Biochemistry of Helodermatid Venom

by Charles Eugene Lidikay

Most people, when asked to name a venomous reptile would think of a snake, and would probably not even realize that there are two species of venomous lizards. The gila monster, Heloderma suspectum, and the Mexican beaded lizard, Heloderma horridum, are the only extant venomous lizards known to exist today. They are large lizards found in the Sonoran Desert of North America, and are the only members of the reptilian family Helodermatidae. Due to their secretive lives, much remained unknown about them up until the last few decades. In fact, many scientists debated as to whether they were venomous. In spite of recent discoveries, much remains unknown about the helodermatids. The venom has been studied as far back as 1891, but it is only recently that much of its biochemistry has been elucidated.

The venom is principally a neurotoxin that causes respiratory failure. It is not as potent as rattlesnake, cobra or black widow venom and would probably require at least half a mL to kill a human. A variety of warm blooded vertebrates are more susceptible than cold blooded vertebrates.

The gila monster's venom apparatus is far different from that of venomous snakes. In both, venom is introduced by means of capillary action via grooves in particular teeth. The main difference between the two is that in the gila monster, the lower rather than the upper teeth are grooved. The groove is not a complete canal, but nevertheless is efficient enough to envenomate prey. These lizards have a characteristic tenacious bite in which they hold on. During this time they tend to gnaw, which seems to aid in the delivery of the venom (Bogert and Martin del Campo, 1956).

The venom is introduced on both sides between the lower jaw and cheek by a duct that serves inferior labial glands. These glands are multi-lobular and together may be able to produce as much as a mL of venom.

Snakes and lizards both comprise the reptilian order squamata, and are thus closely related, so it is not a suprise to find that their venoms are similar. Since many more studies have been performed on snake venoms, I will first discuss them and will finish with a description of helodermatid venom. It is believed that the use of venom is for primarily the capture of prey, rather than defense. Venoms are usually broadly classified in snakes as being mostly myotoxic or neurotoxic. However, this is a generalization, for venoms have multiple physiological, pharmacological and biochemical actions. Venoms contain a complex mixture of bioactive molecules that is species specific. Each component alone may have pronounced effects or may act synergistically with other components of the venom. Some components have more than one bioactivity (e.g. possessing both kinin-like and phospholipase-A2-like activity). Some components may act on various organs of the prey, while others may assist in the penetration and absorption of the venom. In colder climates, snake venom may play an important role in external digestion of prey. This is important since reptiles are ectothermic (i.e. they rely on the environment for body heat). In cold environments, digestion may be too slow, which may lead to putrefaction of ingested prey. Therefore, any external digestion that occurs would be an advantage to snakes living in cold environments.

Studies of whole venom of helodermatids have been used to characterize the toxic nature and to determine the relationship these lizards have with snakes. The whole venom was found to be heat stable and to hydrolyze L-Phe-L-Phe, resembling that of the elapids (cobras and sea snakes). These snakes are the more primitive of the venomous snakes, and it is thought that helodermatids might be more closely related to them (Tu and Murdock, 1967).

The venom is not highly toxic to man. Most bites result only in painful swelling at the localized area of envenomation. There have been isolated cases of fatality, but alcohol intoxication seems to have been the major factor. The action on lab animals (mostly guinea pigs and other small rodents) varies widely. Intravenous injection of whole venom had a LD50 (quantity required to kill fifty percent of test animals) of 2.7 ęg/g whereas that of the purified gilatoxin had a higher LD50 of 2.9. This further illustrates how the components of the venom act together to produce the toxic effects.

Three different toxic elements have been described. The glycoprotein gilatoxin is reported to be the major toxic element (Utaisincharoen, et al, 1993). It has a Mr of 35,000. Another glycoprotein called hemorrhagic toxin (Mr 31,000) has also been reported. Lethal toxin has also been discovered in the venom of H. horridum (Komori, et al, 1988). All three are proteolytic and cause hypertension.

Helodermatid venom also contains phospholipase-A2 (Mr 19,000) (Sosa, et al, 1986). Phospholipase-A2 enzymes are found throughout nature performing a variety of functions. They catalyze the hydrolysis of phospholipids at the second carbon position of glycerol esters. Much research has been performed on snake and helodermatid phospholipase-A2s. Phospholipase-A2 is one of the twenty or so components found in venoms, but it may constitute up to 10% of the dry weight (Tu, 1991).

In fact, many phospholipase-A2 enzymes are actually responsible for lethality. Most lethal phospholipase-A2 enzymes can be characterized by their neuro- and myotoxicities. Neurotoxic phospholipase-A2s act at the presynaptic axon terminal of the peripheral nervous system where they cause an initial increase and then a decrease in acetylcholine (Ach) release (Tu, 1991). Ach is responsible for delivering the impulse from nerve fibers to muscle fibers. The lowering of Ach release caused my neurotoxic phospholipase-A2 may lead to paralysis and respiratory failure as the impulse to contact is no longer able to be transmitted to the muscles. Myotoxic phospholipase-A2 act by depolarizing muscles and cause necrosis of muscle tissue. This necrosis is responsible for much of the pain associated with envenomation. Most phospholipase-A2 activity falls somewhere in between these two extremes, and it is thought that this is the case with helodermatid phospholipase-A2.

Helodermatid venom also contains hyaluronidase (Mr 63,000) specific for the hydrolysis of hyaluronic acid. Hyaluronidase can be found in a multitude of animals. It has been found in many snake venoms. It has been called the spreading factor because it enhances the dispersion of the venom into the tissue of the prey. The tests on the hyaluronidase of helodermatid venoms gave sound proof for its role as a spreading factor. Hemorrhagic protease of a snake was injected along with helodermatid hyaluronidase and showed an increase in hemorrhage relative to the hemorrhagic protease alone.

Phosphomonoesterase and phosphodiesterase have also been isolated from helodermatid venom, but the significance is not yet understood. These may act together with other components of the venom or may have other functions yet to be discovered (Tu, 1991).

Of the proteases present in helodermatid venom, the most studied is the kallikrein-like arginine ester hydrolase (Mr 63,000). It is a member of the serine protease family of enzymes. Kallikreins release kinins which lead to hypotension. Injection of the venom into rabbits caused death by hypotension and it is thought that the presence of the kallikrein-like enzyme is responsible. Kallikreins are also involved in the processing of prohormones and therefore may play a part in the activation of venom precursors (Alagon, et al, 1986).

The venom of helodermatids was found to increase enzyme secretion in a pancreatic acinar preparation, at similar levels as does vasoactive peptide (VIP), or secretin. It also caused a similar increase in intracellular cAMP. It was concluded that the venom contained a biopeptide (secretagogue) that stimulates pancreatic enzyme secretion by interacting with VIP receptors. It was later found that the secretagogue activity of the venom was due to actually two distinct bioactive peptides. Helodermin (Mr 5,900) found in H. horridum is responsible for the increase in levels of cAMP, whereas this function is performed by two distinct proteins in H. suspectum. These two proteins are helospectin-I, and helospectin-II (both with Mr 4,200) (Parker, et al, 1984) Helospectin-I is a 38-residue peptide with the structure: H-S-D-A-T-F-T-A-E-Y-S-K-L-L-A-K-A-L-Q-K-Y-L-E-S-I-L-G-S-S-T-S-P-R-P-P-S-S. Helospectin-II is identical to helospectin-I except that is lacks the last serine.

Increases in enzyme secretion from the pancreatic acini is also due to pancreatic stimulatory factor (PSF, Mr 17,500) (Vandermeers, et al, 1984). This elicits no increase in cellular cAMP levels as it does not interact with VIP receptors. It is thought that PSD interacts with secretin receptors instead (Dehaye, et al, 1983).

These secretagogues have become more specifically referred to as exendins in the helodermatids. That is, peptides that are found in the exocrine secretion (venom) that have endocrine activity (e.g. pancreatic secretagogues). Therefore, helospectin-I and helospectin-II are referred to as exendin-1 and exendin-2, respectively. Exendin-3 was also found (Raufman, et al, 1991). It is 48% homologous to human glucagon with the sequence: H-S-D-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-Q-W-L-K-N-G-P-S-S-G-A-P-P-P-S (Eng, et al, 1990). Exendin-4 has also been described. It is the same as exendin-s except that it has G-E instead of S-D for amino acids 2 and 3, respectively (Eng, et al, 1992). Both of these act similarly on pancreatic preparations by both stimulating increase in cellular cAMP and enzyme secretion. They are thought to play similar roles in the pathology of the venom.

Pancreatic secretagogues are important components of many venoms because they play a major role in their effects. Pancreatitis has been reported after certain scorpion bites (Raufman, et al, 1982). Secretagogues of non-mammals are thought to be precursors of certain bioactive molecules found in mammals. Raufman and coworkers therefore concluded that the secretagogue of helodermatid venom "... is structurally related to but distinct from VIP", the mammalian hormone which stimulates the pancreas to secrete enzymes.

A very interesting new medical breakthrough in diabetes treatment has resulted from the discovery of exendin-3. Amylin Pharmaceuticals, Inc. believes that exendin-3 and its analogs may be beneficial in the treatment of both type I and type II diabetes. It has been found that exendin-3 suppresses the rise in blood glucose levels that occurs after meals in diabetic patients, as well as, stimulating insulin secretion. Exendins also modulate gastric emptying which can slow the entry of glucose into the blood.

In conclusion, the venom of helodermatids is complex and similar to that of snakes. The venoms are composed of a mixture of at least twenty bioactive molecules. The toxic nature is probably not due to just one fraction of the venom, but rather, due to the complex biochemical, pharmacological, and physiological interaction of all the components. Recently, new hope for the treatment of diabetic patients has resulted from research on helodermatid venom. It is interesting to note that a feared lizard is now being looked upon to treat a disease. At one time, these lizards were killed on sight and were drastically reduced in numbers, but hopefully with this new medical breakthrough, biodiversity might be seen as an asset to human health. If these lizards would have been driven to extinction, this new treatment could not even be postulated.


Copyright © 1997 Charles Lidikay and Koni Stone

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