AMER. ZOOL., 23:427-430 (1983)

Venom Toxins: Plausible Evolution from Digestive Enzymes1

ELAZAR KOCHVA,2 ORA NAKAR, AND MICHAEL OVADIA Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

SYNOPSIS Some hydrolytic enzymes are common to the pancreas, the mammalian salivary glands and the snake venom glands. Phospholipase A, which is found in elapid and viperid venoms and in the mammalian pancreas, shows 29 common amino acid residues out of Downloaded from https://academic.oup.com/icb/article/23/2/427/302344 by guest on 23 September 2021 118-125 positions. Presynaptic neurotoxins and other venom toxins are usually composed of 2-3 units or subumts, one of which is a phospholipase. The Vipera palaestmae two- component toxin retains its lethality when the enzyme is replaced by heterologous venom phospholipases, but not by the pig pancreatic enzyme. This toxin is neutralized by a factor found in the blood serum of snakes, which binds to the phospholipase and inhibits its activity. The blood serum of snakes also neutralizes hemorrhagins and inhibits the protease activity of the venom. It is hypothesized that the developing venom glands first produced enzymes that were already secreted by the pancreas and against which inhibitors were present in the blood. These inhibitors facilitated the evolution of enzyme-based toxins by neutralizing any damaging substances that might have escaped from the venom glands.

It has been suggested that enzymes pre- certain elapid venom enzymes (Table 1). ceded toxins during the evolution of the Phospholipases from Viperidae venoms are venom glands in snakes (Strydom, 1977; the most divergent not only in certain areas Gans, 1978) possibly aiding in the digestion of their sequence, but also in the presence of prey and preventing its putrefaction of a seven residue COOH-terminal exten- (Thomas and Pough, 1979; Pough and sion (Heinrikson et al., 1977). Groves, 1983). At least some of these Many venom phospholipases are actually enzymes have their counterparts in the or potentially toxic, sometimes when com- pancreas, a gland which is well developed bined with another subunit or component in all vertebrate animals, including which has no known enzymatic activity. In amphibians and fish. Enzymes of mam- the two-component toxin of Vipera palaes- malian salivary glands are also similar to tinae the enzyme can be replaced by het- those of the pancreas (Hosoi et al., 1981); erologous phospholipases, provided they the primary structure of a-amylase from are taken from viperid or elapid venoms. mouse parotid gland, for instance, differs The pig pancreatic enzyme is not lethal in from the pancreatic enzyme only by 12% this combination, despite its similarity to of its sequence (Hagenbiichle et al., 1980). the elapid phospholipases (Simon et al., Phospholipase A is also a pancreatic 1980, Table 2). These experiments suggest enzyme that is found in Duvernoy's and venom glands of snakes and in Heloderma (cf., Kochva et al., 1980 for references). TABLE 1. Sequence homology of phospholipase A from Thus far, about 20 phospholipases have mammalian pancreas and from snake venoms. been sequenced and all show a high degree Number of Number common Number of homology, the most pronounced being of amino acid of total between the mammalian pancreatic and Source engines residues residues All enzymes 17 29 118-125 All snakes 14 33 118-122 Elapidae + Ungulata 15 35 118-125 Viperidae 6 42 118-122 ' From the Symposium on Adaptive Radiation Within Elapidae 12 42 118-119 a Highly Specialized System: The Diversity of Feeding Mech- Bungarus multicinctus 2 69 118-120 anisms of Snakes presented at the Annual Meeting of Notechis scutatus 3 78 119 the American Society of Zoologists, 27-30 December Ungulata 3 88 123-125 1981, at Dallas, Texas. 2 104 2 Incumbent: The Rose and Norman Lederer Chair Artiodactyla 123-124 in Experimental Biology. Mainly after: Kondo, Toda, and Narita, 1981 427 428 KOCHVA ET AL.

TABLE 2. Substitution of phosphohpase A of Vipera palaestinae two-component toxin by heterologous enzymes.

Mixture injected (/jg) Xon-enzjmatic 1 oxicity Source ofPLA Phospholipas* component (7i dead mice) Vipera palaestinae Viperidae 10 10 45 Pseudocerastes fieldi Viperidae 20 10 83 Waltennnesia aegyptia Elapidae 25 10 83 Sus scrofa Suidae 100 10 0

After: Simon et al., 1980. Downloaded from https://academic.oup.com/icb/article/23/2/427/302344 by guest on 23 September 2021 that the enzyme activity is required but not remained, however: Not only are the ven- sufficient for the lethal effects of the Vipera omous snakes resistant to their own and palaestinae two-component toxin. Indeed, other venoms, but non-venomous snakes Condrea et al. (1981a, b) have shown that are resistant as well and their blood serum the toxic effects of phospholipase can be neutralizes a wide variety of venoms. abolished by a chemical modification of the Kihara et al. (1977a, b), Philpot et al. lysine residues while preserving half or (1978) and others have also shown that the more of the enzymatic activity. When the blood serum of snakes, both venomous and supposedly enzymatic active site is blocked, non-venomous, in addition to neutralizing both enzymatic and lethal activities are the lethality of several venoms also inhibit abolished. their phospholipase and protease activities. In the evolutionary line of venomous Mammalian sera sometimes neutralize the snakes, an additional process appears to be hemorrhagic but not the proteolytic activ- of considerable importance and this ity of Viperidae (Huang and Perez, 1980) involves the well-known resistance of snakes or vice versa (Nakar et al., unpublished). to snake venoms (cf., Omori-Satoh et al., The purified serum factor of Vipera palaes- 1972; Kihara et al., 1977a, b; Ovadia and tinae inhibits more than half of the phos- Kochva, 1977;Philpot^a/., 1978). Inmost pholipase activity of the two-component cases, this resistance could be attributed to toxin and at the same time completely neu- factors found in the blood serum that com- tralizes its lethal effects (Simon etal, 1980). bine with venom components and neutral- Most recently we have obtained similar ize their toxicity. One puzzling question results with snake sera that neutralize the

TABLE 3. Xeutralization of hemorrhagins and inhibition of gelahnases from the venoms of Viperidae and Atractaspis by blood sera ofviperid, elapid, and colubnd snakes.

Hemorrhagin VI pern Erhis Almcl^m Vipera F.rhls .AI/JM Atrarlatpt* Blood serum rnlornln palae^linae colorala rertt\te\ piignddensi* Viperidae Vipera palaestinae Echis color a ta Aspis cerastes Vipera ammodytes Elapidae Xaja nigncollis Walterinnesia aegyptia Colubridae Malpolon monspessulanus Xatnx tessellata Coluber jugularis + + Complete neutralization or inhibition at the dosages tested; + Partial neutralization or inhibition; — Xo neutralization or inhibition. VENOM TOXINS 429

VENOMS

HEMORRHAGINS TWO COMPONENT TOXINS PHOSPHOLIPASE PROTEASE PRESYNAPTIC NEUROTOXINS

\ Downloaded from https://academic.oup.com/icb/article/23/2/427/302344 by guest on 23 September 2021 CARDIOTOXINS POSTSYNAPTIC NEUROTOXINS

PROTEASE INHIBITOR PHOSPHOLIPASE INHIBITOR ANTI-HEMORRHAGIN ANTI-TWO COMPONENT TOXIN

SNAKE BLOOD Fie. 1. Tentative diagram for the evolution of toxins and their antidotes in snakes. hemorrhagic effects and inhibit protease developed from these enzymes in the (gelatinase) activity of Viperidae and Atrac- evolving venomous snakes the blood serum taspis venoms (Table 3; Nakar <>/«/., unpub- inhibitors could function as toxin antidotes lished). Of the nine ophidian sera checked, and prevent any possible damage from all neutralize the hemorrhagin of Viperi- venom components that might have found dae venoms at least partially, while only their way into the blood stream (Fig. 1). two show a marked effect on Atractaspis Thus far, no blood serum antidotes were hemorrhagin. The gelatinase activity is also found against the major toxins of Elapidae generally inhibited, the best results being and the venom of Walterinnesia aegyptia is obtained with the sera of viperid snakes. not even neutralized by its own serum; the We have encountered some difficulties mechanism of the resistance of this snake in assessing the antiproteolytic activity, to its venom remains to be explained (cf., mainly with the sera of Elapidae and Co- Ovadia and Kochva, 1977). lubridae, because of a potentiating effect that appeared at certain ratios of the ACKNOWLEDGMENTS venom-serum mixture (cf., Philpot and We thank Prof. A. Bdolah, Prof. K. Deutsch, 1956). We are now trying to sep- Rondo and Dr. D. J. Strydom for com- arate the different activities of the serum ments, and Mrs. M. Wollberg and Mrs. C. in order to obtain a pure protein inhibitor Meyer for help in the preparation of the and to check whether it is identical with manuscript. the anti-hemorrhagic factor. The points raised above seem to suggest REFERENCES a co-evolutionary trend whereby enzymes Condrea E., J. E. Fletcher, B. E. Rapuano, C. C. Yang, of pancreatic origin were subsequently and P. Rosenberg. 1981a. Effect of modification produced by oral glands of reptiles and of one histidine residue on the enzymatic and mammals. Since the pancreas is a relatively pharmacological properties of a toxic phospho- lipase A, from Naja nigncolhs snake venom and early gland, it may be assumed that enzyme less toxic phospholipases A, from Hemachatus inhibitors were already present in the blood hemachalus and \'aja naja atra snake venoms. serum of reptilian ancestors. When toxins Toxicon 19:61-71. Downloaded from https://academic.oup.com/icb/article/23/2/427/302344 by guest on 23 September 2021 430 KOCHVA ET AL.

Condrea, E.,J. E. Fletcher, B. E. Rapuano, C. C. Yang, Kochva, E., U. Oron, M. Ovadia, T. Simon, and A. and P. Rosenberg. 1981i. Dissociation of enzy- Bdolah. 1980. Venom glands, venom synthesis, matic activity from lethality and pharmacological venom secretion and evolution. In D. Eaker and properties by carbamylation of lysines in Xaja T. Wadstrom (eds.), Xalural toxins, pp. 3-12. Per- nigncolhs and Xaja naja atra snake venom phos- gamon Press, Oxford and New York. pholipases A2. Toxicon 19:705-720. Kondo, K., H. Toda, and K. Narita. 1981. Amino Gans, C. 1978. Reptilian venoms: Some evolutionary acid sequence of phospholipase A from Bungarus considerations. In C. Gans and K. A. Gans (eds.), multicinctus venom. J. Biochem. 89:37-47. Biology of the Reptiha, pp. 1-42. Academic Press, Omori-Satoh, T., S. Sadahiro, A. Ohsaka, and R. London and New York. Murata. 1972. Purification and characterization Hagenbiichle, O., R. Bovey, and R. A. Young. 1980. of an antihemorrhagic factor in the serum of Tn- Tissue-specific expression of mouse a-amylase meresurus flavoviridis, a crotalid. Biochim. Bio- genes: Nucleotide seuqence of isoenzyme raRNAs phys. Acta 285:414-426. from pancreas and salivary gland. Cell 21:179- Ovadia, M. and E. Kochva. 1977. Neutralization of 187. Viperidae and Elapidae snake venoms by sera of Heinrikson, R. L., E. T. Krueger, and P. S. Keim. different animals. Toxicon 15:541-547. 1977. Amino acid sequence of phospholipase Philpot, V. B., Jr. and H. F. Deutsch. 1956. Inhibi- A2-a from the venom of Crotalus adamanteus. A tion and activation of venom proteases. Biochim. new classification of phospholipases A2 based upon Biophys. Acta 21:524-530. structural determinants. J. Biol. Chem. 252:4913- Philpot, V. B., Jr., E. Ezekiel, Y. Laseter, R. Yaeger, 4921. and R. L. Stjernholm.1978. Neutralization of Huang S.-Y. andj. C. Perez. 1980. Comparative study crotalid venoms by fractions from snake sera. of hemorrhagic and proteolytic activities of snake Toxicon 16:603-609. venom. Toxicon 18:421-426. Pough.F. H. and J. D. Groves. 1983. Specializations Hosoi, K., T. Kida, and T. Ueha. 1981. Induction of the body form and food habits of snakes. Amer. of various androgen-dependent esteroproteases Zool. 23:443-454. (trypsin-like and chymotrypsin-like enzymes) by Simon, T., A. Bdolah, and E. Kochva. 1980. The triiodo-L-thyronine in the submandibular glands two-component toxin of Vipera palaestinae: Con- of female mice and mice with testicular femini- tribution of phospholipase A to its activity. Tox- zation.J. Biochem. 89:1793-1798. icon 18:249-259. Kihara, H., S. Matsuda, and M. Yaita. 1977a. The Strydom, D.J. 1977. Snake venom evolution. S. Afr. relationship between Habu (Truneresurus fiavo- J. Sci. 73:70. vxridis) venom proteinase and some inhibitors. Thomas, R. G. and F. H. Pough. 1979. The effect Acta Med. Univ. Kagoshima 19:23-28. of rattlesnake venom on digestion of prey. Tox- Kihara, H., S. Matsuda, and M. Yaita. 1977* Dis- icon 17:221-228. tribution of anti-habu venom factors in various sera. Snake 8:115-120.