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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 l-amino acid oxidase (LAAO) transcripts from the venom gland transcriptomes of NK-M and NK-T in comparison to LAAO sequences of representative venomous snakes.Black, FAD-binding domains; red, substrate-binding domains; blue, helical domains; green, novel C-terminal FAD-binding domain; #, toxins co-expressed in the NK-M and NK-T; ^, toxin transcripts expressed in either source.
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fig-6: Multiple sequence alignment of l-amino acid oxidase (LAAO) transcripts from the venom gland transcriptomes of NK-M and NK-T in comparison to LAAO sequences of representative venomous snakes.Black, FAD-binding domains; red, substrate-binding domains; blue, helical domains; green, novel C-terminal FAD-binding domain; #, toxins co-expressed in the NK-M and NK-T; ^, toxin transcripts expressed in either source.

Mentions: The present study detected the presence of one and eight LAAO transcripts from the venom glands of NK-M and NK-T, respectively. Among these, NKM_LAO01 and NKT_LAO01 reveal a complete sequence with 514 amino acids length (Table 5). These transcripts comprise the three well-defined domains of LAAO and are highly homologous to LAAO reported from other cobras in particular N. atra (UniProtKB: A8QL58) (Pawelek et al., 2000) (Data S3; Fig. 6). The present study is the first report to reveal the full sequence of N. kaouthia LAAO (Data S3; Fig. 6), after the previous reported LAAO for N. naja kaouthia specimen (unspecified origin, Japan Snake Institute) with solely N-terminal sequence (38 amino acid residues) (Sakurai et al., 2001). Multiple sequence alignment shows the snake venom LAAO sequences appear to be highly conserved across different lineages (Fig. 6) and it is noteworthy that the sequence of the major LAAO from NK-M and NK-T venom glands are completely identical (Data S3 and S4). Again, considering that the snake venom LAAO is well conserved and found in the venoms of many lineages, it is most likely evolving under strong purifying selection. LAAO transcripts constitute only about 0.1% of total toxin mRNA, and about 1% of total venom proteins (Tan et al., 2015d). This is another enzyme of low abundance and minimal mutation in most snake venoms, consistent with its rather conserved ancillary function (Du & Clemetson, 2002; Tan et al., 2015e).


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 l-amino acid oxidase (LAAO) transcripts from the venom gland transcriptomes of NK-M and NK-T in comparison to LAAO sequences of representative venomous snakes.Black, FAD-binding domains; red, substrate-binding domains; blue, helical domains; green, novel C-terminal FAD-binding domain; #, toxins co-expressed in the NK-M and NK-T; ^, toxin transcripts expressed in either source.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384570&req=5

fig-6: Multiple sequence alignment of l-amino acid oxidase (LAAO) transcripts from the venom gland transcriptomes of NK-M and NK-T in comparison to LAAO sequences of representative venomous snakes.Black, FAD-binding domains; red, substrate-binding domains; blue, helical domains; green, novel C-terminal FAD-binding domain; #, toxins co-expressed in the NK-M and NK-T; ^, toxin transcripts expressed in either source.
Mentions: The present study detected the presence of one and eight LAAO transcripts from the venom glands of NK-M and NK-T, respectively. Among these, NKM_LAO01 and NKT_LAO01 reveal a complete sequence with 514 amino acids length (Table 5). These transcripts comprise the three well-defined domains of LAAO and are highly homologous to LAAO reported from other cobras in particular N. atra (UniProtKB: A8QL58) (Pawelek et al., 2000) (Data S3; Fig. 6). The present study is the first report to reveal the full sequence of N. kaouthia LAAO (Data S3; Fig. 6), after the previous reported LAAO for N. naja kaouthia specimen (unspecified origin, Japan Snake Institute) with solely N-terminal sequence (38 amino acid residues) (Sakurai et al., 2001). Multiple sequence alignment shows the snake venom LAAO sequences appear to be highly conserved across different lineages (Fig. 6) and it is noteworthy that the sequence of the major LAAO from NK-M and NK-T venom glands are completely identical (Data S3 and S4). Again, considering that the snake venom LAAO is well conserved and found in the venoms of many lineages, it is most likely evolving under strong purifying selection. LAAO transcripts constitute only about 0.1% of total toxin mRNA, and about 1% of total venom proteins (Tan et al., 2015d). This is another enzyme of low abundance and minimal mutation in most snake venoms, consistent with its rather conserved ancillary function (Du & Clemetson, 2002; Tan et al., 2015e).

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.