Tail wags the dog: activity of krait natriuretic peptide is determined by its C-terminal tail in a natriuretic peptide receptor-independent manner.

Natriuretic peptides (NPs) are potent vasoactive hormones, which maintain pressure-volume homoeostasis. Snake venom NPs exhibit distinct biological activity compared with mammalian NPs due to subtle changes in their sequences. We recently identified a new NP from krait venom (KNP), with an unusual 38-residue long C-terminal tail, which has a propensity to form an α-helix. KNP mediates vasodilation via NP receptor (NPR) independent mechanisms on pre-contracted aortic strips in contrast with classical NPs. The infusion of KNP in anaesthetized rats resulted in a prolonged and sustained drop in blood pressure (BP) and heart rate (HR) with no renal effects in contrast with mammalian counterparts. Deletion mutant studies have revealed the presence of two functional segments in KNP, namely Ring and Helix. Although the Ring interacts with NPR, its contribution to the activity of KNP is shown to be negligible as both KNP and Helix elicit equipotent endothelium-dependent vasorelaxation. Further, KNP and Helix signalled through endothelial nitric oxide (NO) to mediate NPR-independent vasodilation. Thus, KNP exhibits non-canonical characteristics through its C-terminal tail, despite a functional NP ring. The present study has altered the paradigm of NP biology through the understanding of structure-function relationships and may serve as a lead for the design of novel hypotensive agents.

Expression and refolding of bioactive α-bungarotoxin V31 in E. coli.

In order to obtain bioactive α-bungarotoxin (αBtx) using recombinant protein technique, a codon-optimized synthetic gene was expressed in fusion with the N-terminal 10-His-tag and C-terminal Strep-tag in Escherichia coli. Further optimization through site-directed mutagenesis enabled moderate expression of the protein without the N-terminal His-tag or the C-terminal Strep-tag. Two such recombinant αBtx (rαBtx) were obtained, both with an additional methionine and a glycine at the N-terminal and one with (G4S1)2-Strep-tag at the C-terminal. The rαBtx proteins were refolded using a novel protocol, which efficiently produced final products with activity similar to its natural counterpart. The protocol could easily be scale up, which produced 0.3-1mg of pure and highly active rαBtx per liter of E. coli culture.

Water-soluble LYNX1 residues important for interaction with muscle-type and/or neuronal nicotinic receptors.

Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the α7 nAChR extracellular ligand-binding domain and the transmembrane domain of α1 glycine receptor (α7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at α7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its "non-classical" binding sites on α7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for α-bungarotoxin and similar snake α-neurotoxins also targeting α7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.

Fluorophore assisted light inactivation (FALI) of recombinant 5-HT3A receptor constitutive internalization and function.

Fluorescent proteins and molecules are now widely used to tag and visualize proteins resulting in an improved understanding of protein trafficking, localization, and function. In addition, fluorescent tags have also been used to inactivate protein function in a spatially and temporally-defined manner, using a technique known as fluorophore-assisted light inactivation (FALI) or chromophore-assisted light inactivation (CALI). In this study we tagged the serotonin3 A subunit with the α-bungarotoxin binding sequence (BBS) and subsequently labeled 5-HT3A/BBS receptors with fluorescently conjugated α-bungarotoxin in live cells. We show that 5-HT3A/BBS receptors are constitutively internalized in the absence of an agonist and internalization as well as receptor function are inhibited by fluorescence. The fluorescence-induced disruption of function and internalization was reduced with oxygen radical scavengers suggesting the involvement of reactive oxygen species, implicating the FALI process. Furthermore, these data suggest that intense illumination during live-cell microscopy may result in inadvertent FALI and inhibition of protein trafficking.

Transcriptomic analysis of the venom gland of the red-headed krait (Bungarus flaviceps) using expressed sequence tags.

BACKGROUND: The Red-headed krait (Bungarus flaviceps, Squamata: Serpentes: Elapidae) is a medically important venomous snake that inhabits South-East Asia. Although the venoms of most species of the snake genus Bungarus have been well characterized, a detailed compositional analysis of B. flaviceps is currently lacking. RESULTS: Here, we have sequenced 845 expressed sequence tags (ESTs) from the venom gland of a B. flaviceps. Of the transcripts, 74.8% were putative toxins; 20.6% were cellular; and 4.6% were unknown. The main venom protein families identified were three-finger toxins (3FTxs), Kunitz-type serine protease inhibitors (including chain B of beta-bungarotoxin), phospholipase A2 (including chain A of beta-bungarotoxin), natriuretic peptide (NP), CRISPs, and C-type lectin. CONCLUSION: The 3FTxs were found to be the major component of the venom (39%). We found eight groups of unique 3FTxs and most of them were different from the well-characterized 3FTxs. We found three groups of Kunitz-type serine protease inhibitors (SPIs); one group was comparable to the classical SPIs and the other two groups to chain B of beta-bungarotoxins (with or without the extra cysteine) based on sequence identity. The latter group may be functional equivalents of dendrotoxins in Bungarus venoms. The natriuretic peptide (NP) found is the first NP for any Asian elapid, and distantly related to Australian elapid NPs. Our study identifies several unique toxins in B. flaviceps venom, which may help in understanding the evolution of venom toxins and the pathophysiological symptoms induced after envenomation.

Protein complexes in snake venom.

Snake venom contains mixture of bioactive proteins and polypeptides. Most of these proteins and polypeptides exist as monomers, but some of them form complexes in the venom. These complexes exhibit much higher levels of pharmacological activity compared to individual components and play an important role in pathophysiological effects during envenomation. They are formed through covalent and/or non-covalent interactions. The subunits of the complexes are either identical (homodimers) or dissimilar (heterodimers; in some cases subunits belong to different families of proteins). The formation of complexes, at times, eliminates the non-specific binding and enhances the binding to the target molecule. On several occasions, it also leads to recognition of new targets as protein-protein interaction in complexes exposes the critical amino acid residues buried in the monomers. Here, we describe the structure and function of various protein complexes of snake venoms and their role in snake venom toxicity.

Kinetic analysis of effects of temperature and time on the regulation of venom expression in Bungarus multicinctus.

Venom gland is a highly efficient venom production system to maintain their predatory arsenal. Venom toxins mRNA has been shown to increase abruptly in snake after venom expenditure, while the dynamics of venom accumulation during synthesis are poorly understood. Here, PacBio long-read sequencing, Illumina RNA sequencing (RNA-seq), and label-free proteome quantification were used to investigate the composition landscape and time- and temperature-dependent dynamics changes of the Bungarus multicinctus venom gland system. Transcriptome data (19.5223 Gb) from six adult B. multicinctus tissues were sequenced using PacBio RS II to generate a reference assembly, and average 7.28 Gb of clean RNA-seq data was obtained from venom glands by Illumina sequencing. Differentially expressed genes (DEGs) mainly were protein processing rather than venom toxins. Post-translational modifications provided the evidence of the significantly different proportions of toxins in the venom proteome with the changing of replenishment time and temperature, but constant of venom toxins mRNA in the venom gland transcriptome. Dynamic of toxins and genes involved in venom gland contraction suggesting the formation of the mature venom gland system would take at least 9 days. In addition, 59 toxin processing genes were identified, peptidylprolyl isomerase B of which underwent positive selection in Toxicofera. These results provide a reference for determining the extraction time of venom, production of polyclonal and monoclonal antibody for precise treatment plans of venomous snakebites, and construction of an in vitro synthesis system for snake venom protein.

Bacterial production and refolding from inclusion bodies of a "weak" toxin, a disulfide rich protein.

The gene for the "weak" toxin of Naja kaouthia venom was expressed in Escherichia coli. "Weak" toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of "weak" toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia "weak" toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain "weak" toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of (125)I-labeled alpha-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (alpha1(2)beta1gammadelta) showed the presence of biological activity of the recombinant "weak" toxin close to the activity of the natural toxin (IC(50) = 4.3 +/- 0.3 and 3.0 +/- 0.5 microM, respectively). The interaction of the recombinant toxin with alpha7 type human neuronal acetylcholine receptor transfected in the GH(4)C(1) cell line also showed the presence of activity close to that of the natural toxin (IC(50) 31 +/- 5.0 and 14.8 +/- 1.3 microM, respectively). The developed bacterial system for production of N. kaouthia venom "weak" toxin was used to obtain (15)N-labeled analog of the neurotoxin.

Enhanced in vivo-imaging in medaka by optimized anaesthesia, fluorescent protein selection and removal of pigmentation.

Fish are ideally suited for in vivo-imaging due to their transparency at early stages combined with a large genetic toolbox. Key challenges to further advance imaging are fluorophore selection, immobilization of the specimen and approaches to eliminate pigmentation. We addressed all three and identified the fluorophores and anaesthesia of choice by high throughput time-lapse imaging. Our results indicate that eGFP and mCherry are the best conservative choices for in vivo-fluorescence experiments, when availability of well-established antibodies and nanobodies matters. Still, mVenusNB and mGFPmut2 delivered highest absolute fluorescence intensities in vivo. Immobilization is of key importance during extended in vivo imaging. Here, traditional approaches are outperformed by mRNA injection of α-Bungarotoxin which allows a complete and reversible, transient immobilization. In combination with fully transparent juvenile and adult fish established by the targeted inactivation of both, oca2 and pnp4a via CRISPR/Cas9-mediated gene editing in medaka we could dramatically improve the state-of-the art imaging conditions in post-embryonic fish, now enabling light-sheet microscopy of the growing retina, brain, gills and inner organs in the absence of side effects caused by anaesthetic drugs or pigmentation.

lynx1, an endogenous toxin-like modulator of nicotinic acetylcholine receptors in the mammalian CNS.

Elapid snake venom neurotoxins exert their effects through high-affinity interactions with specific neurotransmitter receptors. A novel murine gene, lynx1, is highly expressed in the brain and contains the cysteine-rich motif characteristic of this class of neurotoxins. Primary sequence and gene structure analyses reveal an evolutionary relationship between lynx1 and the Ly-6/neurotoxin gene family. lynx1 is expressed in large projection neurons in the hippocampus, cortex, and cerebellum. In cerebellar neurons, lynx1 protein is localized to a specific subdomain including the soma and proximal dendrites. lynx1 binding to brain sections correlates with the distribution of nAChRs, and application of lynx1 to Xenopus oocytes expressing nAChRs results in an increase in acetylcholine-evoked macroscopic currents. These results identify lynx1 as a novel protein modulator for nAChRs in vitro, which could have important implications in the regulation of cholinergic function in vivo.

Tethering naturally occurring peptide toxins for cell-autonomous modulation of ion channels and receptors in vivo.

The physiologies of cells depend on electrochemical signals carried by ion channels and receptors. Venomous animals produce an enormous variety of peptide toxins with high affinity for specific ion channels and receptors. The mammalian prototoxin lynx1 shares with alpha-bungarotoxin the ability to bind and modulate nicotinic receptors (nAChRs); however, lynx1 is tethered to the membrane via a GPI anchor. We show here that several classes of neurotoxins, including bungarotoxins and cobratoxins, retain their selective antagonistic properties when tethered to the membrane. Targeted elimination of nAChR function in zebrafish can be achieved with tethered alpha-bungarotoxin, silencing synaptic transmission without perturbing synapse formation. These studies harness the pharmacological properties of peptide toxins for use in genetic experiments. When combined with specific methods of cell and temporal expression, the extension of this approach to hundreds of naturally occurring peptide toxins opens a new landscape for cell-autonomous regulation of cellular physiology in vivo.

A novel serine protease inhibitor from Bungarus fasciatus venom.

By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.

Engineering varied serine protease inhibitors by converting P1 site of BF9, a weakly active Kunitz-type animal toxin.

Although there were a lot of weakly active animal toxins in the venoms, their values and applications are still mysterious, such as BF9, which is a Kunitz-type toxin isolated from the venom of the elapid snake Bungarus fasciatus. Here, we used BF9 to be a molecular scaffold, and engineered eight BF9-derived peptides by changing P1 site Asn17 of BF9, such as BF9-N17Y and BF9-N17T designed from the polar subfamily, BF9-N17L and BF9-N17G designed from the Non-polar subfamily, BF9-N17D designed from acidic subfamily, and BF9-N17H, BF9-N17K and BF9-N17R designed from basic subfamily. Through enzyme inhibitor experiment assays, we found a potent and selective chymotrypsin inhibitor BF9-N17Y, a potent and selective coagulation factor XIa inhibitor BF9-N17H, and two highly potent coagulation factor XIa inhibitors BF9-N17K and BF9-N17. APTT and PT assays further showed that BF9-N17H, BF9-N17K and BF9-N17R were three novel anticoagulants with selectively intrinsic coagulation pathway inhibitory activity. Considering that natural weakly active animal toxins are also a huge peptide resource, our present work might open a new window about pharmacological applications of weakly active animal toxins, which might be good templates for potent and selective molecular probe and lead drug designs.

Taiwan cobra chymotrypsin inhibitor: cloning, functional expression and gene organization.

A cDNA encoding chymotrypsin inhibitor was constructed from the cellular RNA isolated from the venom glands of Naja atra (Taiwan cobra). The resultant amino acid sequence showed that the mature protein is comprised of 57 amino acid residues with six cysteine residues. Cloned protein was expressed and isolated from the inclusion bodies of E. coli and refolded into a functional protein in vitro. Deleting the first three residues at its N-terminus caused a moderate increase in the inhibitory constant (K(i)) against chymotrypsin. The genomic DNA encoding the chymotrypsin inhibitor was amplified by PCR. The gene shares virtually an identical structural organization with the beta-bungarotoxin B1 chain (a snake Kunitz/BPTI neurotoxic homolog) gene. Moreover, the overall sequence identity of the N. atra chymotrypsin inhibitor and beta-bungarotoxin B1 chain genes was up to 83%. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.

Divergence of genes encoding B chains of beta-bungarotoxins.

The structural organization of the genes encoding B2, B4, B5 and B6 chains of beta-bungarotoxins are reported in this study. These genes shared virtually identical overall organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 and B2 chains were absent in that of B4, B5 and B6 chains. RT-PCR analyses indicated that Bungarus multicinctus venom gland, heart, liver and muscle expressed the RNA transcripts showing sequence similarity with the intronic segment being deleted in B4, B5 and B6 chain genes. This reflects that the ancestral gene of the intronic segment might insert in multiple loci of B. multicinctus genome. Comparative analyses of B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that intron insertions or deletions occur with the evolution of B chains, and that accelerated evolution may diversify the protein-coding sequence of B chain genes same as snake phospholipase A2, neurotoxin and cardiotoxin genes.

Genetic organization of alpha-bungarotoxins from Bungarus multicinctus (Taiwan banded krait): evidence showing that the production of alpha-bungarotoxin isotoxins is not derived from edited mRNAs.

Two genomic DNAs with a size of approximately 2.8 kb, isolated from the liver of Bungarus multicinctus (Taiwan banded krait), encode the precursors of the long neurotoxins, alpha-Bgt(A31) and alpha-Bgt(V31), respectively. Both genes share virtually identical overall organization with three exons separated by two introns, which were inserted in the same positions in the coding regions of the genes. Moreover, their nucleotide sequences share approximately 98% identity. This result indicates that the two genes co-exist in the genome of B.multicinctus, and probably arose from gene duplication. The exon/intron structures of the alpha-Bgt genes were essentially the same as those reported for the short neurotoxins. This reflects that the long and short neurotoxins should share a common evolutionary origin. Comparative analyses on long neurotoxin and short neurotoxin genes showed that the protein coding regions of the exons were more diverse than the introns except for the signal peptide domain. This implies that the protein coding regions of the neurotoxins may have evolved via accelerated evolution. PCR amplification of venom gland cDNA mixtures revealed that only two amino acid sequences corresponding to alpha-Bgt(A31) and alpha-Bgt(V31) could be deduced from the cDNAs. The results of chromatographic analyses and protein sequencing again emphasized the view that, with the exception of alpha-Bgt(A31) and alpha-Bgt(V31), no other alpha-Bgt isotoxins with amino acid substitutions were present in B.multicinctus venom. In contrast to the proposition of Liu et al. ( Nucleic Acids Res., 1998,26, 5624-5629), our findings strongly suggest that each alpha-Bgt isotoxin is derived from the respective gene, and that alpha-Bgt RNA polymorphism does not originate from one single, intronless gene by the mechanism of RNA editing.

[Cloning of alpha-bungarotoxin gene and its prokaryotic expression as a non-fusion protein].

On the basis of the reported amino acid sequence of alpha-bungarotoxin (alpha-BGT), DNA sequence of alpha-BGT was deduced and fourteen partially complementary oligonucleotides were designed and synthesized. A plasmid carrying the coding region of alpha-BGT was obtained by primer extension, PCR and ligation with pMD-18-T. The target fragment was digested with Xba I and EcoR I, recovered and ligated with pET28a(+). The resultant expression vector was transformed into BL21 (DE3), BL21 (DE3) Codon plus, and BL21 (DE3) plysS, respectively. Recombinant alpha-BGT was expressed in BL21 (DE3) and was analyzed by 15% Tris/tricine SDS-PAGE. The result showed that the recombinant protein, mostly found in inclusion bodies, accounted for 11.98% of the total bacterial lysate. The expression capacity could be increased to 16.28% by optimizing expression conditions. Western blotting results showed that the expressed protein had similar immunogenicity with the natural alpha-BGT protein purified from the venom of Krait Bungarus spp. In vivo toxicity assay of purified and renatured proteins in mice showed that LD50 was about 1.28 microg/g.

DNA Aptamers against Taiwan Banded Krait α-Bungarotoxin Recognize Taiwan Cobra Cardiotoxins.

Bungarus multicinctus α-bungarotoxin (α-Bgt) and Naja atra cardiotoxins (CTXs) share a common structural scaffold, and their tertiary structures adopt three-fingered loop motifs. Four DNA aptamers against α-Bgt have been reported previously. Given that the binding of aptamers with targeted proteins depends on structural complementarity, in this study, we investigated whether DNA aptamers against α-Bgt could also recognize CTXs. It was found that N. atra cardiotoxin 3 (CTX3) reduced the electrophoretic mobility of aptamers against α-Bgt. Analysis of the changes in the fluorescence intensity of carboxyfluorescein-labeled aptamers upon binding toxin molecules revealed that CTX3 and α-Bgt could bind the tested aptamers. Moreover, the aptamers inhibited the membrane-damaging activity and cytotoxicity of CTX3. In addition to CTX3, other N. atra CTX isotoxins also bound to the aptamer against α-Bgt. Taken together, our data indicate that aptamers against α-Bgt show cross-reactivity with CTXs. The findings that aptamers against α-Bgt also suppress the biological activities of CTX3 highlight the potential utility of aptamers in regard to the broad inhibition of snake venom three-fingered proteins.

[Molecular cloning and expression of an isotoxin gene, alpha-bungarotoxin, from Bungarus multicinctus].

Abstract: Snake venom contains a number of small proteins,enzymes and other components,which displays a broad spectrum of biological activities. With the ability of specifically binding on acetylcholine acceptor, alpha-bungarotoxins are not only useful molecular probes in investigating the mechanism of neural signal transmission, but also potential pharmic preparations for neural disease treatment. In current research,cDNAs of Bungarus multicinutus venom gland were synthesized using SMART cDNA amplification kit and then, alpha-bungarotoxin genes were cloned and sequenced. Total of 20 clones were sequenced representing 14 isotoxin mRNAs of alpha-bungarotoxins. Among those clones, a novel isotoxin gene was subcloned into two expression plasmids, alpha-BgTX/pQE30a and alpha-BgTX/pGEX-4T-1, and transformed into E. coli. After inducing with IPTG, fused protein of GST-alpha-BgTX was successfully expressed at level of 30% gross proteins of bacteria. More than 25% of fused protein was in the soluble fraction and the rest in inclusion body.

Antibody and T-cell recognition of alpha-bungarotoxin and its synthetic loop-peptides.

Peptides representing the loops and surface regions of alpha-bungarotoxin (BgTX) and control peptide analogs in which these sequences were randomized were synthesized and used to map the recognition profiles of the antibodies and T-cells obtained after BgTX immunization. Also, the abilities of anti-peptide antibodies and T-cells to recognize the immunizing peptide and BgTX were determined. Three regions of BgTX were immunodominant by both rabbit and mouse anti-BgTX antibodies. These regions resided within loops L1 (residues 3-16), L2 (residues 26-41) and the C-terminal tail (residues 66-74) of the toxin. The regions recognized by BgTX-primed T-lymphocytes were mapped in five mouse strains: C57BL/6(H-2b), Balb/c (H-2d), CBA (H-2k), C3H/He (H-2k) and SJL (H-2s). The H-2b and H-2d haplotypes were high responders to BgTX, while the H-2k and H-2s were intermediate responders. The T-cell recognition profile of the peptides varied with the haplotype, consistent with Ir gene control of the responses to the individual regions. The submolecular specificities of antibodies and T-cells were compared in three of the mouse strains (C57BL/6, Balb/c and SJL). In a given mouse strain, there were regions that were strongly recognized by both antibodies and T-cells as well as regions that were predominantly recognized either by antibodies or by T-cells. The peptides were used as immunogens in their free form (i.e. without coupling to any carrier) in two of the mouse strains, Balb/c and SJL. In both mouse strains, the peptides gave strong antibody responses. Antibodies against peptide L2 showed the highest binding to intact BgTX. Antibodies against the other peptides exhibited lower binding activity to the intact toxin, and this activity was dependent on the peptide and the mouse strain. The response of peptide-primed T-cells to a given immunizing peptide was not related to whether this region was immunodominant with BgTX-primed T-cells. The ability of peptide-primed T-cells to recognize the intact toxin varied with the peptide and was dependent on the host strain. These results indicate that anti-peptide antibody and T-cell responses are also under genetic control and that their ability to cross-react with the parent toxin is not only dependent on the conformational exposure of the correlate region in intact BgTX.