07 f02.00i1D 8 1981 Pergaroon Prep Ltd

Toskar, Vol . 19, No . 2, pp. 241-7A7, 1981 Printed in Great Britain

A NEUTRAL DNase IN VENOM SAC EXTRACT OF THE ORIENTAL HORNET VESPA ORIENTALIST CHARACTERIZATION, SPECIFICITY AND MODE OF ACTION* t

B . RING, t J" ISHAYZ and H. SLOR t ' Department of Human Genetics and i Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel (Accepted jor publication 6 October 1980) B. Rnva, J. ISHAY and H . SLOe. A neutral DNase in venom sac extract of the Oriental hornet Vespa arientalis : characterization, spocificity and mode of action . Toxicon 19, 241-247, 1981 .-A neutral DNase was purified by acrylamide gel electrophoresis from venom sac extracts of the Oriental hornet (Vespa arientaGs). The DNase is active optimally at pH 7 in 005 M Tris-HCl buffer. Addition of NaCI inhibited the DNase activity, and with 015 M NaCI the enzyme was completely inhibited. RNA addition also inhibited the enzyme activity. Divalent rations were essential for enzymatic activity. At 1 mM oonoentrations, MgCI= was the best activator while MnCh and CaCl2 activations were, respectively, about 75~ and 25% that of the magnesium salt . EDTA completely inhibited DNase activity. The DNase was characterized as an endonuclease by its ability to nick circular supercoiled PM2 DNA . It is equally active on both native and single-stranded DNA as substrates and produces oligonucleotidea terminated by 5' phosphate and 3' hydroxyl groups. The enzyme is free of RNase and of non-specific phosphodieaterase activities. INTRODUCTION

a social insect living in annual families and is prevalent in the Mediterranean basin and Southeast Asia (ISHAY, 1965). The venom glands of the Oriental hornet consist of two acid glands which secrete their contents into the venom sac and produce a complex venom containing biogenic amines, polypeptides (i.e. various kinins), proteins (primarily enzymes, i.e. poly- and disaccharidases, hyaluronidase, phospholipases, proteases), and toxins . In addition there is one alkaline gland which secretes its products directly into the venom duct, between the venom sac and the stinger (ALLALOUF et al., 1972 ; IKAN and ISHAY, 1973 ; FISCHL et x1.,1974 ; ISHAY et x1.,1974 ; FISCHL et al., 1975 ; ROSENBERG et a1., 1977 ; HABERMANN, 1968 . We have recently al~:overed several DNases in venom sac extract, as well as in the alkaline and acid glands (RING et al., 1978x). The effects ofthese DNases in vivo on invertebrates and vertebrates (RING et al., 1978b) and their efïects in vitro on mammalian blood (SLOR et al ., 1976b) were reported. In this paper we report on the separation of five neutral DNase activities of venom sac extract by polyacrylamide gel electrophoresis. Vespa orientalis is

MATERIALS AND METHODS Hornet collection Hornets were collected by anesthetizing the entire colony at night in their nests, when all workers were inside. The hornets was refrigerated to kcep them in an immobilized state, enhancing ease of handling and dissection . When time did not allow for immediate dissection, the hornets were stored frozen at -20°C . " This research forma part of the M .Sc . thesis of B .R . t Present address : Department of Anatomy and Anthropology, Sackler School of Medicines, Tel-Aviv University, Israel. 241

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B. RING, J. ISHAY and H. SLOB

Venom sac extract

Dissection was accomplished by pulling carefully on the stinger with forceps and removing the exposed venom sacs . The collected sacs were then homogenized (Zivan Homogenize, Israel) at a concentration of 300 mg/ml in saline containing 10'/ sucrose and 1 mg/ml bovine serum albumin (BSA) which was tested to be DNase-free . The homogenate was centrifuged at 6000p for 60 min at 4°C and the supernatant stored at -20°C . DNA preparation

Radiolabeled DNAwasprepared by growingE. coli (T-) in thepresence of labeledthymidine (3H TdR,19 Ci/mM, NuclearCenter, Negev) at aconcentration of 1 pCi/ml.When cultures reached a densityof065at 560 nm,cellswas collected by centrifugation and DNA extracted according to the procedure Of MARMUR (1961) . For "C-labeled DNA, cells was labeled with 0-05 ieCi/ml "C TdR. Specific activities of the DNA was 50,000 cpm/Rg for the'Hlabeled DNA and 36,000 cpm/pg for the r`C-labeled DNA. Single-stranded DNA was prepared by boiling native DNA (in 005 M Tris-HCI, pH 7) for 10 min and fast cooling in an iced water bath. Supawiled PM2 DNA was prepared according to the method of Esre~o et al. (1971) and had a specific activity of 20,000cpm'H/Rg . Oligonucleotidea were prepared by degrading DNA with either DNase I or DNase II, as well as with Peak I venom sac extract DNase to 40-50'/ degradation (acid-soluble) . The precipitate containing the oligonucleotides was dissolved in appropriate buffs (see figure legends) . Acrylamide ge! electrophoresis

Acryhunide gels (10'/)wasprepared in 6 x 110 mm glass tubes and run empty overnight (with0~1 M Tris-HCI, pH 8~5 in both reservoirs), at a cuaent of 4 mA/gel . Fifty microliters of venom sac extract was applied to each gel tube and electrophoresis was carried out for 150 min as previously described (St.oa et ai., 1976a). Tubes was kept frozen at -20°C until ready for elution. Gels were cut into 32-35 slices. Each slice was eluted with Oß5 ml elution buffs for 18 hr at 4°C . Elution buffs consisted of 0-05 M Tris-HCI, pH 7, containing 10'/ suaose and 1 mg BSA (DNase-free) per ml. DNase assay

Reaction mixtures consisted of 120 pl gel elutee, 2000 cpm 'H-DNA, activators or inhibitors at concentrations given in the figure legends, and appropriate buffs to a final volume of 200 pl . When double-label experiments was performed, reaction mixtures wntained 1700 cpm of r`C-labeled single-stranded DNA (dDNA) and 2000 cpm of H-labeled native DNA (nDNA) . Afterincubation for 330 min at 37°C, cold trichloroacetic acid (TCA)wasadded to afinal concentration of 7~5~ . Tubes were chilled in icefor 10 min and then centrifuged at 1500 p for 60 min at 4°C. An aliquot of the acid-soluble supernatant was counted for radioactivity in a water-miscible scintillation fluid (Bray's) in a Packard Tricarb Spectrometer . Endomrclease assay using PM2'H-DNA

Endonuclease activity was assayed by theability of the enzyme to nick circular supercoiled PM2 DNA. Reaction mixtures consisted of 200 pl `endo' buffer (0-06 M Tris-HCl buffer, pH 7~4, 0-05 M NaCI, 5 mM MgCI=), lOpl'Hlabeled DNA (20,000 cpm/Pg, 180,000 cpm/ml) and 10 pl enzyme at a propsdilution (enzyme concentrations was used that could rends acid soluble 50°ô of a similar concentration of linear DNA substrate, undo identical incubation conditions). After incubation at 30°C for a given time, the reaction was laminated by the addition of 0~1 ml denaturing solution (consisting of 0~9 M NaCI, 0~1 M K2 HP0~, 0-025 M EDTA, adjusted to pH 12 with NaOH)and tubes was left at 30°C for 3 min. Tubeswas neutralized to pH 7 by theaddition of0~ 1 M HCI, and their contents filtered through nitrocellulose filters (Schleicha 8c Schuell, 0~45 !~, 25 mm). Filters was rinsed three times with satins : sodium citrate buffer (0-9 M NaCI, 0~6 M sodium citrate, pH 7), dried andradioactivity determined in a toluene-based scintillation fluid in a Packard Tricarb Spectrometer . Since PM2 DNA is naturally in the form of supercoil, it is able to pass through the filter. Endonucleolytic nicks disrupt this structure, and upon denaturation and renaturation, the nicked molecules will remain linear (uncoiled) and thus be trapped on the filters. Since exonucleases must attach to a free end of a DNA molecule in order to degrade the DNA, they cannot degrade circular DNA. Non-specific phosphodiesterase assay

Non-specific phoephodiesterase activity wes determined by incubating a 1 ml sample in 0-05 M Tris-HCl buffs, pH 7, with 1 ml of 1 mM Ca bis-p-nitrophenylphoaphate. After 60 min at 37°C, the reaction was laminated by the addition of an equal volume of 0-05 M NaOH, and the optical density of the yellow color (p-nitrophenol in alkali) was determined at 400 nm on a Gilford spectrophotometer. RNase assay

RNase activity was assayed by the ability of the enzyme to degrade RNA. Incubation oC 1 ml sample in 025 ml of 0-05 M Tris-HCl buffs, pH 7, containing 0-0S M Na=EDTA, was carried out with 025 ml (1 mg/ml) yeast RNA. After 210 minat 37°C the reaction wasterminated by the addition of Uranyl reagent (075% uranyl acetate in 2~5~~ perchloric acid), and centrifugation at 1500 p for30 min at 4°C. The supernatant was diluted 4-fold in distilledwater and the optical density at 260 nm wasdetermined agarnstan appropriate blank. Bovine pancreatic RNase (1 mg/ml) was used as a positive control.

A Neutral DNase of the Oriental Hornet

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Determination of the phosphodiester cleavage site

Oligonucleotides isolated from DNA which was rendered 50'~ acid soluble were prepared as above . DNase II produces 3'P oligonucleotides whereas DNase I produces 5'P oligonucleotides (BERNARDI, 1971 ; LASKOWSKI, 1971). It was previously shown that the exonuclease of snake venom is specific For 5'P-terminal oligonucleotides (SIERAKOWSKA and SHUGAR, 1977). Snake venom exonuclease (Worthington), 125 pg/ml of0~1 M Tris-HCI, pH 8~9, was incubated either with DNase II-produced oligonucleotides (as a negative control) or with DNase Iproduced oligonucleotides (as a positive control), as well as with oligonucleotides produced by the neutral venom sac extract Peak I DNase. After 30 min at 37°C, the reaction was terminated by the addition of perchloric acid to a final concentration of 12%. Radioactivity of the acid-soluble degradation products in the perchlorate-soluble supernatant (after centrifugation as above) was determined in a Packard Tricarb Spectrometer . RESULTS AND DISCUSSION

In the double-label experiments, five distinct neutral DNase activities were observed when venom sac extract was electrophoresed, and gel-slice eluates assayed for their ability to degrade DNA (Fig.1). The extent ofdegradation of both nDNA and dDNA was essentially the same with eluates of Peaks I, II, IV and V; however, Peak II was totally specific for nDNA . Peak I venom sac extract activity was assayed at several pH values on both nDNA and dDNA substrates . As demonstrated in Fig. 2, the enzyme is optimally active at pH 7-0 ; at pH 7~5 the activity against nDNA was about 50'~ that against dDNA . A similar phenomenon occurs also at pH 8. Thus, it seems that the pH activity curve for nDNA is narrower than that for dDNA . The addition of NaCI to Peak I venom sac extract DNase had inhibitory effects on the enzyme activity on both nDNA and dDNA (Fig. 3). At a concentration of 005 M NaCI, enzyme activity was inhibited by about 20%. However, 0~1 M NaCI almost completely

E-TOP

SUCE NUMBER

FIG. I. ELECTROPHORETIC SEPARATION OF NEUTRAL DNASES OF VENOM SAC EXTRACTS. Venom sac extract was electrophoresed as described in Materials and Methods. Gels were sliced into 32 slices and each slice was eluted with elution buffer (see Materials and Methods) . Gel-slice eluates were assayed fortheiractivity against both 14C-single-stranded DNA and'H-native DNA simultaneously, in the presence of 1 mM MgCh. Aliquots of each elutee were also assayed in the presence of 1 mM EDTA, but no activity could be observed in the gel (data not shown). Incubation conditions and details of the assay are described in Materials and Methods. Data is expressed in DNase units. A unit is that amount of enzyme which, under the assay conditions used above, rendered 0~1;~ of the DNA substrate acid-soluble .

244

B. RING, J. ISHAY and H. SLOB 300 260 200 150 100 60

pH 8.6

pH 7.0

pH 7.5

pH 8.0

FIG. Z . EFFECTS OF' pH VALUES ON THE ACTIVITY OF PEAK I VENOM SAC EXTRACi DNASE. Gel slices of Peak 1 venom sac extract DNase were eluted in 0-01 M NaCI, 20°~~ sucrose, 2 mg/ml BSA, and aliquots were buffered with 0~1 M Tris-HCI or Na acetate 0~2 M to pH values shown. MgCl 2 was added to 1 mM final concentration, and incubation with'H-labeled DNA(either native or single stranded DNA) continued for 330 min at 37°C . Acid-soluble degradation products were determined as in Materials and Methods . Data is expressed as DNase units (sce legend to Fig. 1) .

'e >_ a

s

FIG. 3. EFFECTS OF' N8CI ON THE ACTIVITY OF PEAK I VENOM SAC EXTRACT DN83e. Peak I venom sac extract DNase was eluted from acrylamide gels (see Materials and Methods) in 001 M NaCI, 20'/ suaose, 2mg/ml BSA . Aliquots were incubated with ~H-labeled DNA (either native or single-stranded DNA) in 0~OS M Tris-HCl buffer, pH 7, with 1 mM MgCl 2. NaCI was added to a final concentration of 0, 005, 0~1, 015 and 0~2 M. After incubation (see Fig. 2), the acid-soluble DNA degradation products were determined. Data is expressed in DNase units as in Fig. 1 .

A Neutral DNase of the Oriental Hornet

1mM Mp~

FiG. 4.

1mM Mn~

imM G~

245

1mM EDTA

EDTA ON THE ACTIVITY OF PEAK I VENOM SAC EXTRACT DNase. Gel slices of Peak I wen eluted and assayed as in Fig. 3, with the addition of either 1 mM MgClz , 1 mM MnCI,, l mM CaCh or 1 mM Na z EDTA, The substrate was'H-labeled DNA, either native or single-stranded. Incubation and determination of acid-soluble degradation products are detailed in Materials and Methods. Data is expressed as DNase units as in Fig. 1. EFFECTS OF DIVALENT CATIONS AND

inhibited the DNase activity. In preliminary experiments, it was determined that divalent cations are required for the DNase activity. Figure 4 shows the elïects of MgC12, MnCI Z and CaC1 2, as well as ofNa2ECTA, on the activity of the DNase against nDNA and dDNA. The best activation was rendered by MgC12, with somewhat less activation in the presence of MnC12. Both cations activated the DNase against both nDNA and dDNA to the same extent . Activation of the enzyme by CaCIZ was only 25~ that of the Mg-activated DNase when nDNA was used as a substrate. However, when dDNA was used, the Ca-activated DNase was about 50'/ that of the Mg-activated DNase. The addition of EDTA completely inhibited the enzyme activity, demonstrating the dependence ofthis DNase upon activation by divalent canons . As is known, EDTA is a chelating agent and, therefore, neutralizes any elïects of canons such as Mg, Mn, etc. Thus, activity in the presence of EDTA represents activity with no salt addition. No activity was exhibited by the Peak I venom sac extract in non-specific phosphodiesterase and RNase assays . In order to identify the mode of action of this DNase (endonuclease vs exonuclease), PM2 circular supercoiled DNA was used as a substrate. Only endonucleases can nick this substrate . The endonucleases DNase I and DNase II nicked this substrate (176 and 242 cpm, respectively, trapped on filter) while the snake venom exonuclease did not (4 cpm trapped on filter), demonstrating the validity ofthis test as a specific assay for endonucleases. The DNase of venom sac extract Peak I could also nick PM2 DNA (456 cpm trapped) and thus it is characterized as an endonuclease. The site of action of the DNase on the phosphodiester bonds of DNA was also investigated. DNaes are classified as either 3'P formers or as 5'P formers (SIERAKOWSKA 8nd SHUGAR, 1977) according to whether they cleave the phosphodiester bond between the phosphate and carbon 5 of the deoxyribose, or between the phosphate and carbon 3 of the deoxyribose, respectively. Oligonucleotides were produced by the venom sac extract DNase (Peak I) by degrading the DNA substrate to render about 50'/ of its degradation products acid-soluble . DNA degraded in a similar fashion by DNase I as well as by DNase II provided oligonucleotides with 5'P and 3'P, respectively . The oligonucleotides were incubated with snake venom exonuclease which is specific for 5'P oligonucleotides (CORN ând VOLKIN, 1953)

246

B. RING, J . iSHAY and H . SLOR 1 . ACTIVITY SNAKE VENOM EXONUCLEASE ON TABLE OF OLIGONUCLEOTIDFS OBTAINED FROM PARTIALLY DEGRADED DNA (SO~~o ACID-SOLUBLE DEGRADATION PRODUCTS) BY DNe8e9 WITH A KNOWN MODE OF ACTION (DN88e I : DNase II) AND PEAK I VENOM SAC EXTRACT

DNase

'H-labeled substrate

degradation (acid soluble) Mean S .D.

DNase I-produced oligonucleotides

24~9 24U

0-09 0~41

DNase II-produced oligonucleotides

22 1 ~8

015 022

502 441

032 030

Peak I venom sac extract DNase-produced oligonucleotides

zo~o

o~1s

Reaction mixtures consisted of 0~1 ml Tris-HCI, 0~1 M, pH 8~9, 20 pl DNA ofigonucleotides (approximately 800 cpm) and 10 ul of 125 pl/ml snake venom exonuclease . Incubation was carnal out at 37°C for 30 min, and the acid-soluble degradation products determined as in Materials and Methods . Values represent independent experiments in triplicate or duplicate ; each experiment was performed in duplicate .

and the percentage degradation of the oligonucleotides was determined (Table 1). DNase Iproduced oligonucleotides were a good substrate for the snake venom exonuclease, rendering about 24% of the oligonucleotides acid-soluble . DNase II-produced oligonucleotides, however, were a very poor substrate for the exonuclease, and only about 2% of these oligonucleotides were rendered acid-soluble. These results establish the validity of this test as being specific for 5'P oligonucleotides . When the oligonucleotides produced by the venom sac extract DNase were incubated with the snake venom exonuclease, they were degraded to produce about 47~ acid-soluble degradation products. We conclude, therefore, that this DNase is a 5'P former. To our knowledge, this is the first time purification of a DNase from the venom sac extract ofhornets has been accomplished. In additional studies (unpublished results), comparison of the hornet castes with respect to DNase activities in the venom apparatus showed, under most conditions, slightly higher activities among nest-founding queens . Although the queen ceases to function in foraging or defense of the nest after the beginning of the season, her venom is useful in another role : a basic phenomenon in the life cycle ofthe Oriental hornet is the battle between rival queens in the colony, and it seems probable that DNases play a part i this, by destroying genetic material, DNA, thereby causing destruction ofthe egg batch and reproductive capabilities of those queens that manage to remain alive even after being stung. Various attempts to find DNase in the hornet body fluids (hemolymph) extracted from adults and larvae did not reveal any measurable amounts of nucleases. Similar experiments on muscle tissue extracts were also negative (unpublished observations) . The venom sac is composed ofthe bladder (made ofa chitinous membrane), containing the venom, connective tissue and muscles which contract the bladder during stinging (BARB-NEA of al., 1976). It is immersed in hemolymph. Since none of the above components, other than the venom sac extract, has been found to contain detectable amounts of DNases, we assume that the origin of the DNases reported here is from the venom itself. Acknowledgement-This research was supported in part by a grant from the Israel Cancer Association (to H .S .1~

A Neutral DNase of the Oriental Hornet

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REFERENCES ALLALOUF, D., BER, A. andISFIAY, J.(1972) Hyalwonidase activity of extracts of venom sacs of a number of Vespidr~ Hymenoptera. Comp . Biochem. Physiol. 43, 119. ALLALOUF, D., BER, A. and ISFtAY, J. (1975) Properties of testicular hyalwonidase of the honey bees and Oriental hornet : Comparison to insect venom and mammalian testicular hyslwonidase . Comp . Btochem. Physiol. S0, 331. BARB-NEA, L., RosENeERC, P. and ISHAY, J. (1976) The venom apparatus of Vespa orientalis : Morphology and cytology. Toxicon 14, 65 . BERNARDt, G. (1971) Spleen acid deoxyribonuclease. In : The Enzymes, 3rd edn., Vol. 4, p. 271 (HovER, P. D, Ed .). New York : Academic Press. BERNARDt, A. and BERNARDI, G. (1971) Spleen acid exonuclease. In : The Enzymes, 3rd edn., VoL 4, p . 329 (BOYER, P. D., Ed .). New York : Academic Press. CORN, W. E. and VOLKtN, E. (1953) On the structure of ribonucleic acids. I. Degradation with snake venom diesterase and the isolation of pyrimidine diphosphates . J. biol. Chem . 203, 319. ESPFJO, R.T., CANELO, E.S. and StN$FIEtMER, R. L.(1971) Replication of bacteriophage PM2 deoxyribonucleic acid A closed circular double-stranded molecule. J. molee. Biol . 56, 597. FtscttL, J., ISHAY, J. and ROSENBERG, A. (1974) Poly- and disaccharidases in Vespa orientalis (Vespinae, Hymenoptera) . Comp. Biochem. Physiol. 48, 299. FtSCFtL, J., ISHAY, J. end TALMOR, N. (1975) Monosaccharidase activity and pyruvate lactate and carbon dioxide activity of Vespa orientalis hemolymph. Comp . Biochem. Physiol. 50, 71 . HABERMANN, E. (1968) Biochemie, Pharmakologie and Inhaltsstoffe von Hymenopterengiften . Ergeb. Physiol. 60, 220. IKAN, R. and ISHAY, J. (1973) Fra amino acids in hemolymph and venom of the Oriental hornet (Vespa orientalis). Comp. Btochem. Physiol. 44, 949. ISHAY, l. (1965) Entwicklung and Aktivität im Nest von Vespa orientalis L. Deut . Entomol. Zeit ., N.F. 12, 397. ISHAY, J, AVRAM, Z, GRÜNFELD, Y. and GI7TER, S. (1974) Catecholamines in social wasps . Comp . Biochem. Physiol. 48, 369. LASxowstct, M., SR . (1971) Deoxyribonuclease I. In : The Enzymes, 3rd edn., Vol. 4, p.271 (NOYER, P. D., Ed.). New York : Academic Press. MARMUR, J. (1961) InterspeciSc transformation in bacillus . J. molec. Biol. 3, 208. RttvO, B., ABRAMOV, L, ISHAY, J. and SLOR, H. (1978a) Deoxyribonuclease and ribonuclease activities in venom sac extracts from the social wasp Polistes gallicus (Polistinae, Vespidae). Toxicon 16, 77 . RttvO, B., SLOB, H., PERNA, B. and ISHAY, J. (1978b) Deoxyribonucleases of the Oriental hornet (Vespa orientalis) venom : II . Partial characterization and effects in uivo on insects and mammals. Tozicon 16, 473. ROSENBERG, P, ISHAY, J. and GITTER, S. (1977) Phospholipase A and B activities of the Oriental hornet (Vespa orientalis) venom and venom apparatus. Toxicon 15, 141. SIERAROWSRA, H. and SFIUGAR, D. (1977) Mamtnelian nucleolyticenzymes. In : Progress in Nucleic Acids Research and Molecular Biology, Vol. 20, p. 59 (COFiEN, W. E., Ed .). New York : Academic Press. SLOB, H., LEV-SoeE, T. and KALtNA, M. (1976a) A unique DNase associated with mitogen-induced transformation of rat lymphocytes. Exp. Cell Res. 101, 416. SLOB, H., RtNC, B. and ISIiAY, J. (1976b) Nucleases of the Oriental hornet (Vespa orientalis) venom sac extract : I. Acid, neutral and alkaline deoxyribonucka9es and their pharmacological effects on cat blood to tàtro. Toxicon 14, 427.