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Correlation between the surface hydrophobicities and elution orders of Elapid neurotoxins and cardiotoxins on hydrophobic-interaction high-performance liquid chromatography
Toxkon, Vol . 26,14o . 5, pp . 475-483, 1988 . Printed In Great Britain .
0041-0101/88 $3 .00+ .00 ® 1988 Pergamon Preen pk
CORRELATION BETWEEN THE SURFACE HYDROPHOBICITIES AND ELUTION ORDERS OF ELAPID NEUROTOXINS AND CARDIOTOXINS ON HYDROPHOBIC-INTERACTION HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY GERNOT OsTHoFF,I ABRAHAM I. Louwl" and CARows J. REINECK' 'National Chemical Research Laboratory, Council for Scientific and Industrial Research, P . O. Box 395, Pretoria 0001, Republic of South Africa, and =Biochemistry Department, Potchefstroom University for CHE, Potchefstroom 2520, Republic of South Africa (Accepted forpublication
27
October
1987)
G. OsTHoFF, A.
I. Louw and C. J. Retrdc~. Correlation between the surface hydrophobicities and elution orders of Elapid neurotoxins and cardiotoxins on hydrophobic-interaction highperformance liquid chromatography. Toxicon 26, 475-483, 1988 - Hydrophilic and hydrophobic regions were predicted for Elapid neuro- and cardiotoxins . The contribution of these regions to the retention times of neuro- and cardiotoxins on hydrophobic-interaction HPLC was assessed from the known surface accessibilities of amino acid side-chains within these regions. Differences in retention times between neuro- and cardiotoxins on hydrophobic-interaction HPLC could be attributed to differences in hydrophobicity of regions 6-12 and 22-26 between these two types of toxins . Smaller differences in retention times between cardiotoxins were due to the variable hydrophobicities of regions 1-4 and 26-36.
INTRODUCTION MANY OF THE functional
sites of proteins are contained in domains or regions which are surface accessible. These domains can be epitopes for the interaction with antibodies (WILSON et al., 1984), binding sites for hormones, e.g. insulin (SAUNDERS, 1981) or sites that interact with lipid bilayers to fulfil the function of membrane proteins (RATNAM et al., 1986) or membrane-active proteins (DuFouRQ et al., 1978; BOUGIS et al., 1983). It has become important to identify these protein surface regions when designing drugs (BLAKE and Li, 1975; YOKOYAMA et al., 1980) or synthetic vaccines (ARNON, 1986). Because of the accumulating amount of protein primary structure data derived from gene sequencing, parallelled by a scarcity of knowledge of polypeptide conformation, theoretical methods have become important for predicting secondary structure (CHOU and FASMAN, 1974x, 1974b; GARNIER et al., 1978) and surface regions such as antigenic determinants (HOPP and WOODS 1981 ; KYTE and DOOLITTLE, 1982; KARPLUS and SCHULz, 1985). These prediction methods make controversial assumptions and the results frequently are in conflict with experimental evidence. (KABscH and SANDER, 1983; THORNTON et al., 1986). Cardiotoxins and short neurotoxins from cobra venoms are two families of homologous single-chain globular proteins (mol. wt -21 6800). These two toxin families `Current address: Biochemistry Department, University of Pretoria, Pretoria 0002, Republic of South Africa . 475
476
G . OSTHOFF et al.
share a number of common structural features, such as invariant amino acid residues and disulphide bond locations (KARLssoN, 1978). Although the common tertiary structure of short neurotoxins (Low et al., 1976; TSERNOGLOU and PETSKo, 1977) and their biological action (KARLSSON, 1978) are known, less is understood of cardiotoxin conformation and its mode of action. A number of studies have been directed at discovering the surface accessibilities and functional roles of the invariant amino acid residues of neuro- and cardiotoxins . Surface regions in snake venom toxins have been identified by crystallography (Low et al., 1976; TSERNomou and PETsKo, 1977) as well as by nuclear magnetic resonance (n.m .r .) (LAUTERWEIN et al., 1977; STEINMETz et al., 1981), circular dichroism (CD) spectroscopy (VISSER and LOÜw, 1978), photochemically induced nuclear polarization (photo-CIDNP) (MUSzKAT et al., 1984), and proteolysis (DuFouRQ et al., 1978; BoUGIS et al., 1983), and fluorescence studies (VINCENT et al., 1978) of lipid bound cardiotoxins and monoclonal antibodies (MIINEz, 1986) . In addition surface accessibilities of amino acid side chains have been probed by various chemical modifications (CARLSSON and LOUw, 1978, 1987 ; CARLSSON, 1980, 1983, 1987) as well as by photo-CIDNP studies (MuszKAT et al., 1984) . Comparative reports on the hydrophobicities of neurotoxins and cardiotoxins focus largely upon the prevalence of hydrophobic amino acid residues in cardiotoxins in comparison to neurotoxins (LAUTERWEIN and WÜTHRICH, 1978) as well as the high binding affinity of cardiotoxins to lipid membranes (BouGis et al., 1983) . It has been deduced from the latter studies that the peptide segment, which include amino acid residues 6 -12 in the polypeptide chain, interact with the hydrophobic core of model membranes . Information on other hydrophobic surface groups which may contribute to the biological activity of cardiotoxins is, however, not available. Using CD spectroscopy we have recently shown that the conformation of neuro- and cardiotoxins is not affected by the salt -buffer eluting medium used for hydrophobicinteraction HPLC (HIC-HPLC) (OSTHOFF et al., 1987) . The results of this study have suggested that HIC-HPLC could be employed to identify the surface hydrophobic regions in snake venom neuro- and cardiotoxins . In the present study the contribution of predicted hydrophobic regions to the retention times of individual toxins on HIC-HPLC was evaluated from published data on the surface accessibility of certain amino acid residues within these regions . MATERIALS AND METHODS The following toxins were used for the investigation : Nqja nivea cardiotoxins V°1 and VH2 (Hams and VILJOEN, 1976) and neurotoxin P (Hmmes, 1971) ; Ngja hgje annulifera cardiotoxin V °1(WEt9E et at., 1973) and neurotoxin CM-14 VouwiRT, 1975); NOM mehmokuaa cardiotoxin V°I (CARusoN and JouEEaT, 1974) and neurotoxin d (Hum, 1972) ; Naja ngja Aaouthia cardiotoxin CM-7+7A (JouaERT and TAuAARD, 1980) ; Nqja mossambka mosmrnbiea cardiotoxins V°l, V°2 (Louw 1974a) and V°4 (Louw, 1974b). The HIC-HPLC was carried out on a TSK Phenyl 5-PW column (BioRad Laboratories) with a linear gradient of (NHJSO4 as described previously (OsTHoFF et W., 1987) . As before Nqja mosiambica mossambiea c ardiotoxin V°I was used as a reference standard in all chromatographic runs, to which the retention times of individual toxins were compared. Hydrophilicity/hydrophobicity profiles of short neurotoxins and cardiotoxins were predicted by the method of HGPP and WooDs (1991) . Each amino acid residue in a sequence was assigned the published hydrophilicity index value, and these were repetitively averaged down the length of the polypeptide chain . An average of six amino acid residues was used for all toxins . Other averages were also investigated, but averages below six gave a scattered plot, while averages higher than six resulted in broad profiles with obscured detail . The surface access bilities of predicted hydrophilic and hydrophobic regions were deduced from the availability of amino acid side chains within these regions to chemical modification (CARSON and Louw, 1978, 1987; CARIssoN, 1980, 1983, 1987) and monoclonal antibodies (Mtno3z, 1986) as well as crystallographic (Low et at., 1976; TsEaNoGLou and Pumo, 1977) and photo-CIDNP studies (Mus=AT et al ., 1984) .
Surface Accessible Regions in Elapid Toxins
477
SEQUENCE POSITION FiO . 1 . HYDROPHILICITY PROFILES OF CARDIOTOXINS AND NEUROTOXINS .
Naja nivea ß (short neurotoxin); (--) Nqja hgje annalifera CM 14 (short neurotoxin) (not drawn where coincident with ( )) ; (-) Nqja nivea V"1 (cardiotoidn); (0) accessible amino acid resid in short neurotoxins; (~) inaccessible amino acid residues in short neurotoxins ; accessible amino acid residues in short cardiotoxins . (
)
(UM)
RESULTS and DISCUSSION
The hydrophilicity/hydrophobicity profiles of the short neurotoxins and cardiotoxins were segregated into three groups for clarity and are plotted in Figs. 1 and 2. Both kinds of toxins show a similar distribution with respect to their predicted hydrophilic and hydrophobic regions at residues 40 - 60; the neurotoxins have hydrophilic regions at amino acid positions 43 - 48 and 54 - 60, and hydrophobic regions at positions 41- 42 and 49 - 53 (Fig. 1). The cardiotoxins have hydrophilic regions at positions 42 - 46 and 56 - 60 and a hydrophobic region at positions 47 - 55 (Figs. 1 and 2). The surface accessibilities of these regions were indicated by their interaction with monoclonal antibodies and by the susceptibilities of amino acid side chains within these regions to chemical modification . Monoclonal antibodies raised against various short neurotoxins interact with sites including residues 46, 48, 53, 57 and 62 (MgNEZ, 1986) (Fig. 3). In addition, crystallographic data for erabutoxins a and b (Low et al., 1976; TSERNOGLOu and PETSKO, 1977) show residue 47 to be accessible while He 52 and Asn 61 are inaccessible . For cardiotoxins it has been shown that residues 45, 47, 49, 51, 52, 57 and 58. are accessible to monoclonal antibodies (MP-NEz, 1986) (Fig . 3). Further support for the accessibility of Tyr 51 in cardiotoxins has come from chemical modification studies (CARLSSON, 1980) and photo-CIDNP (MuszKAT et al., 1984). Since the hydrophilic and hydrophobic properties and surface accessibility of region 40 - 60 appear to be similar for
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FIO . 2 . HYDROPHILICITY PROFILES OF SNAKE VENOM CARDIOTOXINS .
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TABLE 1 . RETENTION TIMES OF SNAKE VENOM TOXINS BY
HIC-HPLC
Toxins Retention times (min)* Neurotoxins N. melanoleuca dt 2.87 N. h. annulifera CM-14t 2.95 N. nivea ßt 2.82 Cardiotoxins N. m. mosswnbica V"4 18 .02 t 0.06 N. n. kaouthia CM-7 + 7A 21 .72 t 0.23 N. melanoleuca V"1* 23 .39 t 0.06 N. m. massambica V"2* 23 .54 :± 0.15 N. nivea V"1* 23 .59 :j- 0.13 N. h. annuljjera V"1* 23 .65 t 0.30 N. m. mossambica V"1 26 .33 * 0.36 N. nivea V"1 28 .25 t 0.08 *Average of three normalized retention times with mean deviation for three runs (see Materials and Methods). 'Coinjection of neurotoxins gave a retention time of 2.88 t 0.06 min for all three toxins . *Coinjection of these cardiotoxins gave a retention time of 23 .61 t 0.19 min for all four toxins .
TABLE
2.
CORRELATION OF SURFACE HYDitopHOBICITms AND RETENTION TIMES OF CARDIOTOXINS
Cardiotoxin N. m. mossambioa V"4 N. n. kaouthia CM7 +7A N. melanoleuca V"1 N. m. mossambica V"2 N. nivea V"1 N. h. annul(jera V"1 N. m. moz ambica V"l N. nivea V"2 0, Hydrophilic . 1, Hydrophobic .
1-4 0 0 0 1 0 0 1 1
Hydrophobicity in region : 6-12 22-26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
26-36 0 1 1 0 1 1 1 1
Retention time (min) 18 .02 21 .72 23 .39 23 .54 23 .59 23 .65 26 .33 28 .25
t 0.06 t 0.23 t 0.06 t 0.15 t 0.13 t 0.30 t 0.36 t 0.08
neuro- and cardiotoxins, HIC-HPLC was not expected to be able to discriminate between the surface hydrophobicities of this domain in the individual toxins . Residue positions 1-40 of neuro- and cardiotoxins, in contrast, differ with respect to the distribution of their hydrophilic and hydrophobic regions (Figs. 1 and 2). The neurotoxins are predicted to have hydrophilic regions at amino acid positions 8 -13, 18 - 20, 22 - 32 and 34 - 40, and a hydrophobic region at positions 1- 7. The cardiotoxins on the other hand, are predicted to have hydrophilic regions at positions 13 - 18 and 36-38. Variable hydrophilic regions in cardiotoxins coincide to some degree with the corresponding regions on neurotoxins at residue positions 1- 4 and 26 - 36. However, all the cardiotoxins are predicted to have hydrophobic regions at positions 6-12 and 22 - 26 (Figs. 1 and 2). Region 6-12 has been shown to interact with membrane lipids (VINCENT et al., 1978; BOUGIS et al., 1983) and is therefore accessible at the surface. The corresponding region in neurotoxins which is predicted to be hydrophilic (Fig. 1), does not interact with membrane lipids (VINCENT et al., 1978). Surface accessible amino acid residues within region 1- 40 have been identified by the binding of monoclonal antibodies
Surface Accessible Regions in Elapid Toxins
481
to residues 7, 12, 15, 16, 18, 21, 23, 30, 32, 33 and 36 in neurotoxins (MgNEZ, 1986). It is also evident from crystallographic data that Ser 9, . Lys 27, Trp 29, Asp 31, Arg 33 and Gly 34 are accessible while Tyr 25 is buried (Low et al., 1976 ; TSERNogr oU and PETSKO, 1977). Furthermore, photo-CIDNP of various neurotoxins has showabt#at Trp 29 and His 32 are also accessible, while His 6, Tyr 25 and His 26 are not (MUstkAT et al., 1984). Monoclonal antibodies showed that residues 1, 11, 12, 23, 25 and 27-31 are accessible in cardiotoxins (MÉNEz, 1986). Photo-CIDNP has also shown that Tyr 11, 22, and 25 are accessible (MUSZKAT et al., 1984). Chemical modification experiments have provided supplementary evidence that Tyr 22 and 25 are accessible (CARLSSON, 1980), as well as Met 24 and 26 (CARLSSON and LOUw, 1978 ; CARLSSON, 1987) and Arg 36 (CARLSSON, 1983). It is thus apparent from the above and Figs . 1 and 2 that residue positions 6-12 and 22-26 in cardiotoxins are largely hydrophobic in character and surface accessible . The corresponding areas in neurotoxins are also surface accessible but, in contrast, mainly hydrophilic. It is thus expected that this difference in surface hydrophobicity between neurotoxins and cardiotoxins will be reflected by their respective retention times on HICHPLC . It is evident from the results in Table 1 that the retention times of neurotoxins and cardiotoxins differ from each other by as much as 15 - 25 min. These results not only corroborate our own conclusions (Figs. 1 and 2) but also the general assumption that cardiotoxins have a higher hydrophobicity than neurotoxins (LAUTERWEIN and WOTHRICH, 1978) . Whereas the individual retention times of the neurotoxins are exceptionally close to each other, the retention times of individual cardiotoxins differ by a maximum of 10 min from one another. From a correlation between the surface accessibilities of hydrophobic regions in cardiotoxins and their retention times on HIC-HPLC it is indicative that the differences in their retention times are due to the variable contribution of regions 1- 4 and 26 - 36 to their total surface hydrophobicity (Table 2) . The increase in retention time also correlates with a transition from hydrophobicity in either of regions 1- 4 or 26 - 36 to hydrophobicity in both these regions . Combining the data on predicted hydrophilicity/hydrophobicity profiles with the HICHPLC retention times, we could deduce that regions 1- 4, 6 -12 and 22 - 36 are surface accessible in the cardiotoxins and short neurotoxins. Although crystallographic data for short neurotoxins have indicated that Tyr 25 is buried (Low et al., 1976 ; TSERNOGLOU and PETSKO, 1977), the remainder of the amino acid residues within region 22 - 32 have been shown by other physico-chemical methods to be surface accessible (Low et al., 1976 ; TSERNOGLOU and PETSKO, 1977 ; MISNEZ, 1986). A correspondence in the hydrophobicity and surface accessibility of region 22-26 among cardiotoxins is suggestive of an interaction between this region and biological membranes. The importance of this region was indicated by the complete loss of biological activity obtained upon chemical modification of Met 24 and Met 26 in cardiotoxins (CARLSSON and Louw, 1978). It appears that this work can be usefully extended to other homologous series of proteins to identify surface accessible regions, provided that HIC-HPLC conditions do not lead to protein denaturation . Multi-subunit proteins are reported to undergo such effects on TSK-5-PW columns (INGRAHAM et al., 1985) and are therefore unsuitable subjects for this type of study. Acknowledgements - The authors are grateful to Drs F.H .H . CARLSSON and R. C. CLARK for comments on the draft manuscript, and Professor L. Vmatt for introducing us to predictive methods in protein chemistry.
482
G. OSTHOFF et cal. REFERENCES
ARNoN, R. (1986) Synthetic peptides as the basis for future vaccines. Trends biochem. Sci. 11, 521 . Bt.Aim, J. and L i, C. H. (1975) The synthesis and biological activity of [165,182,189-5carbamidomethylcyrteinel-human growth hormone-(140-191). Int. J. Prpt. and Prot. Res. 7, 495. Bores, D. P. (1971) The aminoacid sequence of toxins a andßfrom Ngja nhwa venom and thedisulphide bonds of toxin aJ. biol. Cheat. 246, 7383. Bores, D. P. (1972) The amino add sequence of toxins b and d from Ngia melanoleuca venom. J. Not. Chem . 247,2866 . Bores, D. P. and VtuoHN, C. C. (1976) The amino acid sequence of three non-curarimimdic toxins from Nqja ntven venom. Biochbn. blophys. Acts 446, 1. Bouois, P., TmstEa, M., VON RtersctioTEN, J., RocHAT, H., FAucoN J. F. and DupouRQ, D. (1983) Are interactions with phospholipids responsible for pharmacological activities of cardiotoxins? Mol. cell. Biochem. 55, 49 . CARLssoN, F. H. H. (1M) The preparation of 3-nitrotyrosyl derivatives of three Elapid venom cardiotoxins . Btochim. blophys. Acts 6U, 460. CARmsoN, F. H. H. (1983) The modification of arginine-36 in Elapid venom ardiotoxin using 1,2cyclohexanedione . Biochhn. biophys. Acts 746, 18 . CARtssON, F. H. H. (1987) The selective S-alkylation of a methionine residue in an Elapid venom ardiotoxin. Ins. J. Blochem.19, 915. CARtssoN, F. H. H. and JOUDERT, F. J. (1974) The isolation and purification of three cytotoxin homologues from the venom of the forest cobra (Naja melanoleuca) and the complete amino add sequence of toxin V°l. Biochbn. biophys. Acts 336, 453. CARLssoN, F. H. H. and Louw, A. 1. (1978) The oxidation of methionine and its effect on the properties of cardiotoxin V % from Naja melartolaura venom. Biochim. biop". Acts 534, 322. CARISSON, F. H. H. and Louw, A. l. (1987) Properties of some 3-nitrotyrosyl Elapid venom ardiotoxins . Int. J. Biochem. 19, 9. CHou, P. Y. and FAsmAN, G. D. (1974x) Conformational parameters for amino adds in helical, ß-sheet and random coil regions calculated from proteins. Biochemistry 74, 211. CHou, P. Y. and FAsum, G. D. (1974b) Prediction of protein conformation. Biochemistry 74, 222. Dut+ouRQ, J. and FAUCON, J. F. (1978) Specific binding of a ardiotoxin from Naja maxsambica momambka to charged phospholipids detected by intrinsic fluoroscence . Biochemitry 17, 1170. DuFroN, M. J. and Hit=, R. C. (1983) Conformational properties of the neurotoxins and cardiotoxins isolated from Elapid snake venoms. CRC Grit. Rev. Biochem. 14, 113. GARNmR, J., OsmuTHo"E, D. J. and RonsoN, B. (1978) Analysis of the accuracy and implication of simple methods for predicting the secondary structure of globular proteins . J. motet. Biol. 12", 97. Hopp, T. P. and WOODS, K . R. (1981) Prediction of protein antigenic determinants from amino add sequences. Proc. natn. Aced. Sci. U.S.A . 78, 3824. INORAHAm. R. H., LAu, S. Y. M., TANEJA, A. K . and HODoEs, R. S. (1985) Denaturation and the effects of temperature on hydrophobic-interaction and reversed-phase high-performance liquid chromatography of proteins. J. Chromat. 327, 77 . JouiuatT, F. J. (1975) The amino add sequences of three toxins (CM-10, CM-12 and CM-14) from Nqja haje annultfera (Egyptian cobra) venom. Hoppe-Seyhers Z. physiol. Chem . 356, 53 . JounEaT, F. J. and TAuAARD, N. (1980) The complete primary structure of three cytotoxins (CM-6, CM-7 and CM-7A) from Naja raja kaouthia (Siamese cobra) snake venom. Toxicon 18, 455. KAEscH, W. and SANDER, C. (1983) How good are predictions of protein secondary structure? FEBS Lett . 155, 179. KARL soN, E. (1978) Chemistry of vrotein toxins in snake venoms . In : Handbook of Experimental Pharmacology, Vol. 52, p. 159 (LEE, C. Y. Ed.) Berlin: Springer . KARPLUs, P. A. and ScHut.z, G. E. (1985) Prediction of chain flexibility in proteins . Naturwissenschaften 72, 212. KYTF, J. and DOOLrrrLE, R. F. (1982) A simple method of displaying the hydropathic character of a protein. J. moles. Biol. 157, 105 . LAUTERwmN, J. and WOTHwcH, K. (1978) A possible structural basis for the different modes of action of neurotoxins and cardiotoxins from snake venoms . FEES Lett. 93, 181 . LAUTERWEIN, J ., WOTtmcH, K., ScHwEtrz, H., VINCENT, J. P. and LAzDUNsxi, M. (1977) Studies of a neurotoxin and a cardiotoxin from Nqja massambka massombka: amide proton resonance. Biochem. biophys. Res. Commun . 76, 1071 . Louw, A. I. (1974x) The purification and properties of five non-neurotoxic polypeptides from Naja mossanbka momambka venom. Biochim. blophys. Aria 336, 470. Louw, A. 1. (1974b) The complete amino add sequence of cytotoxin V"4 from the venom of Naja mauambica massambla. Bioehem. Biophys. Res. Commun . 58, 1022 .
Surfaa Accessible Regions in Elapid Toxins
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Low, B., PREnoN, H. S., SATO, A., ROSEN, L. S., $EARL, J. E., RuDKO, A. D. and RICHARDSON, J. S. (1976) Three dimensional structure of erabutoxin b neurotoxic protein: inhibitor of acetylcholine receptor. Proc. nata . Acad. Sci. U.S.A . 73, 2991 . MENEZ, A. (1986) Molecular immunology of snake toxins . Pharmac. Ther. 30, 91 . MuszKAT, K. A., KArr, I., HAYAm, K. and TAwYA, N. (1984) Photochemically induced nuclear polarization study of exposed tyrosines, tryptophans and histidines in pottsynaptic neurotoxins and in membranotoxins of Elapid and Hydrophid snake venoms . Biochemistry 23, 4913 . OSTHOFF, G., Louw, A. I. and VISSER, L. (1987) Reversed-phase and hydrophobic interaction highperformance liquid chromatography of Elapid cardiotoxins . Analyt. Blochem . 164, 315. RATNAM,. M., LE-NGuYEN, D., RIVIER, J., SARGENr, P. B. and UNDmom, J. (1986) Transmembrane topography of nicotinic acetylcholine receptor . Immunochemical tests contradict theoretical predictions based on hydrophobicity profiles . Biochemistry 25, 2633 . SAuNDERs, D. J. (1981) Structure-function relationship in insulin . Blosci. Rep. 1, 485. STEINmETz, W. E., MooNEN, C., KumAR, A., LAwuNsV1, M., VISOR, L., CARLSSON, F. H. H. and WOTMCH, K. (1981) 'H Nuclear-magnetic-resonance studies of the conformation of cardiotoxin V`2 from Nqja mossambica mossambica. Eur. 1. Biochem . 120, 467. THORNTON, J. M., EDwARDB, M. S., TAYLOR, W. R. and BARLow, D. J. (1986) Location of continuous antigenic determinants in the protruding regions of proteins . EUBO 1. 5, 409. TsERNoGLou, D. and Pmwo, G. A. (1977) Three-dimensional structure of neurotoxin a from venom of the Philippines sea snake. Proc. natn . Acad. Sci. U.S.A . 74, 971 . VINCENT, J. P., BALERNA, M. and LAZOuNsia, M. (1978) Properties of association of cardiotoxin with lipid vesicles and natural membranes. FEBS Lett . 85, 103. VLssER, L. and Louw, A. 1. (1978) The conformation of cardiotoxins and neurotoxins from snake venoms . Blochim. biophys. Acts 533, 80. WELSE, K. H. K., CARLSSON, F. H. H., JouBERT, F. J. and STRYDGM, D. J. (1973) The purification of toxins V"1 and V°2, two cytotoxin homologues from banded Egyptian cobra (Ngja hgje annuljjena) venom, and the complete amino acid sequence of toxin V°l . Hoppe-Seyler's Z. pkysiol. Chem . 354, 1317 . WILSON, 1. A., NimAN, H. L., HOUGHTEN, R. A., CHERENsoN, A. R., CONNOLLY, M. L. and LERNER, R. A. (1984) The structure of an antigenic determinant in a protein. Cell 37, 767. YoKOHAMA, S., TuxusmmA, D., KuPFERBERG, J. P., KEzDY, F. J. and KAISER, E. T. (1980) The mechanism of activation of lecithin : cholesterol acyltransferase by apolipoprotein A-1 and an amphiphilic peptide. 1. biol. Chem . 255, 7333 .
0041-0101/88 $3 .00+ .00 ® 1988 Pergamon Preen pk
CORRELATION BETWEEN THE SURFACE HYDROPHOBICITIES AND ELUTION ORDERS OF ELAPID NEUROTOXINS AND CARDIOTOXINS ON HYDROPHOBIC-INTERACTION HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY GERNOT OsTHoFF,I ABRAHAM I. Louwl" and CARows J. REINECK' 'National Chemical Research Laboratory, Council for Scientific and Industrial Research, P . O. Box 395, Pretoria 0001, Republic of South Africa, and =Biochemistry Department, Potchefstroom University for CHE, Potchefstroom 2520, Republic of South Africa (Accepted forpublication
27
October
1987)
G. OsTHoFF, A.
I. Louw and C. J. Retrdc~. Correlation between the surface hydrophobicities and elution orders of Elapid neurotoxins and cardiotoxins on hydrophobic-interaction highperformance liquid chromatography. Toxicon 26, 475-483, 1988 - Hydrophilic and hydrophobic regions were predicted for Elapid neuro- and cardiotoxins . The contribution of these regions to the retention times of neuro- and cardiotoxins on hydrophobic-interaction HPLC was assessed from the known surface accessibilities of amino acid side-chains within these regions. Differences in retention times between neuro- and cardiotoxins on hydrophobic-interaction HPLC could be attributed to differences in hydrophobicity of regions 6-12 and 22-26 between these two types of toxins . Smaller differences in retention times between cardiotoxins were due to the variable hydrophobicities of regions 1-4 and 26-36.
INTRODUCTION MANY OF THE functional
sites of proteins are contained in domains or regions which are surface accessible. These domains can be epitopes for the interaction with antibodies (WILSON et al., 1984), binding sites for hormones, e.g. insulin (SAUNDERS, 1981) or sites that interact with lipid bilayers to fulfil the function of membrane proteins (RATNAM et al., 1986) or membrane-active proteins (DuFouRQ et al., 1978; BOUGIS et al., 1983). It has become important to identify these protein surface regions when designing drugs (BLAKE and Li, 1975; YOKOYAMA et al., 1980) or synthetic vaccines (ARNON, 1986). Because of the accumulating amount of protein primary structure data derived from gene sequencing, parallelled by a scarcity of knowledge of polypeptide conformation, theoretical methods have become important for predicting secondary structure (CHOU and FASMAN, 1974x, 1974b; GARNIER et al., 1978) and surface regions such as antigenic determinants (HOPP and WOODS 1981 ; KYTE and DOOLITTLE, 1982; KARPLUS and SCHULz, 1985). These prediction methods make controversial assumptions and the results frequently are in conflict with experimental evidence. (KABscH and SANDER, 1983; THORNTON et al., 1986). Cardiotoxins and short neurotoxins from cobra venoms are two families of homologous single-chain globular proteins (mol. wt -21 6800). These two toxin families `Current address: Biochemistry Department, University of Pretoria, Pretoria 0002, Republic of South Africa . 475
476
G . OSTHOFF et al.
share a number of common structural features, such as invariant amino acid residues and disulphide bond locations (KARLssoN, 1978). Although the common tertiary structure of short neurotoxins (Low et al., 1976; TSERNOGLOU and PETSKo, 1977) and their biological action (KARLSSON, 1978) are known, less is understood of cardiotoxin conformation and its mode of action. A number of studies have been directed at discovering the surface accessibilities and functional roles of the invariant amino acid residues of neuro- and cardiotoxins . Surface regions in snake venom toxins have been identified by crystallography (Low et al., 1976; TSERNomou and PETsKo, 1977) as well as by nuclear magnetic resonance (n.m .r .) (LAUTERWEIN et al., 1977; STEINMETz et al., 1981), circular dichroism (CD) spectroscopy (VISSER and LOÜw, 1978), photochemically induced nuclear polarization (photo-CIDNP) (MUSzKAT et al., 1984), and proteolysis (DuFouRQ et al., 1978; BoUGIS et al., 1983), and fluorescence studies (VINCENT et al., 1978) of lipid bound cardiotoxins and monoclonal antibodies (MIINEz, 1986) . In addition surface accessibilities of amino acid side chains have been probed by various chemical modifications (CARLSSON and LOUw, 1978, 1987 ; CARLSSON, 1980, 1983, 1987) as well as by photo-CIDNP studies (MuszKAT et al., 1984) . Comparative reports on the hydrophobicities of neurotoxins and cardiotoxins focus largely upon the prevalence of hydrophobic amino acid residues in cardiotoxins in comparison to neurotoxins (LAUTERWEIN and WÜTHRICH, 1978) as well as the high binding affinity of cardiotoxins to lipid membranes (BouGis et al., 1983) . It has been deduced from the latter studies that the peptide segment, which include amino acid residues 6 -12 in the polypeptide chain, interact with the hydrophobic core of model membranes . Information on other hydrophobic surface groups which may contribute to the biological activity of cardiotoxins is, however, not available. Using CD spectroscopy we have recently shown that the conformation of neuro- and cardiotoxins is not affected by the salt -buffer eluting medium used for hydrophobicinteraction HPLC (HIC-HPLC) (OSTHOFF et al., 1987) . The results of this study have suggested that HIC-HPLC could be employed to identify the surface hydrophobic regions in snake venom neuro- and cardiotoxins . In the present study the contribution of predicted hydrophobic regions to the retention times of individual toxins on HIC-HPLC was evaluated from published data on the surface accessibility of certain amino acid residues within these regions . MATERIALS AND METHODS The following toxins were used for the investigation : Nqja nivea cardiotoxins V°1 and VH2 (Hams and VILJOEN, 1976) and neurotoxin P (Hmmes, 1971) ; Ngja hgje annulifera cardiotoxin V °1(WEt9E et at., 1973) and neurotoxin CM-14 VouwiRT, 1975); NOM mehmokuaa cardiotoxin V°I (CARusoN and JouEEaT, 1974) and neurotoxin d (Hum, 1972) ; Naja ngja Aaouthia cardiotoxin CM-7+7A (JouaERT and TAuAARD, 1980) ; Nqja mossambka mosmrnbiea cardiotoxins V°l, V°2 (Louw 1974a) and V°4 (Louw, 1974b). The HIC-HPLC was carried out on a TSK Phenyl 5-PW column (BioRad Laboratories) with a linear gradient of (NHJSO4 as described previously (OsTHoFF et W., 1987) . As before Nqja mosiambica mossambiea c ardiotoxin V°I was used as a reference standard in all chromatographic runs, to which the retention times of individual toxins were compared. Hydrophilicity/hydrophobicity profiles of short neurotoxins and cardiotoxins were predicted by the method of HGPP and WooDs (1991) . Each amino acid residue in a sequence was assigned the published hydrophilicity index value, and these were repetitively averaged down the length of the polypeptide chain . An average of six amino acid residues was used for all toxins . Other averages were also investigated, but averages below six gave a scattered plot, while averages higher than six resulted in broad profiles with obscured detail . The surface access bilities of predicted hydrophilic and hydrophobic regions were deduced from the availability of amino acid side chains within these regions to chemical modification (CARSON and Louw, 1978, 1987; CARIssoN, 1980, 1983, 1987) and monoclonal antibodies (Mtno3z, 1986) as well as crystallographic (Low et at., 1976; TsEaNoGLou and Pumo, 1977) and photo-CIDNP studies (Mus=AT et al ., 1984) .
Surface Accessible Regions in Elapid Toxins
477
SEQUENCE POSITION FiO . 1 . HYDROPHILICITY PROFILES OF CARDIOTOXINS AND NEUROTOXINS .
Naja nivea ß (short neurotoxin); (--) Nqja hgje annalifera CM 14 (short neurotoxin) (not drawn where coincident with ( )) ; (-) Nqja nivea V"1 (cardiotoidn); (0) accessible amino acid resid in short neurotoxins; (~) inaccessible amino acid residues in short neurotoxins ; accessible amino acid residues in short cardiotoxins . (
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RESULTS and DISCUSSION
The hydrophilicity/hydrophobicity profiles of the short neurotoxins and cardiotoxins were segregated into three groups for clarity and are plotted in Figs. 1 and 2. Both kinds of toxins show a similar distribution with respect to their predicted hydrophilic and hydrophobic regions at residues 40 - 60; the neurotoxins have hydrophilic regions at amino acid positions 43 - 48 and 54 - 60, and hydrophobic regions at positions 41- 42 and 49 - 53 (Fig. 1). The cardiotoxins have hydrophilic regions at positions 42 - 46 and 56 - 60 and a hydrophobic region at positions 47 - 55 (Figs. 1 and 2). The surface accessibilities of these regions were indicated by their interaction with monoclonal antibodies and by the susceptibilities of amino acid side chains within these regions to chemical modification . Monoclonal antibodies raised against various short neurotoxins interact with sites including residues 46, 48, 53, 57 and 62 (MgNEZ, 1986) (Fig. 3). In addition, crystallographic data for erabutoxins a and b (Low et al., 1976; TSERNOGLOu and PETSKO, 1977) show residue 47 to be accessible while He 52 and Asn 61 are inaccessible . For cardiotoxins it has been shown that residues 45, 47, 49, 51, 52, 57 and 58. are accessible to monoclonal antibodies (MP-NEz, 1986) (Fig . 3). Further support for the accessibility of Tyr 51 in cardiotoxins has come from chemical modification studies (CARLSSON, 1980) and photo-CIDNP (MuszKAT et al., 1984). Since the hydrophilic and hydrophobic properties and surface accessibility of region 40 - 60 appear to be similar for
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TABLE 1 . RETENTION TIMES OF SNAKE VENOM TOXINS BY
HIC-HPLC
Toxins Retention times (min)* Neurotoxins N. melanoleuca dt 2.87 N. h. annulifera CM-14t 2.95 N. nivea ßt 2.82 Cardiotoxins N. m. mosswnbica V"4 18 .02 t 0.06 N. n. kaouthia CM-7 + 7A 21 .72 t 0.23 N. melanoleuca V"1* 23 .39 t 0.06 N. m. massambica V"2* 23 .54 :± 0.15 N. nivea V"1* 23 .59 :j- 0.13 N. h. annuljjera V"1* 23 .65 t 0.30 N. m. mossambica V"1 26 .33 * 0.36 N. nivea V"1 28 .25 t 0.08 *Average of three normalized retention times with mean deviation for three runs (see Materials and Methods). 'Coinjection of neurotoxins gave a retention time of 2.88 t 0.06 min for all three toxins . *Coinjection of these cardiotoxins gave a retention time of 23 .61 t 0.19 min for all four toxins .
TABLE
2.
CORRELATION OF SURFACE HYDitopHOBICITms AND RETENTION TIMES OF CARDIOTOXINS
Cardiotoxin N. m. mossambioa V"4 N. n. kaouthia CM7 +7A N. melanoleuca V"1 N. m. mossambica V"2 N. nivea V"1 N. h. annul(jera V"1 N. m. moz ambica V"l N. nivea V"2 0, Hydrophilic . 1, Hydrophobic .
1-4 0 0 0 1 0 0 1 1
Hydrophobicity in region : 6-12 22-26 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
26-36 0 1 1 0 1 1 1 1
Retention time (min) 18 .02 21 .72 23 .39 23 .54 23 .59 23 .65 26 .33 28 .25
t 0.06 t 0.23 t 0.06 t 0.15 t 0.13 t 0.30 t 0.36 t 0.08
neuro- and cardiotoxins, HIC-HPLC was not expected to be able to discriminate between the surface hydrophobicities of this domain in the individual toxins . Residue positions 1-40 of neuro- and cardiotoxins, in contrast, differ with respect to the distribution of their hydrophilic and hydrophobic regions (Figs. 1 and 2). The neurotoxins are predicted to have hydrophilic regions at amino acid positions 8 -13, 18 - 20, 22 - 32 and 34 - 40, and a hydrophobic region at positions 1- 7. The cardiotoxins on the other hand, are predicted to have hydrophilic regions at positions 13 - 18 and 36-38. Variable hydrophilic regions in cardiotoxins coincide to some degree with the corresponding regions on neurotoxins at residue positions 1- 4 and 26 - 36. However, all the cardiotoxins are predicted to have hydrophobic regions at positions 6-12 and 22 - 26 (Figs. 1 and 2). Region 6-12 has been shown to interact with membrane lipids (VINCENT et al., 1978; BOUGIS et al., 1983) and is therefore accessible at the surface. The corresponding region in neurotoxins which is predicted to be hydrophilic (Fig. 1), does not interact with membrane lipids (VINCENT et al., 1978). Surface accessible amino acid residues within region 1- 40 have been identified by the binding of monoclonal antibodies
Surface Accessible Regions in Elapid Toxins
481
to residues 7, 12, 15, 16, 18, 21, 23, 30, 32, 33 and 36 in neurotoxins (MgNEZ, 1986). It is also evident from crystallographic data that Ser 9, . Lys 27, Trp 29, Asp 31, Arg 33 and Gly 34 are accessible while Tyr 25 is buried (Low et al., 1976 ; TSERNogr oU and PETSKO, 1977). Furthermore, photo-CIDNP of various neurotoxins has showabt#at Trp 29 and His 32 are also accessible, while His 6, Tyr 25 and His 26 are not (MUstkAT et al., 1984). Monoclonal antibodies showed that residues 1, 11, 12, 23, 25 and 27-31 are accessible in cardiotoxins (MÉNEz, 1986). Photo-CIDNP has also shown that Tyr 11, 22, and 25 are accessible (MUSZKAT et al., 1984). Chemical modification experiments have provided supplementary evidence that Tyr 22 and 25 are accessible (CARLSSON, 1980), as well as Met 24 and 26 (CARLSSON and LOUw, 1978 ; CARLSSON, 1987) and Arg 36 (CARLSSON, 1983). It is thus apparent from the above and Figs . 1 and 2 that residue positions 6-12 and 22-26 in cardiotoxins are largely hydrophobic in character and surface accessible . The corresponding areas in neurotoxins are also surface accessible but, in contrast, mainly hydrophilic. It is thus expected that this difference in surface hydrophobicity between neurotoxins and cardiotoxins will be reflected by their respective retention times on HICHPLC . It is evident from the results in Table 1 that the retention times of neurotoxins and cardiotoxins differ from each other by as much as 15 - 25 min. These results not only corroborate our own conclusions (Figs. 1 and 2) but also the general assumption that cardiotoxins have a higher hydrophobicity than neurotoxins (LAUTERWEIN and WOTHRICH, 1978) . Whereas the individual retention times of the neurotoxins are exceptionally close to each other, the retention times of individual cardiotoxins differ by a maximum of 10 min from one another. From a correlation between the surface accessibilities of hydrophobic regions in cardiotoxins and their retention times on HIC-HPLC it is indicative that the differences in their retention times are due to the variable contribution of regions 1- 4 and 26 - 36 to their total surface hydrophobicity (Table 2) . The increase in retention time also correlates with a transition from hydrophobicity in either of regions 1- 4 or 26 - 36 to hydrophobicity in both these regions . Combining the data on predicted hydrophilicity/hydrophobicity profiles with the HICHPLC retention times, we could deduce that regions 1- 4, 6 -12 and 22 - 36 are surface accessible in the cardiotoxins and short neurotoxins. Although crystallographic data for short neurotoxins have indicated that Tyr 25 is buried (Low et al., 1976 ; TSERNOGLOU and PETSKO, 1977), the remainder of the amino acid residues within region 22 - 32 have been shown by other physico-chemical methods to be surface accessible (Low et al., 1976 ; TSERNOGLOU and PETSKO, 1977 ; MISNEZ, 1986). A correspondence in the hydrophobicity and surface accessibility of region 22-26 among cardiotoxins is suggestive of an interaction between this region and biological membranes. The importance of this region was indicated by the complete loss of biological activity obtained upon chemical modification of Met 24 and Met 26 in cardiotoxins (CARLSSON and Louw, 1978). It appears that this work can be usefully extended to other homologous series of proteins to identify surface accessible regions, provided that HIC-HPLC conditions do not lead to protein denaturation . Multi-subunit proteins are reported to undergo such effects on TSK-5-PW columns (INGRAHAM et al., 1985) and are therefore unsuitable subjects for this type of study. Acknowledgements - The authors are grateful to Drs F.H .H . CARLSSON and R. C. CLARK for comments on the draft manuscript, and Professor L. Vmatt for introducing us to predictive methods in protein chemistry.
482
G. OSTHOFF et cal. REFERENCES
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