Natural and Selected Synthetic Toxins - American Chemical Society

Chapter 19

Lethal Toxins of Lizard Venoms That Possess Kallikrein-Like Activity

Downloaded by STANFORD UNIV GREEN LIBR on September 23, 2012 | http://pubs.acs.org Publication Date: December 20, 1999 | doi: 10.1021/bk-2000-0745.ch019

Anthony T. Tu Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870

The venoms of the lizard genus Heloderma contain many different types of proteins. Two types of Heloderma toxins, horridum toxin and gila toxin, were isolated in our laboratory. There are some similarities and some differences between these two toxins. For example, both toxins have kallikrein enzymatic activity, and the primary structure is also similar; moreover, they both have hypotensive effects when injected into rats. Gila toxin has a higher molecular weight and consists of 245 amino acid residues, while horricum toxin contains 210 residues. Both horridum and gila toxins are hemorrhagic, yet only horridum toxin causes exophthalmia. In this review article the chemical and several biological activities of these two toxins are compared extensively.

Among lizards, only two species, Heloderma horridum and Heloderma suspectum, have been found to be venomous (Figure 1A and B). Heloderma venom, like most venoms, is a complex mixture of proteins with diverse biological activity. Several enzymes and bioactive compounds have been isolated. These properties include phospholipase A2, kallikrein-like enzymes such as gila toxin and helodermatine, arginine ester hydrolases and hyaluronidase (7). Lizard venom proteins such as extendin-3 and extendin-4 interact with vasoactive intestinal peptide receptors (2,3). Other physiologically active proteins include helodermins and helospectins « 5, 6). Lethal activities have been reported for gila toxin, horridum toxin, and another toxin called "lethal toxin" (7, 8, 9, 10, 11). However, aside from being lethal, gila toxin possesses kallikrein-like activity, and horridum toxin has ester hydrolase activity, both of which can potentially be valuable pharmacological agents.

©2000 American Chemical Society

In Natural and Selected Synthetic Toxins; Tu, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

283

284 Isolation of Gila Toxin and Horridum Toxin


Analysis of venom fractions of Heloderma suspectum suspectum and H. horridum horridum venoms revealed that both gila toxin and horridum toxins were found in the second fraction of Sephadex G-75 fractionation (Figure 2A). Both toxins were eventually purified to a homogeneous state by subsequent three-step purifications. Molecular weight determination by SDS-PAGE indicates that homogeneous gila toxin has a higher molecular weight than that of horridum toxin (Figure 3). Primary Structure Both gila toxin and horridum toxins are glycoproteins (7, 12). The carbohydrate portion of horridum toxin has been better characterized than that of gila toxin. For horridum toxin, mannose, glucose, galactose, and N-acetylglucosamine are the monosaccharides present, with a total of 4 mol of carbohydrates/mol of protein. Horridum toxin is quite different from gila toxins isolated from Heloderma venoms in primary structure and exophthalmic effect. The primary structure of apoprotein portions of two toxins was identified and is shown in Figure 4. Horridum toxin has 210 residues, whereas gila toxin has 245, with the major differences being in the Cterminus. Within thefirst210 residues, only seven sites of residue substitution exist, at positions 25, 55, 107, 111, 112, 144, and 209 (Figure 4). CD spectroscopy demonstrated similar secondary structure between the two proteins, as follows: horridum toxin: 11% a-helix, 41% 0-structure, 26% 0-turns, and 25% unordered structure; gila toxin: 6% a-helix, 42% /3-structure, 27% 0-turns, and 25% unordered structure (Figure 5). Proteolytic Activity Both toxins possess proteolytic activity, and both substrate specificity and sequence homology studies indicate that they are both kallikrein-like proteases. Both proteins hydrolyze kininogen and produce bradykinin, a hypotensive peptide (Figures 6 and 7). Degradation products of high molecular weight kininogen following incubation with horridum toxin are shown in Figure 8. Both gila toxin and horridum toxin hydrolyze arginine esters (BAEE and TAME) and do not hydrolyze tyrosine ester such as ATEE (Table 1). They do not hydrolyze chromophogenic substrates for thrombin, factor X activation enzyme, or plasmin. The fact that kallikrein-like activity is inhibited by DFP but not by EDTA suggests that both toxins are serine type protease rather than metalloenzyme. Since bradykinin is a well known hypotensive agent, hypotensive activity should be observed following animal injection with gila toxin or horridum toxin; indeed, injection of gila toxin into a rat does show pronounced effect of hypotensive action (Figure 9). Bradykinin is also known to stimulate uterus smooth muscle and to induce contractions. Testing fraction B^ (isolated after incubation of kinogen with gila toxin) using rat uterus revealed that the production of rat uterus contractions is



285

À

B

Figure 1. A. Heloderma horridum horridum from Mexico. B. Heloderma suspectum from southern Utah.



286

Cilatoxin Figure 2. Comparison offractionationpatterns for gila toxin and horridum toxin.



287

Horridum Toxin Figure 2. Continued



288

Figure 3. Homogeneity of (A) horridum toxin and (B) gila toxin on SDS-PAGE (10% gels).



289

20 L H R S E G S G Q Q E C D E T Q H P W L A L G G Q E C D E T G H P W L A L L H R S E G 8

Horridum toxin Qilatoxln

50

Horridum toxin Gllatoxin

L T A A H C E E L L T A A H C E E L

N R D N R D

G P M K I G P M K I

Horridum toxin Gllatoxin

70 80 R G D E Q V K V A A V K K C Y P A T A G T R G D E Q V K V A A V K K C Y P A T A G T

Horridum Toxin Gilatoxin

V L V L


M N N 0 L L M N N D L L

C F G M I C F G M ï

N R N V L N R N V L

Y N C N Y V N T Y N C N Y V N T

110 120 100 R E L F P M L F K L D E Y V D Y N E R V A V D Y N E R V A R E L F P M L if., K L D

130 S L P T S P A S L S L P T S P A S L 160 E I E I


P D V P V C P D V P V C


190 C A G V D F G G K O S C A G V D F G G K D S

Gilatoxin

S W G F

Gilatoxin

245 G T T C P

N I N I

30

Üji W S Q V L 1 W S Q V L

G A E G A E

ISO 140 C S V L G W G T T -,9 • P D D V T L P D O V T L C S V L G W G T T

Éjj!

180 N K L N K L

170 N N A V C Q V A R D L W K N N A V C Q V A R D L W K

200 210 C K G D S G G P L V C D N Q L T G i. ' V C K G D S G G P L V C D N Q L T Qitïii V

N C E Q G E K Y G Y I

K L I

230 K F N F W I

ON

I

I

240 Q G

Figure 4A. Alignment of amino acid residues of horridum toxin and gila toxin. Residues that differ are shaded.



Figure 4B. Alignment of amino acid residues for horridum toxin, human pancreatic kallikrein, human plasma kallikrein, and trypsin. Residues which shaded are identical to horridum toxin. Alignments were made to maximum homology.


291


4.000E+03

Figure 6. Degradation of high molecular weight kininogen with plasma kallikrein (left) and with gila toxin (right) examined by SDS-PAGE.



292

Figure 7. Degradation of high molecular weight kininogen by horridum toxin.

Kininogen (114 kD)

H (63 kD)

gjj^radykinin

L (58 kD)

[ ML (45 kD) QF(13kD)

Figure 8. Diagram showing the hydrolysis products of kininogen when incubated with horridum toxin.



240 200 0 54 0 0 0 0 Yes No

245 209 0 52 0 0 0 0 Yes No

A c t i v i t y of g i l a toxin (units/mg)

Unit i s the amount of enzyme which hydrolyzes 1 jxmol of substrate per minute.

N-Benzoylarginine e t h y l ester (BAEE) N-Tosylarginine methyl ester (TAME) N-Acetyl-L-tyrosine e t h y l ester (ATEE) #-Benzoyl-Phe~Val-Arg-pNA (thrombin-like) Benzoyl-Ile-Glu-Gly-Arg-pNA (factor X A) N- Benz oy1 - Val - Leu - Lys - pNA (human plasmin) Val-Leu-Arg-pNA (urinary k a l l i k r e i n ) Ile-Phe-Lys-pMA (plasmin-like) I n h i b i t e d by DFP I n h i b i t e d by EDTA

Substrate

A c t i v i t y of horridum toxin (units/mg)

Proteolytic Activity of Horridum Toxin and Gila Toxin Toward Synthetic Substrates and Effects of Inhibitors

TABLE 1


294


mm

min

mm

min

Figure 9. The effect of gila toxin on rat blood pressure. A, blood pressure after injection with normal saline. B, blood pressure after intravenous injection of gila toxin (0.5 fxg/g; 20% of LD ). Arrow indicates the point of sample injection. 50



295 concentration-dependent, indicating that the peptide is indeed bradykinin (Figure 10). Neither gila toxin nor horridum produced a fibrin clot when incubated with fibrinogen (data not shown). Fibrinogen has been used as a substrate by many investigators to study the action of venom proteases because these proteases often affect the blood coagulation pathway. The nature offibrinogenhydrolysis gives some insight into the substrate specificity of horridum toxin. Fibrinogen is composed of three polypeptide chains: Aa, Bj3, and 7. It is therefore of interest to determine which of the chains are hydrolyzed by horridum toxin. Horridum toxin and gila toxin hydrolyzed the A a chain of fibrinogen faster than the B/î chain (Figure I I A , C). The 7 chain was resistant to hydrolysis for 12 h, although hydrolysis did occur after 18 h or incubation. Atroxase hydrolyzed both the A a and Bj3 chains, but not the 7 chain (Figure 1 IB). The manner of hydrolysis by horridum toxin differed from that of atroxase, but was similar to that of gila toxin (Figure 11 A,B,C), which hydrolyzed all three chains. Although gila toxin hydrolyzed kininogen, it did not hydrolyze the substrate for kallikrein, Val-Leu-Arg-pNA. Plasmin-like activity was also absent, as neither substrate tested, N-benzoyl-Val-Leu-Lys-pNA and N-benzoyl-Ile-Phe-Lys-pNA, was hydrolyzed. Comparison to Other Kallikreins Kallikreins exist as a tissue-type and as a plasma-type protein. A notable difference between tissue-type kallikreins and plasma-type kallikreins is the presence of residues 91-100 (Figure 4 A, B), MSLLENHTRQ, in tissue-type kallikreins and not in plasma-type kallikreins. This segment is frequently called the "kallikrein loop." Horridum toxin has segment 91-100,although the sequence is different from that of tissue-type kallikrein. Other similarities between horridum toxin and tissue-type kallikrein include the conservation of the cystine residues, the proline residues, and some large domains, which are characteristic of serine proteases. These sequences include the LTAAHC region at position 40-45, GWG at 155-157, GGKD at 209-212, and GDSGGPL at 216-222. In fact, the C-terminus has a high degree of homology with trypsin (a serine protease). Nevertheless, the Cys -Val motif seems to be a feature of tissue kallikreins and is absent from plasma kallikreins. The Cys in other tissue kallikreins is substituted by Trp in horridum toxins, whereas the Trp in human kallikrein is substituted by Cys in the toxins. However, the enzymatic activity is conserved, and there appears to be room for those residues in the structure. The absence of some characteristic features of plasma kallikreins, such as the GLPLQ region at 48-52, Cys , and Lys -His , and Trp -Arg , also further substantiates the classification of horridum toxin as a tissue kallikrein. His , Asp , and Ser play important roles in catalysis by serine proteases. All these residues are present in horridum toxin as well, further corroboration that horridum toxin is also a serine protease. Taken together, the evidence indicates that horridum toxin and gila toxin are different types of kallikreins: structurally similar to tissue type, but having the substrate specificity of plasma kallikrein. 174

175

29

55

138

225

226

231

232

44

218


109

296

A cm 3-

0.01 y g Buo

w Y

2-


1-

B cm

0.02 p g B40

321-

c cm 321-

j

bradykinin (positive control )

BKy/

y

0 cm 3-

kininogen (negative control)

2-

c

Figure 10. Stimulated contraction of isolated rat uterus smooth muscle. A, effect of 0.01 jLtg/g of 1*40 is the purified product bradykinin released from HMW kininogen by the action of gila toxin. W is the point of washing of muscle with buffer. B, effect of 0.02 /ig of B^. C, effect of 0.02 ^g of bradykinin (positive control). D, effect of filtrate (potential spontaneously released product) from incubation of kininogen alone (negative control). The X-axis represents time.



297

Figure 11. SDS-PAGE analysis of fibrinogen (2%) hydrolysis products after incubation for the indicated time intervals with (A) gila toxin, (B) atroxase, (C) horridum toxin.


298


Cleavage of Angiotensin I and II by Gila Toxin Incubation of angiotensin I with gila toxin resulted in the degradation of angiotensin I, a hypertensive peptide originating from angiotensinogen. At zero time, only angiotensin I was visible (as peak a, Figure 12). As incubation continued, digestion of angiotensin I is demonstrated by the appearance of a new peak, peak b (Figure 12 Bb, Cb, Db). The amino acid sequence of peak b was determined and found to be Val-Tyr-Ile-His-Pro-Phe-His-Leu, demonstrating that the arginylvaline bond of angiotensin I was cleaved by gila toxin. Gila toxin also hydrolyzed angiotensin II and released a dipeptide from the NH -terminal end (data not shown). The cleavage of angiotensin I may be a contributing factor for the prolonged hypotensive action of gila toxin. 2

Toxicology Both gila toxin and horridum toxins are lethal and have an L D of 2.9 and 2.6 ptg/g in mice, respectively. Fractionation of the toxins from crude venom did not significantly increase toxicity. The action of horridum toxin on biological systems is significantly different from gila toxin. Horridum toxin causes exophthalmia, whereas gila toxin does not (Figure 13). The presence of two proteins with similar sequences but different biological activities is not unexpected in Heloderma venom. Other peptides, such as extendin-3 and extendin-4, differ only in two amino acids but have different interactions with their receptors. 50

Discussion Lizard envenomation causes dizziness, hypotension, tachycardia, tissue damage, and ecchymosis. Anaphylaxis, severe periorbital swelling, lymphangitis, and lymphoadenites were also observed in lizard poisoning. Since a crude venom contains many varieties of proteins, it is hard to define which venom component induces which biological effect. So far, two toxins were isolated from lizard venom in our laboratory. Gila toxin is nonhemorrhagic but lethal to mice. Horridum toxin is hemorrhagic, lethal, and causes exophthalmia. Venom lethality is apparently caused by gila toxin or horridum toxin. One surprising fact is that both gila toxin and horridum toxin are proteolytic enzymes especially like kallikrein. Kallikrein is a relatively natural constituent of all animals. The amino acid sequence of both gila toxin and horridum toxin has high homology with kallikrein from mammarian source. Then a question is why the lizard kallikrein is so toxic while the mammarian enzyme is nontoxic. This question has to be solved eventually in the future by further investigation. Another question is why horridum toxin exhibited exophthalmia while gila toxin did not, although the two toxins have some similarity in their amino acid sequences. Again, there is no answer at the moment.


299


A

H

a •(

B

a

H

t

M?

2

-«b

L

'.VU.

M

M aH

a*-

I

!§

Figure 12. HPLC chromatograms of angiotensin I cleavage by gilatoxin. Each chromatogram represents angiotensin I (a) and degradation products (b) after 0-, 1-, 2-, and 6-h incubation times (A-D, respectively). Chromatography was performed by using a linear gradient of 0-50% acetonitrile in water containing 0.1% trifluoroacetic acid for 50 min at a flow rate of 1 ml/min.



300

Figure 13. The exophthalmic effect induced by intravenous injection of horridum toxin. (A) control (saline) ami (B) horridum toxin.


301


Literature Cited 1. Tu, A. T. In Handbook of Natural Toxins; Tu, A. T., Ed.; Marcel Dekker: New York, 1991, Vol. 5; pp. 755-773. 2. Eng, J.; Kleinman, W. A.; Singh, L.; Singh, G.; Raufman, J. P. J. Biol. Chem. 1992, 267, 7402-7405. 3. Raufman, J. P.; Jensen, R. T.; Sutliff, V. E.; Pisano, J. J.; Gardner, J. D. Am. J. Physiol. 1982, 242, G471-G474. 4. Vandermeers, A.; Vandermeers-Piret, M . C.; Vigneron, L.; Rathe, J.; Stievenart, M . ; Winand, J.; Christophe, J. Eur. J. Biochem. 1991, 196, 537544. 5. Vandermeers, A.; Gourlet, P.; Vandermeers-Piret, M . C.; Cauvin, A.; DeNeif, P.; Rathe, J.; Sroboda, M . ; Robberecht, P.; Christophe, J. Eur. J. Biochem. 1987, 164, 321-377. 6. Parker, D. S.; Raufman, J. P.; O'Donohue, T. L.; Bledsoe, M . ; Yoshida, H.; Pisano, J. J. J. Biol. Chem. 1984, 259, 11751-11755. 7. Utaisincharoen, P.; Mackessy, S. P.; Miller, R. A.; Tu, A. T. J. Biol. Chem. 1993, 268, 21975-21983. 8. Hendon, R. A.; Tu, A. T. Biochemistry 1981, 20, 3517-3522. 9. Nikai, T.; Imai, K.; Sugihara, H.; Tu, A. T. Arch. Biochem. Biophys. 1988, 264, 270-280. 10. Komori, Y.; Nikai, T.; Sugihara, H. Biochem. Biophys. Res. Comm. 1988, 154, 613-619. 11. Komori, Y.; Nikai, T.; Sugihara, H. Biochim. Biophys. Acta 1988, 967, 92102. 12. Datta, G.; Tu, A. T. J. Peptide Res. 1997, 50, 443-450.


Natural and Selected Synthetic Toxins - American Chemical Society

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