Fig 1.
Immunoreactivity of Philippine Cobra Antivenom (PCAV) toward isolatedα-neurotoxins (short-chain and long-chain alpha-neurotoxins) of Asian elapids.
PCAV binding was measured by indirect ELISA against N. philippinensis venom (homologous venom control), short-chain α-neurotoxins (SNTX) from indicated species, and long-chain α-neurotoxins (LNTX). Bars represent mean absorbance at 450 nm ± SEM (n = 3). Statistical significance was assessed by one-way ANOVA with multiple comparisons versus the homologous N. philippinensis venom control; ns, not significant; p < 0.05, *p < 0.01, **p < 0.001. Abbreviations of genera: N: Naja, for true cobra; H: Hydrophis, for sea snake; L: Laticauda, for sea krait; O: Ophiophagus, for King Cobra.
Fig 2.
Immunoreactivity of Naja kaouthia Antivenom (NkMAV), Neuro Bivalent Antivenom (NBAV) and Serum Anti Bisa Ular (SABU) toward venoms and isolated alpha-neurotoxins (SNTX and LNTX) of Asian elapids by indirect ELISA.
Values were expressed as means ± SEM from three independent experiments. Abbreviation of genus: N: Naja, for true cobra; H: Hydrophis, for sea snake; L: Laticauda, for sea krait; O: Ophiophagus, for King Cobra. Values were expressed as means ± SEM from three independent experiments. Different letters indicate significant differences among antivenoms within each venom or toxin antigen group (two-way ANOVA followed by Tukey’s post-hoc test, p < 0.05).
Fig 3.
Hierarchical clustered heatmaps showing the immunoreactivity of regional antivenoms toward (A) whole cobra venoms and (B) isolatedα-neurotoxins (SNTX and LNTX).
Color intensity corresponds to row z-scored ELISA absorbance (A₄₅₀) values, with red indicating higher-than-average and blue lower-than-average binding for each antivenom. Abbreviations: PCAV, Philippine Cobra Antivenom; NKMAV, Naja kaouthia Monovalent Antivenom; NBAV, Neuro Bivalent Antivenom; SABU, Indonesian polyvalent antivenom; SNTX, short-chain alpha-neurotoxin; LNTX, long-chain alpha-neurotoxin.
Fig 4.
Multiple sequence alignment of short-chain alpha-neurotoxins (SNTXs) from selected elapid species included in the immunoreactivity study.
Loops I–III and the C-terminal region are indicated above the alignment. Conserved cysteine residues forming the characteristic disulfide-bonded scaffold of three-finger toxins are shown. Solid arrows indicate primary residues directly implicated in nicotinic acetylcholine receptor (nAChR) binding, whereas dashed arrows denote supporting residues contributing to receptor interaction. The boxed region highlights sequence variability within the receptor-binding motif located in loop II. Conservation scores are shown below the alignment.
Fig 5.
Multiple sequence alignment of representative and database-derived SNTX sequences across elapid lineages including cobras, mamba, coral snake, sea snake, sea krait, Mallee black-back snake (Australian species, Suta nigriceps) and a synthetic SNTX (ScNTX) exhibiting the highest similarity to the SNTX of S. nigriceps, in addition to experimentally tested SNTXs in immunoreactivity study.
Loops I–III and the C-terminal region are indicated above the alignment. Conserved cysteine residues forming the disulfide-bonded scaffold characteristic of three-finger toxins are shown. Residues are shaded according to BLOSUM62 similarity. Solid arrows denote primary residues implicated in nicotinic acetylcholine receptor (nAChR) binding, whereas dashed arrows indicate supporting residues contributing to receptor interaction. The boxed region highlights sequence variability within the receptor-binding motif located in loop II. Conservation scores are shown below the alignment.
Fig 6.
Comparison of representative short-chain neurotoxins (SNTX) and long-chain neurotoxins (LNTX) across elapid lineages, including species whose toxins were experimentally tested in immunoreactivity study.
SNTXs from cobras and sea snake are aligned with LNTXs from cobra, king cobra, and krait. Loops I–III and the C-terminal region are indicated above the alignment. Conserved cysteine residues forming the characteristic disulfide-bonded scaffold of three-finger toxins are shown; note the additional cysteine pair in loop II of long-chain neurotoxins, corresponding to an extra disulfide bond. Residues are shaded according to BLOSUM62 similarity. Solid arrows indicate primary residues implicated in nicotinic acetylcholine receptor (nAChR) binding, whereas dashed arrows denote supporting residues contributing to receptor interaction. Purple boxes highlight sequence variability within the receptor-binding motif of the two cobra SNTXs. Conservation scores are shown below the alignment.
Fig 7.
Phylogenetic tree of short neurotoxins (SNTXs) from representative lineages of elapid snakes.
The dataset includes mambas (Dendroaspis polylepsis, Dendroaspis viridis), coral snakes (Micrurus frontalis, Micrurus laticollaris), sea snakes (Hydrophis curtus, Hydrophis schistosus), an Australian elapid (Suta nigriceps), and true cobras (Naja spp.). Cobra sequences comprise Asian cobras of the subgenus Naja (Naja sputatrix, Naja kaouthia, Naja sumatrana, Naja atra, Naja philippinensis, Naja samarensis), African cobras of the subgenus Uraeus (Naja nivea, Naja haje), Boulengerina (Naja melanoleuca), and subgenus Afronaja (Naja pallida). The sequence 7LUW_ScNTX represents the previously reported synthetic short neurotoxin construct [33]. Sequences marked with an asterisk (*) denote basal position of SNTX variants bearing the distinctive epitope motif “WWS----TII”, in contrast to the derived “RWR----YRT/I” motif found in other Southeast/East Asian cobras. The tree was inferred using the maximum likelihood method implemented in PhyML (v3.1/3.0 aLRT), and visualized using TreeDyn (v198.3).
Fig 8.
Linear epitope prediction of SNTXs.
Linear B-cell epitope propensity and surface accessibility of four short α-neurotoxins (SNTXs): P59276 (Naja kaouthia), P80958 and P60770 (Naja atra), and ScNTX (synthetic construct), were predicted using the IEDB analysis resource. (A) BepiPred-2.0 (threshold = 0.5). (B) Emini surface accessibility scale (threshold = 1.0). Predicted epitope regions are highlighted in yellow. (C) Loop II motif variants (WWS----TII, RWR----YRT, TWR----TII) associated with antigenic divergence.
Fig 9.
Structure-based epitope prediction of SNTXs.
Structure-based epitope prediction of the same four SNTXs using DiscoTope 2.0 and corresponding PDB structures (1JE9, 1ONJ, 1COE, and 7LUW). (A) DiscoTope scores (threshold = –7.7; red line), with positive residues shown in green. (B) Structural mapping of predicted epitope residues (yellow) on toxin models in Jmol mode. (C) Loop II motif variants (WWS----TII, RWR----YRT, TWR----TII) associated with antigenic divergence.
Table 1.
Composition of short- and long-chain alpha-neurotoxins from representative Afro-Asian cobras (Naja spp.), and marine elapid species. Values represent the percentage abundance of short-chain and long-chain α-neurotoxins relative to total venom protein, as reported in the cited quantitative proteomic studies. Ranges indicate values reported from different geographical populations or independent studies. ND, not detected.