J. Van Rietschoten

Structure-function relationships for cardiotoxins interacting with phospholipids

Four cardiotoxins (CTX I-IV) from Naja mossambica mossambica were compared for their ability to interact with phospholipid vesicles and their capacity to bind erythrocytes. It is concluded that the affinity of the toxins always increases in the order: I approximately equal to II less than III less than IV. The binding is specific for charged lipids even in lipid mixtures. Proteolytic attack of the free and lipid-bound cardiotoxin indicates that at least the first loop Leu1-Thr13 is at the lipid contact. Tryptic and synthetic peptides constitutive of this loop are shown to interact with lipids. Arg5 residue increases the affinity toward the bilayer. The Raman spectra of lipid-bound cardiotoxin indicate a secondary and tertiary structure mainly similar to that of the free toxin. On charged lipids cardiotoxins induce a decrease of the enthalpy and an increase of disorder without change in the transition temperature; at saturating amounts of toxin the transition is abolished. In binary mixtures of phosphatidylcholine and charged lipids the observed effects can be accounted by a phase separation induced by the toxin.

Convergent solid phase peptide synthesis. II. Synthesis of the 1–6 apamin protected segment on a NBB-resin. Synthesis of apamin

Abstract The article deals with the use of the NBB-resin for synthesis of protected segments followed by solid phase segment condenstaion. Solid phse synthesis on a NBB-resin of the segment 1–6 of apamin yielded either the (1–6) apamin-OH segment after photolysis or (1–6) apamin-NH-NH 2 after hydrazinolysis. The two protected segments were purified on Sephaex LH-20 followed by Bio-Beads S-X1 chromatography and respectively coupled onto a resin on which the 7–18 sequence of apamin was assembled stepwise with the standard solid phase procedure. On a portion of the resin, stepwise synthesis was continued to complete apamin. After HF treatment, deprotection of the cysteines, formation of the disulfide bonds and purification, biologically active apamin was obtained in the three cases.

Convergent solid phase peptide synthesis. I. Synthesis of protected segments on a hydroxymethylphenyloxymethyl resin using the base labile FMOC α-amine protection. Model synthesis of LHRH.

Abstract Convergent solid phase peptide synthesis has been applied to yield LHRH. The segments 1–6 and 7–10 of LHRH were synthesized on a hydroxymethylphenyloxymethyl resin using the base labile Fmoc protecting group on the α-amines. The side chains were protected by HF labile groups. Purification of the segments was performed on Sephadex LH-20 columns and by HPLC on Silica Gel 60 columns. The two segments were then assembled on an α-aminobenzyl resin to yield entire sequence of LHRH. After HF treatment and standard purification on Sephadex G-15 and carboxymethylcellulose CM-52 the desired LHRH was obtained. Synthesis of the segments by the same strategy on carbazoyloxymethylphenyloxymethyl resin showed up unexpected difficulties.

Immunochemistry of scorpion α-toxins: Study with synthetic peptides of the antigenicity of four regions of toxin II of Androctonus australis Hector

Sequences 19-29 and 28-39 of toxin II of the North African scorpion Androctonus australis Hector have been synthesized. These two peptides correspond to the highest peaks in the hydrophilicity profile of toxin II and were thus believed to account for a significant proportion of toxin antigenicity. Affinity chromatography of solid-phase-bonded peptides allowed us to purify two sub-populations from the total IgGs raised against the native toxin. They both still bound to 125I-toxin II and showed a restricted heterogeneity in their specificity. Solid-phase immunoassays confirmed the antigenicity of these synthetic peptides and also that of two other previously described synthetic replicates of the antigenic regions of toxin II: sequences (5-14) S-S (60-64) and 50-59. The location of the four antigenic regions relative to the postulated location of the receptor-binding site of the toxin is discussed.

Convergent solid phase peptide synthesis. v. synthesis of the 1-4, 32-34, and 53-59 protected segments of the toxin ii of androctonus australis hector.

Abstract Two protected peptide segments corresponding to the sequence 32-34 and 53-59 of toxin II of the north African scorpion Androctonus australis Hector have been synthesized on a photolabile Nbb-resin using the TFA-labile Boc α-amino protection and HF-labile side chain protecting groups. A third protected peptide corresponding to segment 1-4 has been synthesized on the same resin but with a t-butyl group for β protection of aspartic acid and a Z group on the lysine side chain. For this last segment a combination of Boc and Fmoc groups for α -amino protection has been used successfully on the Nbb-resin. After photolysis the crude peptides have been treated by solvent extraction and semi-preparative HPLC to yield highly purified segments. These syntheses show the flexibility of the convergent solid phase approach and how segments with different and independent protecting groups can be assembled by solid phase peptide procedure.

Immunochemistry of scorpion α-toxins: Purification and characterization of two functionally independent IgG populations raised against toxin II of Androctonus australis Hector

We report the isolation and characterization of two IgG populations specific to two synthetic peptides corresponding to two antigenic sites of toxin II of the North African scorpion Androctonus australis Hector. Firstly, thanks to the use of: (1) antigenic homology studies between toxin II of A. australis Hector and toxin III of Buthus occitanus tunetanus, (2) chemical modification of toxin II of A. australis Hector, and (3) prediction of the localization of the four major antigenic sites of scorpion alpha-toxins by the method developed by Hopp and Woods [Proc. natn. Acad. Sci. U.S.A. 78, 3824-3828 (1981)], we have established that the region around the disulfide bridge between cysteines 12 and 63 as well as the stretch of residues 50-59 probably each enclosed an antigenic site. Secondly, the synthetic replicates of these regions linked to Sepharose allowed us to isolate, by immunoaffinity chromatography, two IgG populations from the whole anti-toxin II of A. australis Hector IgGs. Finally, each of these two IgG populations was shown to be specific to one antigenic site as evidenced by the multideterminant effect on the slopes of binding curves developed by Berzofsky et al. [Biochemistry 15, 2113-2121 (1976)]. Furthermore, these two IgG populations were found to be functionally independent and this could be related to the fact that the two regions carrying the two antigenic sites are not close to each other in space and that there is neither steric hindrance nor cooperative effects between them. The association constant of these site-specific IgG populations was calculated and found to be equal to 1.18-5.14 X 10(9) l/mole for IgG anti-site 1 and 1.16-5.62 X 10(9) l/mole for IgG anti-site 2 respectively by Sips [J. chem. Phys. 16, 490-495 (1948)], Scatchard [Am. N.Y. Acad. Sci. 51, 660-772 (1949)] and Steward and Petty [Immunology 23, 881-887 (1972)] representations. The index of heterogeneity of 0.9 for anti-P1 and anti-P2 indicates the purification of essentially homogeneous affinity IgG populations.

Solid phase synthesis and HPLC purification of the protected 1-12 sequence of apamin for rapid synthesis of apamin analogues differing in the C-terminal region

Apamin is a bee neurotoxin, active in the central nervous system. It is an 18-peptide whose amino acid residues 13 and 14 play an essential role for binding to its receptor and for displaying toxicity. In order to accelerate the preparation of apamin analogues differing in the C-terminal region, a new strategy was set up involving solid phase synthesis of the 1-12 segment, which, after purification, can be solid phase coupled with different 13-18 sequences. The formula of the 12-membered protected peptide is: Boc-Cys(Acm)-Asn-Cys(Acm)-Lys(Z)-Ala-Pro-Glu(Bzl)-Thr(Bzl)-Ala-Leu- Cys(Acm)-Ala-OH. It has been assembled on the photosensitive resin, α-(4-bromomethyl-3-nitro-benzamido)benzylcopoly(styrene-1%divinyl- benzene) by conventional solid phase technique with Boc-amino acids. Boc-Leu was coupled by the method of Suzuki. Photolysis of batches of 1 gram of peptide-resin In trifluoroethanol/methylene chloride (20:80) yielded 89% of cleavage. The procedure for segment purification: organic extractions (ether, chloroform and precipitation from DMF with water), gel filtration on Sephadex LH-60, and finally semi-preparative HPLC on C18 in DMF/H2O (82:18) gave excellent results and an overall purification yield of 55%. After characterization, the purified 1-12 segment was coupled with three analogous 13-18 apamin sequences assembled on benzhydrylamine resins with yields of 77, 94, and 96%. After HF cleavage, deprotection and oxidation of the cysteines, the three peptides, apamin, p-aminophenylalanine-13- apamin, and p-aminophenylalanine-14-apamin were purified on carboxy-methylcellulose CM-52 and C18 HPLC. The purified peptides (yield 14-17%), after chemical characterization, were tested for toxic activity on mice and binding on synaptic membranes. The two analogues were about 100 times less toxic to mice than apamin and about 1000 times less potent in the binding assay.

Convergent solid phase peptide synthesis. VII: Good yields in the coupling of protected segments on a solid support

Abstract Protected peptides corresponding to the 64 amino acids of the whole sequence of toxin II of Androctonus australis Hector have been assembled on a solid support. Solid phase sequencing has proved to be extremely useful for the evaluation of the coupling yields, which have all been nearly quantitative.

Maurotoxin, a four disulfide bridges scorpion toxin acting on K+ channels

Maurotoxin, a toxin from the venom of the Tunisian chactoid scorpion Scorpio maurus, has been purified to homogeneity by gel filtration/reversed-phase HPLC, and characterized. It is a basic and C-terminal amidated 34-residue polypeptide cross-linked by four disulfide bridges. From Edman sequencing results, only six different pairings between the first six half-cystines were retained whereas a disulfide bridge was predicted between the two half-cystines in positions 31 and 34. Modelling based on the structure of charybdotoxin favored two different pairings, one of which possessed two disulfides in common with the general motif of scorpion toxins. The solid-phase technique was used to obtain synthetic maurotoxin, sMTX. The half-cystine pairings of sMTX were determined by enzymatic cleavage and were found to be Cys3 Cys24, Cys9-Cys29, Cys13-Cys19, and Cys31-34, in agreement with experimental data obtained with natural maurotoxin. Both natural and synthetic maurotoxins were lethal to mice following intracerebroventricular injection (LD50, 80 ng/mouse). They blocked the Kv1.1, Kv1.2, and Kv1.3 channels expressed in Xenopus oocytes with almost identical half-effects (IC50) in the range of 40, 0.8 and 150 nM, respectively. They also competed with 125I-apamin (SKca channel blocker) and 125I-kaliotoxin (Kv channel blocker) for binding to rat brain synaptosomes with IC50 of about 5 and 0.03 nM. As the natural and synthetic maurotoxins exhibit indistinguishable physicochemical and pharmacological properties, they are likely to adopt the same half-cystine pairing pattern which is unique among known scorpion toxins. However, this disulfide organization is different from those reported for Pandinus imperator and Heterometrus spinnifer toxins 1 (Pi1 and HsTx1), two novel four-disulfide bridged K+ channel-acting scorpion toxin sharing about 50-70% sequence identity with maurotoxin.

Localization of the toxic site of naja mossambica cardiotoxins: Small synthetic peptides express an in vivo lethality

Cardiotoxins are small basic proteins which cause heart failure when they are injected in vivo. In order to better understand their molecular mode of action, short peptides designed on the model of the first loop of the molecule of cardiotoxin IV from Naja mossambica mossambica venom have been synthetized by the solid-phase procedure of Merrifield. These peptides express lethality in mouse when they are injected intravenously. Taking into account the respective molecular weights, they are 3.5 to 5% as toxic as the cardiotoxin. Furthermore, the symptomatology they induce is undistinguishable from that induced by cardiotoxins. These results strongly support our previous hypothesis that the first loop of the molecule is the toxic site of cardiotoxins.