TOXIN REVIEWS Vol. 22, No. 4, pp. 651–662, 2003
Variations of Phospholipases A
2in the Geographic
Venom Samples of Pitvipers
Inn-Ho Tsai,*Ying-Ming Wang, and Yi-Hsuan Chen
Institute of Biological Chemistry, Academia Sinica, and Institute of Biochemical Sciences, National Taiwan University,
Taipei, Taiwan
CONTENTS
ABSTRACT . . . 652 I. INTRODUCTION. . . 652 II. RESULTS . . . 653
A. PLA Variations in the Venom of Calloselasma
rhodostoma and Crotalus v. viridis . . . 653 B. PLA Variations in the Venom of Protobothrops
mucrosquamatus . . . 654 C. PLA Variations in the Venom of Trimeresurus stejnegeri . . . 656
*Correspondence: Inn-Ho Tsai, Institute of Biological Chemistry, Academia Sinica, and Institute of Biochemical Sciences, National Taiwan University, P. O. Box 23-106, Taipei, Taiwan; Fax: + 886-2-23635038; E-mail: bc201@gate.sinica.edu.tw.
651
DOI: 10.1081/TXR-120026919 0731-3837 (Print); 1525-6057 (Online)
III. CONCLUSION . . . 659 ACKNOWLEDGMENTS . . . 659 REFERENCES . . . 660
ABSTRACT
The geographic variations of phospholipases A2(PLAs) in the venom of
four medically important pit vipers were investigated. We have studied the PLAs by HPLC-purification, cDNA cloning and sequencing, mass characterization, and functional classification. We found that: 1) Anti-platelet acidic PLA isoforms in the venoms of Calloselasma rhodostoma from five southeastern Asian countries, and those of the Crotalus v. viridis from seven American States are differentially expressed depending on locality. The variations could be attributed to their distinct specificities towards the platelets of different prey, and to possible adaptation for playing other functional roles. In contrast, structures of the myonecrotic and the edema-inducing basic PLAs in both venoms were relatively conserved. 2) A special type of the acidic anti-platelet PLA is present in the venom of some Protobothrops species. Its expression level is diminished in the snake of the southern or the tropical ranges. 3) The venom of Bamboo tree vipers (Trimeresurus stejnegeri) in Taiwan and China showed extraordinary geographic variations in their acidic and basic PLAs. The high RNA-polymorphism of their venom proteins may have been derived from interbreeding between several ancestral pit viper species. In addition, migration, isolation of different populations and rapid evolution of the venom proteins to adapt for diversified diets may have resulted in further variations in this venom species.
Key Words: Pitviper venom; Phospholipase A2; Geographic variations
of venom; Molecular cloning.
I. INTRODUCTION
The multigene snake venom families have gone through gene-duplication and accelerated evolution to generate variants with adapted functions (Kordis et al., 2002). While ontological, seasonal, and geographic variations of the venom proteins are recognized, the details and underlying causes remain to be elucidated. In order to characterize the intra-species venom variations, not only carefully pooled, but also many individual venoms should be collected from various localities, and efficient protein analyses should be performed on many samples (Chippaux et al., 1991).
The secreted phospholipase A2 (PLA) is a well-characterized enzyme
family found in almost all viperid venoms (Danse et al., 1997; Kini, 1997). It has been known that many pit viper venoms contain distinct subtypes or isoforms of PLAs (Tsai, 1997), and show geographic variations. For ex-amples, the Lys49 – PLA homologs were absent in the venom of Okinawa T. flavoviridis but present in the habu venoms on other neighboring islands (Chijiwa et al., 2000); contents of Mojave-toxin-like PLA in the venom of Crotalus s. scutatus and Crotalus h. horridus were varied with different localities (Glenn et al., 1983, 1994); the proportion of two acidic venom PLAs in C. rubber changed from North to South in Baja Peninsula (Mexico) (Straight et al., 1992), and variations related with a form of local adaptation was found in B. asper venom (Sasa and Barrantes, 1998).
It is important to know the geographic variations of the venom for the purpose of antivenin manufacture, snakebite treatment and conservation management. Besides genetic background of the species, microhabitat sep-aration and adaptation to diet or ecology may gradually result in variations in snake venom toxins (Creer et al., 2002; Daltry et al., 1998). How and why are venom PLAs varied according to its locality are the questions to be addressed at the molecular level. We have used a comparative proteomic approach coupled with cloning and complete sequencing of the venom PLAs. The primers used in our PCR experiments were reliable for cloning most of the viper venom PLAs from the fresh venom glands (Tsai et al., 2001, 2003). Other alternative primers have also been designed based on distinct sequences at the N-terminal of the PLAs of interest.
II. RESULTS
A. PLA Variations in the Venom of Calloselasma rhodostoma and Crotalus v. viridis
During a typical reversed phase-HPLC purification of the venom PLAs under pH < 3, the basic PLAs were eluted earlier than the acidic PLAs, which often form dimers in buffer solution of pH > 4.2 (Tsai et al., 2001). The acidic anti-platelet PLAs in the venoms of Malayan pit viper (Cal-loselasma rhodostoma) from four southeastern Asian Nations (Table 1) are differentially expressed depending on locality (Tsai et al., 2001). These var-iations may be attributed to the adaptive evolution and distinct specificities of the PLAs towards platelets of different preys (Table 2).
Another example of the geographic variation was observed for the venom PLA of the prairie rattlesnake (Crotalus v. viridis) from seven American States (Tsai et al., 2003). Venoms of C.rhodostoma and C.v. viridis, contain only a single form of basic PLAs playing myonecrotic and/or edema-inducing role
(Tsai et al., 2001, 2003), but contain several acidic PLAs with E6 substitution (Tables 1 and 3). Cvv – E6a is more potent in the inhibition of human platelets than Cvv – E6e, while the reverse is true for the inhibition of rabbit platelets. Another acidic PLA, Cvv – E6f, may cause edema in the rat paw (Tsai et al., 2003). The functions of Cvv – E6 b, c, and d are not clear and a role for the digestion of prey is suspected. Notably, Cvv – E6e and Cvv – E6f are only expressed in the southern range (e.g. Texas, New Mexico, and Arizona), while Cvv – E6b and Cvv – E6c are only found in the northern range of the rattlesnake (e.g. South Dakota, Wyoming, and Colorado). The content of the myotoxic PLA with N6 substitution increased as the range of the rattlers moved toward Southeast in accord with their adaptation to the diets consisting more small mammals. Our results on venom PLA correlates well with those derived from phylogenetic analyses of mtDNA of the C.viridis complex (Anaya et al., 1992).
B. PLA Variations in the Venom of Protobothrops mucrosquamatus
The venoms of P. mucrosquamatus (formerly Trimeresurus mucros-quamatus) in Taiwan contain three or four PLAs depending on the sam-ple site. Only the habu snakes from northern Taiwan contain an acidic
Table 2. The inhibition potency of three venom PLAs of C. rhodostoma on the aggregation of rabbit and the human platelets induced by ADP.
PLA variant
Rabbit platelet Human platelet IC50(nM)
H1E6 70 880
S1E6a 142 428
S1E6b 110 520
Table 1. Proportion of PLAs in different geographic pooled venom samples of C. rhodostoma.
Venom origin Vietnam Thailand Malaysia West Java
PLA Relative abundance (%)
W6 55 63 59 41
H1E6 8 14 5 0
S1E6a 17 6 9 5
Table 3. Masses and enzymatic activities of the acidic PLAs of C. v. viridis venom. PLA Mass Enzyme activity (m mol/min/ m g enzyme) N-terminal sequences E6a 13467 351 ± 1 6 S L V Q F E T L I M K I A G R S G L L W Y S A E6b 13660 1129 ± 2 0 N L V K S E6c 13817 840 ± 6 N L V K S E6d 13782 680 ± 5 M V K FS E6e 13633 1306 ± 4 2 N L V K S E6f 13876 518 ± 2 5 MM I V K F G N6 14200 280 ± 8 N L NK M KMMTKKNAF P F TS Masses were determined by ESI-MS. The initial rates toward 3 m M dipalmitoyl phosphatidylcholine and 3mM sodium deoxycholate were measured twice in the presence of 10 mM Ca 2+ at 37 °C.
anti-platelet PLA with Arg6 substitution (designated as TmPL-III) (Tsai et al., 2001). This PLA is absent in all the ten individual venom samples from southern Taiwan we analyzed. The mRNAs encoding a PLA very similar to TmPL-III were found in both venom glands of C. rhodostoma (Tsai et al., 2000) and Protobothrops jerdonii (Lu et al., 2002) and the protein sequences are shown in Figure 1. It appears that the expression of this PLA reduced remarkably as the range of P. mucrosquamatus moved toward South or became tropical, and this PLA is not expressed in all the geographic venom samples of C. rhodostoma, a tropical species (Tsai et al., 2001).
C. PLA Variations in the Venom of Trimeresurus stejnegeri
Previous analyses on the mtDNA and venom proteins revealed un-usually high geographic diversity in the populations of bamboo-tree viper (Creer et al., 2001 – 2003). Two linage of Taiwanese T. stejnegeri (pre-viously named T. gramineus) have been identified. The smaller linage was restricted to the north and east coasts, whereas the larger linage occupied all but the northern range of the species (Creer et al., 2001). Although five PLAs (i.e. K49a, A1, A2, A5 in Table 4a and Tgr-PLA-IV) have been reported in the pooled venom of Taiwanese T. stejnegeri (Fukagawa et al., 1992; Nakai et al., 1995; Oda et al., 1991), we further identified about 8 novel PLAs from about 20 geographic venom samples of this species and compiled in Table 4a.
Recent results of a survey on the masses of the PLAs from 104 venom samples of Taiwanese T. stejnegeri collected from 38 sites identified about 22 PLA isoforms (Creer et al., 2003). By matching the PLA-mass results with our molecular data (Table 4a), the PLA-profiles for the venom samples from different localities were summarized in Table 4b. Apparently, the geographic variations are rather complicated and individual differences were also observed. In general, K49a, Ts-3 and 4 are found in the northern coast population; Ts-R6, G6, A2 and A5 are found mainly in the T. stejnegeri venom from southwestern Taiwan; Ts-A1 and K49a are common in the eastern population; and Ts-A1 and K49a may have evolved into Ts-A6 and K49c, respectively, in T. stejnegeri on the two off-shore islands (Table 4b).
In addition, we have identified about 6 distinct venom PLAs from T. stejnegeri collected from three southeastern Chinese provinces (data not shown). Only two of them are identical to those from Taiwanese T. stejnegeri venom. Thus, there are great venom differences between the Taiwanese and the Chinese T. stejnegeri. A cladogram or phylogentic tree, based on selected amino acidic sequences in the acidic PLAs from Asiat-ic viper venoms, suggested that the venom genes of T. stejnegeri were
Figure 1. Alignment of the amino acid sequences of the acidic PLAs with Arg-6 substitution from P. mucrosquamatus (Tsai et al., 2001), C. rhodostoma (Tsai et al., 2000) and P. jerdonii (Lu et al., 2002). The residues identical to those in the first line are denoted with a dot, gaps (– ) are introduced to be consistent with the common numbering system for the group II PLAs.
Table 4.
a. Inventory of the PLA variants found in the venom samples of Taiwanese T. stejnegeri.
PLA variant Mass N-terminal sequences 1 23
Ts-R6 13689 HLLQLRKMIKKMTNKEPILSYGK Ts-K49a 13892 SVIELGKMIFQETGKNPATSYGL Ts-K49b 13929 GVIELTKMFVQEMGKNALTSYSL Ts-K49c 13876 SVIELGKMIFQETGKNPATSYGL Ts-G6 13805 SLLEFGRMIKEETGKNPLSSYIS Ts-A1 13734 HLMQFETLIMKVAGRSGVWYYGS Ts-A2 13779 NLLQFENMIRNVAGRSGIWWYSD Ts-A3 13750 SLIQFETLIMKVAKKSGMFSYSA Ts-A4 13925 SLIQFETLIMKVAKKSGMFSYSA Ts-A5 13711 NLMQFETLIMKVAGRSGVWYYGS Ts-A6 13939 HLMQFENMIKKVTGRSGIWWYGS Ts-A7 13905 HLLQFETMIIKMTKQTGLFSYSF
b. Geographic variations of PLAs in the venom of Taiwanese T. stejnegeri (Creer et al., 2003).
Geographic region Sample site Composition of PLA variantsa Northern Taiwan Taipei K49a, A3, A4, (A1)
Taoyaun A3, K49a, G6
Western Taiwan Miaoli A1, K49a, G6
Taichung A1 or A3, (G6, or A2) Nantou A5 or A3, (A6, R6, or G6)
Southwestern Taiwan Chiai R6, G6, A3
Kaohsiung R6 or A1, G6, (A5, or K49a) Pingtung A5, K49a, (K49b, A2, or A6)
Eastern Taiwan Ilan K49a, A1
Hualien A1, K49a
Taitung K49a, A1or A3 Pacific Islands Green Island A6, K49c
Lanyu A6, K49c
Abbreviations of the PLAs: Ts, T. stejnegeri venom; R6, PLA with Arg6 substitution; A1 A7, the acidic PLAs. Masses were determined by ESI-MS spectrometry, and the amino acid residues 6 are bolded.
a
PLA isoforms listed are those found in the majority of the samples from that locality, and in the order of the Mass peak intensity (by MALDI-TOF) (Creer et al., 2003). The PLAs found in only one venom or less-representative venom are listed in parentheses.
possibly resulted from combination or merger of the genes from several ancestral species. Fast evolution and adaptation to diversified diets, migra-tion and separated populamigra-tions may also contribute to the venom variamigra-tions.
III. CONCLUSION
Depending on the natural history and degree of ecological diversity of the snake, the venom compositions show more or fewer geographic variations. Venom variations are obviously not restricted to the venom PLA, e.g., variations in the venom proteases of rattlesnake have been shown (Minton and Weinstein, 1986), and venom serine proteases present in T. stejnegeri of Taiwan apparently are different from those of China (Chang and Huang, 1995; Zhang et al., 1998). However, venom PLAs are con-venient and informative windows for comparison of the variations. In the cases of P. mucrosquamatus and C.rhodostoma, variations in the venom PLAs are manifested mainly in their different proportions, while an on-and-off switch for the expression of individual PLA is common in the cases of C.v. viridis and T. stejnegeri. Usually, the acidic PLAs of the pitviper venoms have evolved with greater variations than the basic PLAs. This is probably because the functional specificities toward the preys’ platelets are diversified among the acidic PLAs (Tsai et al., 2003). In contrast, the basic myotoxic, edema-inducing PLAs possibly have wider specificity and demand less structural variations. However, contents of the basic isoforms often are increased as the pit vipers move to hotter-drier localities, probably as an adaptation to diets containing more small mammals.
Our results provide clues for the fast evolution and high diversity of venom proteins of pit vipers. PCR-assisted cDNA cloning and sequencing, as well as high-throughput proteomic tools have greatly facilitated the venom research. Ecological study has also helped to correlate the venom diversity with the prey-environment. But it remains difficult to explain the venom variations in terms of the diet-ecology of the snakes. More studies on the functional roles and specificities of venom components are essential for deciphering the meaning of the venom diversity. Hopefully, clarification of the functional subtypes of the toxins may also improve their usages for medical or pharmaceutical purposes.
ACKNOWLEDGMENTS
We thank Professors S. P. Mackessey, E. Rael and A. T. Tu for their gifts of C. v. viridis venom or glands, Professor M. C. Tu, Q. C. Wang and
Mr. T. S. Tsai for the gifts of T. stejnegeri venom and glands, and Dr. W. H. Chou for allowing us to read the pre-print before publication (Creer et al., 2003). The research has been supported by grants from National Science Council, and Academia Sinica, Taiwan, ROC.
REFERENCES
Anaya, M., Rael, E. D., Lieb, C. S., Perez, J. C., Salo, R. J. (1992). Antibody detection of venom protein variation within a population of the rattlesnake Crotalus v. viridis. J. Herpetol. 26:473 – 482.
Chang, M. C., Huang, T. F. (1995). Characterization of a thrombin-like enzyme, grambin, from the venom of Trimeresurus gramineus and its in vivo antithrombotic effect. Toxicon 33:1087 – 1098.
Chijiwa, T., Deshimaru, M., Nobuhisha, I., Nakai, M., Ohawa, T., Oda, N., Nakashima, K., Fukumaki, Y., Shimohigashi, Y., Hattori, S., Ohno,
M. (2000). Regional evolution of venom-gland phospholipase A2
isoenzymes of Trimeresurus flavoviridis snakes in the southwester islands of Japan. Biochem. J. 347:491 – 499.
Chippaux, J. P., Wolliams, V., White, J. (1991). Snake venom variability: methods of study, results and interpretation. Toxicon 29:1271. Creer, S., Malhotra, A., Thorpe, R. S., Chou, W.-H. (2001). Multiple
causation of phylogeographical pattern as revealed by nested clade analysis of the bamboo viper (Trimeresurus stejnegeri) within Taiwan. Mol. Ecol. 10:1967 – 1981.
Creer, S., Chou, W. H., Malhotra, A., Thorpe, R. S. (2002). Offshore insular variation in the diet of the Taiwanese bamboo viper Trimeresurus stejnegeri (Schmidt). Zool. Sci. 19:907 – 913.
Creer, S., Malhotra, A., Thorpe, R. S., Sto¨cklin, R., Favreau, P., Chou, W.-H. (2003). Genetic and ecological correlates of intraspecific variation in pitviper venomcomposition detected using matrix-assisted laser de-sorption time-of-flight mass spectrometry (MALDI-TOF-MS) and isoelectric focusing. J. Mol. Ecol. 56:317 – 329.
Daltry, J. C., Wu¨ster, W., Thorpe, R. S. (1998). Intraspecific variation in the feeding ecology of the crotaline snake Calloselasma rhodostoma in Southeast Asia. J. Herpetol. 32:198 – 205.
Danse, J. M., Gasparini, S., Me´nez, A. (1997). Molecular biology of snake
venom phospholipase A2. In: Kini, R. M., ed. Venom Phospholipase A2
Enzyme: Structure, Function and Mechanism. Chichester, UK: Wiley & Sons, pp. 29 – 71.
Fukagawa, T., Matsumoto, H., Shimohigashi, Y., Ogawa, T., Oda, N., Chang, C. C., Ohno, M. (1992). Sequence determination and characterization
of a phospholipase A2isozyme from Trimeresurus gramineus (green
habu snake) venom. Toxicon 30:1331 – 1341.
Glenn, J. L., Straight, R. C., Wolfe, M. C., Hardy, D. L. (1983). Geographical variation in Crotalus scutulatus scutulatus (Mojave rattlesnake) venom properties. Toxicon 21:119 – 130.
Glenn, J. L., Straight, R. C., Wolt, T. B. (1994). Regional variation in the presence of canebrake toxin in Crotalus horridus venom. Comp. Biochem. Physiol. 107C:337 – 346.
Kini, R. M. (1997). Phospholipase A2, a complex multifunctional protein
puzzle. In: Kini, R. M., ed. Venom Phospholipase A2 Enzymes:
Structure, Function and Mechanism. Chichester, UK: Wiley & Sons, pp. 1 – 28.
Kordis, D., Krizaj, I., Gubensek, F. (2002). Functional diversification of animal toxins by adaptive evolution. In: Me´nez, A., ed. Perspectives in Molecular Toxinology. Chichester, UK: Wiley & Sons, pp. 403 – 419.
Lu, Q. M., Jin, Y., Wei, J. F., Li, D. S., Zhu, S. W., Wang, W. Y., Xiong,
Y. L. (2002). Characterization and cloning of a novel phospholipase A2
from the venom of Trimeresurus jerdonii snake. Toxicon 40:1313 – 1319.
Minton, S. A., Weinstein, S. A. (1986). Geographic and ontogenic variation in venom of the western diamondback rattlesnake (Crotalus atrox). Toxicon 24:71 – 80.
Nakai, M., Nakashima, K. I., Ogawa, T., Shimohigashi, Y., Hattori, S., Chang, C. C., Ohno, M. (1995). Purification and primary structure of a
myotoxic lysine-49 phospholipase A2with low lipolytic activity from
Trimeresurus gramineus venom. Toxicon 33:1469 – 1478.
Oda, N., Nakamura, H., Sakamoto, S., Liu, S. Y., Kihara, H., Chang, C. C.,
Ohno, M. (1991). Amino acid sequence of a phospholipase A2from
the venom of Trimeresurus gramineus (green habu snake). Toxicon 29:157 – 166.
Sasa, M., Barrantes, R. (1998). Allozyme variation in populations of Bothrops asper (Serpentes: Viperidae) in Costa Rica. Herpetologica 54:462 – 469.
Straight, R. C., Glenn, J. L., Wolt, T. B., Wolfe, M. C. (1992). North – south
regional variation in phospholipase A2 activity in the venom of
Crotalus ruber. Comp. Biochem. Physiol. 103B:635 – 639.
Tsai, I. H. (1997). Phospholipases A2of Asian snake venoms. J. Toxicol.,
Toxin Rev. 16:79 – 114.
Tsai, I. H., Wang, Y. M., Au, L. C., Ko, T. P., Chen, Y. H., Chu, Y. F. (2000).
Phospholipases A2 from Callosellasma rhodostoma venom gland:
three-dimensional-modelling and chemical modification of the major isozyme. Eur. J. Biochem. 267:6684 – 6691.
Tsai, I. H., Chen, Y. H., Wang, Y. M., Liau, M. Y., Lu, P. J. (2001). Dif-ferential expression and geographic variation of the venom
phospho-lipases A2 of Calloselasma rhodostoma and Trimeresurus
mucro-squamatus. Arch. Biochem. Biophys. 387:257 – 264.
Tsai, I. H., Wang, Y. M., Chen, Y. H., Tu, A. T. (2003). Geographic var-iations, cloning, and functional analyses of the venom acidic
phos-pholipases A2of Crotalus v. viridis. Arch. Biochem. Biophys. 411:289 –
296.
Zhang, Y., Gao, R., Lee, W. H., Zhu, S. W., Xiong, Y. L., Wang, W. Y. (1998). Characterization of a fibrinogen-clotting enzyme from Tri-meresurus stejnegeri venom and comparative study with other venom proteases. Toxicon 36:131 – 142.
