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Comparative venom gland transcriptomics of Naja kaouthia (monocled cobra) from Malaysia and Thailand: elucidating geographical venom variation and insights into sequence novelty

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ABSTRACT

Background: The monocled cobra (Naja kaouthia) is a medically important venomous snake in Southeast Asia. Its venom has been shown to vary geographically in relation to venom composition and neurotoxic activity, indicating vast diversity of the toxin genes within the species. To investigate the polygenic trait of the venom and its locale-specific variation, we profiled and compared the venom gland transcriptomes of N. kaouthia from Malaysia (NK-M) and Thailand (NK-T) applying next-generation sequencing (NGS) technology.

Methods: The transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T.

Results and discussion: The toxin transcripts showed high redundancy (41–82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin’s fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the exclusively and abundantly expressed cytotoxin CTX-3 in NK-T. The findings suggested correlation with the geographical variation in proteome and toxicity of the venom, and support the call for optimising antivenom production and use in the region. Besides, the current study uncovered full and partial sequences of numerous toxin genes from N. kaouthia which have not been reported hitherto; these include N. kaouthia-specific l-amino acid oxidase (LAAO), snake venom serine protease (SVSP), cystatin, acetylcholinesterase (AChE), hyaluronidase (HYA), waprin, phospholipase B (PLB), aminopeptidase (AP), neprilysin, etc. Taken together, the findings further enrich the snake toxin database and provide deeper insights into the genetic diversity of cobra venom toxins.

No MeSH data available.


Multiple sequence alignment of phospholipase A2 (PLA2) transcripts from the venom gland transcriptomes of NK-M and NK-T in comparison to PLA2 sequences of representative venomous snakes.Red, conservative disulphide bonds; black, additional disulphide bond; blue, residues of pancreatic loops; #, toxins co-expressed in the NK-M and NK-T; ^, toxin transcripts expressed in either source.
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fig-3: Multiple sequence alignment of phospholipase A2 (PLA2) transcripts from the venom gland transcriptomes of NK-M and NK-T in comparison to PLA2 sequences of representative venomous snakes.Red, conservative disulphide bonds; black, additional disulphide bond; blue, residues of pancreatic loops; #, toxins co-expressed in the NK-M and NK-T; ^, toxin transcripts expressed in either source.

Mentions: A total of two and one PLA2 transcripts were identified from NK-M and NK-T venom glands respectively. Among these, NKM_PLA01 and NKT_PLA01 are the predominantly expressed forms of PLA2 and they are homologous to Group-IA acidic PLA2-1 (UniProtKB: P00596) isolated from the venom of N. kaouthia from Thailand (Joubert & Taljaard, 1980) (Fig. 3). From the sequence obtained, NKT_PLA01 was identical to the previously reported sequence (UniProtKB: P00596), while NKM_PLA01 is highly homologous with only one amino acid differing between the two, at the non-functional-critical site (Data S3; Fig. 3). Snake venom PLA2 commonly exists in multiple isoforms and exhibits great diversity in biological properties (Kini, 2003), however, it has not been easy to pinpoint the residues or segments of PLA2 that govern the pharmacological effects (Doley, Zhou & Kini, 2009). In agreement with the proteomic study and purified toxin characterisation (Tan et al., 2015d, 2016a), the predominant form of PLA2 in N. kaouthia venom is the acidic, enzymatic PLA2, shown with conserved Asp-49 residue in the current study. The acidic Asp-49-PLA2, however, was not lethal in mice even at a dose of >10–20 times of the median lethal dose of whole venom (Tan et al., 2016a). This is consistent with the report of non-toxic property of various acidic-type PLA2s isolated from the venoms of Indian N. kaouthia (Joubert & Taljaard, 1980) and Pakistani N. naja (Wong, Tan & Tan, 2016), but diverged markedly from the highly lethal, neutral/basic venom PLA2s characterised for other Southeast Asian cobras including Naja sumatrana and N. sputatrix (Leong et al., 2015; Tan & Arunmozhiarasi, 1989). The acidic PLA2s hence may serve a secondary role of ancillary function, for instance, potentiating the toxic actions of other venom components, including CTXs/cardiotoxins (Gasanov, Dagda & Rael, 2014), SVMPs (Bustillo et al., 2015) and weak neurotoxin (Mukherjee, 2010) to enhance tissue damages which are crucial for prey digestion but clinically deleterious as it complicates local tissue necrosis. On the other hand, although the mRNA level of the predominant form of PLA2 was relatively low (∼2%) within the venom gland, the PLA2 protein content in N. kaouthia venom is disproportionately higher (12–14% of total venom proteins) (Laustsen et al., 2015; Tan et al., 2015d). A possible explanation for this is that the mRNA of the PLA2 may have longer half-life, and thus a low mRNA level is sufficient to produce the necessary PLA2 protein (Vogel & Marcotte, 2012).


Comparative venom gland transcriptomics of Naja kaouthia (monocled cobra) from Malaysia and Thailand: elucidating geographical venom variation and insights into sequence novelty
Multiple sequence alignment of phospholipase A2 (PLA2) transcripts from the venom gland transcriptomes of NK-M and NK-T in comparison to PLA2 sequences of representative venomous snakes.Red, conservative disulphide bonds; black, additional disulphide bond; blue, residues of pancreatic loops; #, toxins co-expressed in the NK-M and NK-T; ^, toxin transcripts expressed in either source.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384570&req=5

fig-3: Multiple sequence alignment of phospholipase A2 (PLA2) transcripts from the venom gland transcriptomes of NK-M and NK-T in comparison to PLA2 sequences of representative venomous snakes.Red, conservative disulphide bonds; black, additional disulphide bond; blue, residues of pancreatic loops; #, toxins co-expressed in the NK-M and NK-T; ^, toxin transcripts expressed in either source.
Mentions: A total of two and one PLA2 transcripts were identified from NK-M and NK-T venom glands respectively. Among these, NKM_PLA01 and NKT_PLA01 are the predominantly expressed forms of PLA2 and they are homologous to Group-IA acidic PLA2-1 (UniProtKB: P00596) isolated from the venom of N. kaouthia from Thailand (Joubert & Taljaard, 1980) (Fig. 3). From the sequence obtained, NKT_PLA01 was identical to the previously reported sequence (UniProtKB: P00596), while NKM_PLA01 is highly homologous with only one amino acid differing between the two, at the non-functional-critical site (Data S3; Fig. 3). Snake venom PLA2 commonly exists in multiple isoforms and exhibits great diversity in biological properties (Kini, 2003), however, it has not been easy to pinpoint the residues or segments of PLA2 that govern the pharmacological effects (Doley, Zhou & Kini, 2009). In agreement with the proteomic study and purified toxin characterisation (Tan et al., 2015d, 2016a), the predominant form of PLA2 in N. kaouthia venom is the acidic, enzymatic PLA2, shown with conserved Asp-49 residue in the current study. The acidic Asp-49-PLA2, however, was not lethal in mice even at a dose of >10–20 times of the median lethal dose of whole venom (Tan et al., 2016a). This is consistent with the report of non-toxic property of various acidic-type PLA2s isolated from the venoms of Indian N. kaouthia (Joubert & Taljaard, 1980) and Pakistani N. naja (Wong, Tan & Tan, 2016), but diverged markedly from the highly lethal, neutral/basic venom PLA2s characterised for other Southeast Asian cobras including Naja sumatrana and N. sputatrix (Leong et al., 2015; Tan & Arunmozhiarasi, 1989). The acidic PLA2s hence may serve a secondary role of ancillary function, for instance, potentiating the toxic actions of other venom components, including CTXs/cardiotoxins (Gasanov, Dagda & Rael, 2014), SVMPs (Bustillo et al., 2015) and weak neurotoxin (Mukherjee, 2010) to enhance tissue damages which are crucial for prey digestion but clinically deleterious as it complicates local tissue necrosis. On the other hand, although the mRNA level of the predominant form of PLA2 was relatively low (∼2%) within the venom gland, the PLA2 protein content in N. kaouthia venom is disproportionately higher (12–14% of total venom proteins) (Laustsen et al., 2015; Tan et al., 2015d). A possible explanation for this is that the mRNA of the PLA2 may have longer half-life, and thus a low mRNA level is sufficient to produce the necessary PLA2 protein (Vogel & Marcotte, 2012).

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The monocled cobra (Naja kaouthia) is a medically important venomous snake in Southeast Asia. Its venom has been shown to vary geographically in relation to venom composition and neurotoxic activity, indicating vast diversity of the toxin genes within the species. To investigate the polygenic trait of the venom and its locale-specific variation, we profiled and compared the venom gland transcriptomes of N. kaouthia from Malaysia (NK-M) and Thailand (NK-T) applying next-generation sequencing (NGS) technology.

Methods: The transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T.

Results and discussion: The toxin transcripts showed high redundancy (41–82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin’s fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the exclusively and abundantly expressed cytotoxin CTX-3 in NK-T. The findings suggested correlation with the geographical variation in proteome and toxicity of the venom, and support the call for optimising antivenom production and use in the region. Besides, the current study uncovered full and partial sequences of numerous toxin genes from N. kaouthia which have not been reported hitherto; these include N. kaouthia-specific l-amino acid oxidase (LAAO), snake venom serine protease (SVSP), cystatin, acetylcholinesterase (AChE), hyaluronidase (HYA), waprin, phospholipase B (PLB), aminopeptidase (AP), neprilysin, etc. Taken together, the findings further enrich the snake toxin database and provide deeper insights into the genetic diversity of cobra venom toxins.

No MeSH data available.