BACKGROUND
toxin proteins appeared more than  <dig> million years ago in the common ancestor of venomous reptiles such as snakes and their saurian cousins  <cit> .
the prevailing thoughts on venom evolution agree that toxin genes were initially recruited from genes of proteins with normal physiological functions, followed by repeated gene duplication that creates redundancy, allowing a gene copy to be selectively expressed in the venom gland.
the ‘free’ copies subsequently underwent neofunctionalization through positive selection and molecular adaptation at accelerated rates, driven primarily by changes in the ecological niche, diet and predator–prey arms race  <cit> .
besides gene duplications, alternative splicing and alterations of domain sturctures are other mechanisms capable of generating novel toxin genes  <cit> .
the emergence of paralogous groups of multigene families across taxonomic lineages is accompanied by the occurrence of multiple isoforms within each major toxin family, resulting in diverse functional variations of venom proteins that have great impacts on medical management and toxinological research  <cit> .
the immerse variety of toxins also serves as a source of leads for drug discovery.
from a clinical standpoint, the phenomenon gives rise to diverse snakebite syndromes and potentially suboptimal antivenom effectiveness.
the phenomenon complicates snakebite management that relies heavily on the understanding of venom complexity and the availability of effective antivenom.
in most developing and under-developed countries, snake envenomation remains a neglected tropical disease and a disease of poverty  <cit> .
malaysia, as a tropical country that is home to many venomous snake species, shares similar public health concerns.
the problem affects not only the rural populations but also the suburban regions due to rapid urbanization as the human populations increasingly encroach upon snake habitat .
among the native snakes in malaysia, the king cobra  is a known cause of envenomation  <cit> , partly due to its popularity among snake hobbyists.
the king cobra is the world’s largest venomous snake , capable of delivering huge amounts of venom per bite  <cit>  .
clinically king cobra bites cause neurotoxicty, paralysing respiratory muscles and leading to rapid death if untreated.
in surviving victims, the bites can result in extensive tissue necrosis and crippling deformity, adding to the heavy toll of human suffering in socioeconomically deprived countries.
ophiophagus hannah is widely distributed across the indian subcontinent, southeastern asia and the southern part of china  <cit> .
as the generic name ophiophagus  suggests, its diet consists primarily of snakes, although small mammals are also eaten.
the ophiophagus is a monotypic genus with only one species recognized currently.
however, its vast geographical distribution accompanied by morphological differences suggests potential taxonomic divergence and variation in venom composition.
in fact, the venom of king cobra has been studied since the early 1970’s.
various components have been isolated and characterized, including l-amino acid oxidases  <cit> , metalloproteinases  <cit> , three finger toxins   <cit> , phospholipases a <dig>   <cit> , ohanin  <cit> , kunitz-type protease inhibitors  <cit>  and factor x activator  <cit> .
nonetheless, the significance of the origin and intraspecific variation of king cobra venom has only been recently documented.
in a study with venom samples sourced from a few countries, differences in venom properties were noted between chinese and southeast asian king cobras  <cit> .
a genomic study of an indonesian king cobra  by vonk et al.
<cit>  indicated great diversity in the pla <dig> and 3ftx gene families in a single specimen.
indeed, intraspecific variations of snake venom are attributed to multiple factors such as diet, geographical distribution, ontogeny, season/climate changes etc.
<cit> , and the phenomenon is hence highly relevant to the king cobra in view of its extremely vast distribution.
producing regional or local antivenom would be ideal; unfortunately the production of an affordable and effective antivenom tailored to different areas by local manufacturers would be hampered by financial and technical constraints.
thus, a new approach has been recently proposed by the toxinological community to focus on the investigation of specific venom-gland transcriptomes and venom proteomes for the development of pan-regional antivenoms  <cit> .
vonk et al.
<cit>  reported the venom-gland transcriptome and venom proteome of a specimen from the indonesian island of bali.
however, the possibility of intraspecific venom variations from king cobra in the distant malayan peninsula cannot be excluded.
in addition, the paper reported the numbers of toxin subtypes identified in the venom proteome without the relative toxin abundances.
earlier, vejayan et al.
<cit>  reported 2-dimensional gel electrophoresis findings of the malaysian king cobra venom .
unfortunately, their mass spectrometric analysis  yielded a large number of unidentifiable proteins, whilst only five toxin protein families were determined  as well as one non-toxin protein, thioredoxin.
the study verified the presence of some previously isolated toxins but shed little light on the venom proteome.
another recent report on king cobra venom of probable thai origin profiled the venom as well, detecting  <dig> protein types/families , but again without the estimation of relative toxin abundance  <cit> .
the 1d electrophoretic gel findings of the thai king cobra venom appeared similar to that reported by chang et al.
<cit> , suggesting that 3ftxs are the major component of king cobra venoms of different localities.
however, the diversity in toxin sequences and isoforms as well as variations in toxin abundance could not be ascertained.
to remedy the current lack of venom-gland transcriptomic and venom proteomic data for the malaysian king cobra, the current study employed next-generation sequencing technology  and nano-liquid chromatography-tandem mass spectrometry  for a deeper insight into the toxin diversity of king cobra venom.
RESULTS
sequencing and transcriptome assembly
from the primary output statistics of transcript sequencing, a total of  <dig> , <dig> pairs of clean reads passed the illumina quality filter for further de novo assembly using the short reads assembling program, trinity  <cit> .
trinity created  <dig>  contigs , connected to form  <dig>  unigenes  in total as shown in fig.
<dig>  blastx alignment  between the unigenes and sequences in the ncbi non-redundant  protein database yielded  <dig>  annotated unigenes .
after filtering low-frequency transcripts , the assemblies were reduced from  <dig>  to  <dig>  transcripts and categorized into unidentified , non-toxin  and toxin  groups.
of these,  <dig> toxin transcripts encode full-length toxin sequences.
although the toxin group accounted for  <dig> transcripts only, it is abundantly expressed as much as  <dig>  % of the total expression , whereas the unidentified and non-toxin groups were  <dig>  % and  <dig>  % respectively .
toxin transcripts were expressed at extremely higher redundancies  compared to non-toxin transcripts  .fig.
<dig>
de novo transcriptome assembly with short reads assembling program, trinity.
a length distribution of contigs from reads assembly.
b length distribution of unigenes generated from connected contigs
fig.
<dig> the malaysian ophiophagus hannah venom gland mrna expression profile.
pie charts mainly display the percentage abundance of toxin transcripts.
toxins transcripts accounted for  <dig>  % of the total fpkm.
three-finger toxins are the most abundant toxin family in the venom gland .
3ftx, three-finger toxin; svmp, snake venom metalloproteinase; pla <dig>  phospholipase a2; crisp, cysteine-rich secretory protein; kun, kunitz-type proteinase inhibitor; laao, l-amino acid oxidase; cvf, cobra venom factor; igf, insulin-like growth factor; np, natriuretic peptide; svsp, snake venom serine protease; pde, phosphodiesterase; ngf, nerve growth factor; ache, acetylcholinesterase; plb, phospholipase-b; dpp iv, dipeptidylpeptidase iv; ctl, c-type lectin; vegf, vascular endothelial growth factor
venom-gland transcriptome
the identifiable toxin transcripts  represent  <dig> protein families related to venom toxic functions.
the toxin constituents, as indicated by fpkm, are dominated by 3ftxs .
the toxin transcript abundances are followed by snake venom metalloproteases , phospholipases a <dig> , cysteine-rich secretory proteins , kunitz-type proteinase inhibitors  and l-amino acid oxidases .
a variety of toxin or protein mrnas is expressed in low abundances , including cobra venom factors , vespryn, natriuretic peptides , snake venom serine proteases , phosphodiesterases , nerve growth factors , acetylcholinesterases , neprilysins, hyaluronidase, phopholipases b , cystatins, dipeptidylpeptidase iv , 5’nucleotidases , waprins, c-type lectins , insulin-like growth factors , and vascular endothelial growth factors  .
the sequences and parameter details of the transcripts were sorted according to gene families and compiled in additional file  <dig>
venom proteome
when examined under reducing  <dig> % sds-page, moh venom showed three major protein bands each with a distinct molecular mass range: high , intermediate , and low .
the gel as shown in fig.
<dig> was divided into  <dig> sections of equal sizes in preparation for in-gel digestion and nano-lcms/ms, and the protein abundances from each section were estimated by gel densitometry for in-gel protein amount correlation  <cit> .
proteins were detected by nano-lcms/ms from all gel sections including the pale-staining ones  notwithstanding their low abundances.
the moh venom proteins mainly migrated to section  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  accounting for approximately  <dig> %  of the total venom protein .fig.
<dig> densitometry quantitative measurement of sds-page gel using myimageanalysis software.
a sds-page gel sections cutting scale with protein marker range from ~10kda to 170kda.
b gel densitometry section  <dig> to  <dig> displayed in percentages
a customized reference database  was used in data mining to identify peptides sequenced by tandem mass spectrometry.
a total of  <dig> toxins were identified from the moh venom;  <dig> of these were matched to the transcripts.
all toxins were assigned to  <dig> protein families.
3ftxs and svmps were the most abundantly expressed toxins in the proteome, accounting for  <dig>  % and  <dig>  %, respectively, of the venom.
this was followed by crisp , laaos , vespryn , pla <dig>  and cvfs .
protein families with an abundance of less than  <dig> % each were categorized into “others” and considered as low-abundance proteins  .
the sequences and parameter details of the proteins identified were sorted according to gel sections and protein families as compiled in additional file  <dig> fig.
<dig> protein expression profiles of malaysian o. hannah venom with a total of  <dig> unique toxins peptides identified encoded for  <dig> protein families.
three-finger toxins are the most abundant toxin family in the moh venom .
3ftx, three-finger toxin; laao, l-amino acid oxidase; svmp, snake venom metalloproteinase; crisp, cysteine-rich secretory protein; cvf, cobra venom factor; pla <dig>  phospholipase a2; 5’nuc, 5’nucleotidase; kun, kunitz-type protease inhibitor; svsp, snake venom serine protease; pde, phosphodiesterase; ngf, nerve growth factor; plb, phospholipase-b; ache, acetylcholinesterase
based on the proteomic data, transcript sequences of translated major toxins were selected for sequence comparison with related toxins.
the sequence alignments of the major transcripts were illustrated in the following figures according to their respective toxin families: 3ftxs , svmps , crisps , laaos , vespryns , pla2s  and cvfs .
figure  <dig> shows the phylogenetic tree of the major 3ftxs of moh.fig.
<dig> sequence alignments of three-finger toxin  transcripts of malaysian o. hannah venom-gland transcriptome.
a malaysian o. hannah 3ftxs were aligned to show disulfide bonding .
b l-3ftxs were aligned and compared to sequences from balinese king cobra  <cit>  and public database .
c s-3ftxs were aligned and compared to sequences from balinese king cobra  <cit>  and and public database
fig.
<dig> phylogenetic tree of selected 3ftx transcripts of malaysian o. hannah constructed with mega  <dig> using the maximum likelihood method and presented by figtree  <dig> .
.
neurotoxin- <dig> androctonus australis used as outgroup
comparison between transcriptome and proteome
in this study, data from the venom-gland transcriptome  and the venom proteome  derived from the same specimen did not show significant correlation .
table  <dig> summarized the quantitative results of the transcripts and proteins by toxin family for the malaysian king cobra studied.
in addition, the “–omic” information available for the balinese king cobra  <cit>  was included along in table  <dig> for comparison purposes.
of note, the transcripts by protein family for the balinese specimen were denoted as present or absent as quantitative data on transcript abundances were not available  <cit> .
in addition, the expression of the percentage of toxin family in the venom proteome is different between the two studies: this current study  reports the relative abundance of protein in each toxin family ; whereas the other   <cit>  indicated the relative number of toxin subtypes without estimation of protein abundance.
sequences of toxin transcripts of the two king cobras were compared and compiled in additional file  <dig> table  <dig> comparison of the venom-gland transcriptomes and the venom proteomes of king cobra  from malaysia  and indonesia
athe relative abundance of a particular transcript was expressed in percentage of total fpkm of all toxins.
values shown are percentages sorted according to toxin gene families
bthe detection of transcript sorted according to toxin gene family.
exact quantitative values were not provided in the original report.
√ indicates detection
cthe relative abundance of a particular toxin was expressed in percentage of total mean spectral intensities of all toxins .
values shown are percentages sorted according to toxin protein families.
the numbers of subtype within each family are indicated in brackets
dthe relative ‘presence’ of a particular toxin was expressed in percentage of the total number of all toxin subtypes .
values shown are percentages sorted according to toxin protein families.
the numbers of subtype within each family are indicated in brackets
discussion
venom-gland transcriptome
high redundancy was observed overall in the toxin genes, indicating that multiple toxin isoforms are highly expressed in the venom gland in contrast to non-toxin genes.
the phenomenon supports the hypothesis that the toxins were products of repeated gene duplications which underwent molecular adaptation for subsequent neofunctionalization: the evolutionary mechanism proposed to be behind the astonishing molecular diversity of snake venoms  <cit> .
sixty three of the  <dig> toxin transcripts show high homology with sequences previously reported for ophiophagus hannah, while the other sequences  are at variable degrees of similarity to toxins from other taxa, consistent with the view that toxin genes expanded from a limited pool of protein families among the advanced snakes  <cit> .
the moh venom-gland transcriptome reflects the use of venom as an innovated predatory strategy.
the role of 3ftxs as the principal toxins in moh venom is supported by the fact that they are the most abundantly expressed and the most isomerically diverse toxin genes.
the 3ftx mrna expression level in our study appears to be higher  than that reported by vonk et al.
<cit>  in a balinese specimen  .
a total of  <dig> unique 3ftx transcripts are identified and classified as short-chain , long-chain  and weak  neurotoxin isoforms, as well as neurotoxin-like hypothetical proteins  .
in king cobra envenomation, the neurotoxins are thought to be the major pathogenic cause of rapid death mediated through neuromuscular paralysis and respiratory failure, attributable to the curare-like neurotoxins of the 3ftx family  <cit> .
from the venom-gland trasncriptome, high expression levels were also noted for the genes of svmps and pla2s .
svmps and pla <dig> are two enzymes commonly present in snake venoms across the elapidae and viperidae  <cit> .
the svmp genes of moh appear highly diverse ; the multiple isoforms are suggestive of the vast biological activities of the enzyme.
the pla <dig> transcripts, on the other hand, are comprised of only  <dig> distinct sequences, representing a finding of low sequence diversification similar to that in laaos .
the enzymatic products of these genes are not usually involved directly in lethality but play ancillary roles in causing tissue inflammation and destruction .
meanwhile, approximately  <dig> % of the non-toxin genes appear unrelated to envenomation.
most of these are housekeeping genes associated with cellular metabolisms, and they are transcribed at very low fpkm levels, marked with low redundancies.
venom proteome
when used in combination, the next generation transcriptomic and proteomic analyses can provide a powerful approach to unveil the molecular complexity of venom toxins.
this approach was adopted in the current study for a better correlation of the two processes of gene expression and protein synthesis in the venom glands of the malaysian king cobra.
among the many proteomic methods, we employed a sequential method of 1d-sds-page followed by in-gel digestion and nano-esi-lcms/ms.
a simpler method of whole venom in-solution lcms/ms is time-saving, cost-friendly, and has been tested out in several venomic studies  <cit> ; however, additional steps of venom protein separation such as sds-page prior to lcms/ms are known to be effective to “de-complex” the venom and thus greatly enhance the protein identification by lcms/ms  <cit> .
in this study, the 1d-sds-page pattern of moh venom is similar to the observation reported in chang et al.
<cit> .
the authors deduced that the prominent bands of king cobra venoms were represented by laaos and svmps , crisps , pla2s and 3ftxs , without reporting their relative abundances.
our approach in examining the protein composition throughout the electrophoretic passage has made it possible to estimate the relative abundance of these toxins, in addition to the detection of several other proteins.
while the genomic disclosure by vonk et al.
<cit>  has been an excellent reference for the evolution and toxin diversity of the balinese king cobra venom, the individual toxin abundance has not been reported.
our current study that revealed the relative abundance of toxins in moh venom is thus hoped to provide further insights and correlation between the tissue transcriptome and toxin expression profiles in king cobra.
it is however worth noting that toxin variations can still exist between venoms of different geographical origins.
it is interesting to note that 3ftxs and pla <dig> in moh venom made up approximately  <dig> % of the overall abundance, apparently less than in the venoms of local elapids such as naja cobras and kraits, where the total abundance of these two major toxins can be as high as 70- <dig> %  <cit> .
instead, a substantial amount of svmps , crisp  and laaos  were found expressed in the moh venom.
a hypothesis for the difference could be that the non-neurotoxin components act synergistically in moh venom in order to subdue neurotoxic elapids such as cobras, which are known to be resistant to neurotoxins  <cit> .
comparison of transcriptome and proteome
the expression of mrnas and protein abundance are not necessarily identical due to various cellular regulatory processes e.g., post-transcriptional, translational and protein degradation that control the steady-state protein abundances  <cit> .
moreover, the correlation between transcriptomic and proteomic abundances in snake venom has traditionally been poor  <cit> .
it should be noted that in the correlation study, the protein expression is ideally measured based on peptide unit, analogous to the analysis for rna expression described by mortazavi et al.
<cit>  in order to correct for the molecule size of messenger transcript and protein.
using this approach, aird et al.
<cit>  reported a significant correlation of the data  for two east asiatic crotalids, although some variances in the results remained unexplained possibly due to biological as well as analytical factors  <cit> .
our study on the moh specimen, however, showed no significant correlation between the venom-gland transciptome and the venom proteome.
the correlation may be influenced by many factors, one of which is the time span between venom collection and gland tissue harvesting.
in this study, the venom glands had been stimulated by venom milking several days prior to their harvesting as a step to maximise the mrna synthesis.
admittedly, toxin components can be synthesized at different rates and hence the respective metrics of mrna and protein expressions taken at one particular time point may not be well correlated.
in addition, extensive post-translational modifications occur in many complex toxins such as svmps; hence the mature proteins produced may not be necessarily congruent to the mrna expressed.
these factors may explain some of the variances in the data presented here and elsewhere  <cit> .
in the following section, we further discuss the expression of moh toxins according to their protein families.
major transcripts and venom constituents
three-finger toxins
three-finger toxins  comprise a family of non-enzymatic polypeptides of 60- <dig> amino acid residues with four or five disulfide bridges  <cit> .
3ftxs are usually the principal toxins found in the venoms of elapid snakes such as king cobra, naja cobras, coral snakes, kraits, mambas and sea snakes  <cit> .
the genes may have been recruited before the divergence of different venomous snakes, as low levels of 3ftx transcripts have been reported in the venom-gland transcriptomes of some viperids and colubrids .
the classification of 3ftxs in general is made based on the number and position of disulfide bonds.
short-chain 3ftxs  possess only four disulfide bonds, while long-chain 3ftxs  have five with an additional disulfide bond at the distal end of loop ii.
“non-conventional 3ftxs”  also have five disulfide bonds, with the fifth bond located within loop i of the molecule  <cit>  .
the molecular diversity of 3ftx genes of moh is well reflected by the presence of multiple unique 3ftx transcripts.
the two main 3ftxs of moh, moh_ftx <dig> and moh_ftx <dig>  share sequence consensus to most curare-like α-neurotoxins as shown in fig.
5b.
moh_ftx <dig> is similar to an uncharacterized 3ftxs  in the database with an unknown mechanism of action.
l-3ftxs constitute the majority of 3ftx transcripts in the venom gland, notably moh_ftx <dig>  .
interestingly, moh_ftx <dig>  a long-chain neurotoxin, has an n-terminal peptide sequence  not seen in other l-3ftxs but identical to that of a weak neurotoxin, wntx- <dig> .
the sequence in the central core of moh_ftx <dig> is nevertheless similar to most other long-chain neurotoxins.
it is known that the functional sites of long-chain neurotoxins e.g., α-cobrotoxin/elapitoxin from naja kaouthia reside mainly in the second loop of the toxin, with important residues of k <dig>  d <dig>  r <dig> and k <dig>  while the first loop is not involved in the toxin function  <cit> .
moh_ftx <dig> and moh_ftx <dig> were found to contain all the critical residues except that the major l-3ftxs, moh_ftx <dig> showed substitution of k <dig> with glutamine and k <dig> with asparagine.
the effect of variations on these critical residues were unknown.
among the s-3ftx transcripts identified, the three most abundantly expressed isoforms  are homologous to neurotoxin-like protein  <dig>  hypothetical protein l345_ <dig>  and muscarinic toxin-like protein  <dig> homolog, respectively .
these transcripts belong to the 3ftx “orphan group” ; with the exception of the muscarinic toxin-like protein, the functions of the other two neurotoxin-like proteins are not well understood  <cit> .
a similar type of transcript has also been reported in balinese o. hannah  <cit> , suggesting that some atypical 3ftxs are conserved .
site-directed mutagenesis and nicotinic cholinergic receptor  binding studies using the short-chain α-neurotoxin, erabutoxin a , revealed that the functional sites of the molecule are distributed over the tips of all three loops in the toxin, including s <dig> and q <dig> in loop i and k <dig>  w <dig>  d <dig> and r <dig> that play important roles in short-chain neurotoxin selectivity and affinity to nachr  <cit> .
in contrast, the functional site of l-3ftxs lies mostly within the second loop of the toxin.
the lack of the critical resides q <dig> in moh_ftx06; q <dig> and w <dig> in moh_ftx07; q <dig> and k <dig> in moh_ftx <dig>  implies their potential structural-function differences in the moh venom.
although they are categorized as s-3ftxs , the unusual n-terminal amino acid sequences presented in these transcripts may influence the toxin-receptor binding as the tip of the first loop of the typical short-chain neurotoxin, ea has been shown critical for its binding with the receptor  <cit> .
three other s-3ftx transcripts encode a typical short-chain neurotoxin sntx <dig> , a type a muscarinic toxin  and a cytotoxin, cardiotoxin- <dig> , all at extremely low expression levels .
their roles in envenomation have not been elucidated through functional studies.
on phylogenetic analysis, s-3ftx, l-3ftx and nc-3ftx were grouped into separate clusters on the phylogenetic tree .
genes for both the s-3ftxs identified in moh venom proteome are basal in the phylogenetic relationship.
the finding supports that 3ftx genes of moh evolved from short-chain isoforms which later adapted and diverged into genes encoding non-conventional 3ftxs and long-chain 3ftxs.
the phylogenetic analysis also showed that the main l-3ftx, moh_ftx <dig> is more related to the long-chain neurotoxin oh55/ <dig> of the balinese specimen  <cit> , but distantly diverged from α-cobrotoxin/elapitoxin, indicating their distinctness from the long-chain neurotoxins of naja cobra .
on the venom proteome, the number of expressed moh 3ftxs in this study  is less than that reported for the balinese specimen   <cit>  .
it is however comparable to the number of 3ftxs reported for the thai specimen   <cit>  and is more diverse than that of the other two malaysian king cobra samples studied using chromatography separation   <cit>  and 2de method   <cit> .
the discrepancies could be due to the use of the sensitive nano-lcms/ms in our study, or geographical variations of the venom within malaysia .
the current study further showed that the l-3ftxs and s-3ftxs account for  <dig>  % and  <dig>  % of total 3ftx abundance in the proteome, while the non-conventional 3ftxs constitute the least  .
in term of all protein abundance, the amount of 3ftxs in moh venom is hence  <dig>  %, a finding comparable to chang et al.
<cit>  , although the 3ftx subtypes varied between these two studies.
while chang et al.
<cit>  concluded that s-3ftxs constituted  <dig> % w/w of their malaysian venom sample with l-3ftxs constituting only  <dig> % w/w, we demonstrated a lesser extreme distribution of s-3ftx:l-3ftx with a ratio of 1: <dig> .
the major form of l-3ftx by toxin abundance is nonetheless same in both studies, viz.
oh <dig> .
similarly, the 3ftxs in our moh venom appear to be primarily neurotoxins of variable isoforms, with a very minimum content of cytotoxin or cardiotoxin , a finding comparable to chang et al.
<cit>  .
interestingly, muscarinic toxin-like proteins  and several nc-3ftxs were expressed in moh venom at trace amounts; these isoforms have not been reported in the previous studies of malaysian specimens  <cit> .
the cytotoxin and non-conventional 3ftxs of low abundance in moh venom likely play minimal role in the lethal action of the venom in view of their minute amount and the higher ld <dig> value  compared to alpha-neurotoxins  <cit> .
the proteomic presence of long neurotoxin oh <dig> in the moh venom also contrasts with the previous conclusion  <cit>  which proposed this to be one of the unique 3ftx markers exclusive for chinese king cobra  and not supposed to be present in the southeast asian specimens.
our finding hence indicates that the 3ftx profile of king cobra may be more complex and diverse than previously thought.
nonetheless, the presence of a large number of 3ftx variants in high abundance also implies the possibilities of variable protein structure folding, toxin-receptor interaction and potency.
in addition, the immunogenicity of the venom is likely determined by the abundant 3ftxs; the small molecular sizes and diverse epitopes of 3ftxs can be a challenge to the production of a high efficacy antivenom.
using a computerized model to analyse long ntx sequences retrieved from uniprotkb database, danpaiboon et al.
<cit>  predicted that a total of  <dig> amino acids in a king cobra long ntx  interacted with the human single-chain variable antibody fragments specific to n. kaouthia long ntx  at the following  <dig> amino acids: c <dig>  w <dig>  g <dig>  r <dig>  k <dig> in loop  <dig>  and r <dig>  n <dig> in loop  <dig>  hence supporting the apparently weak cross-neutralization effect of nkln-huscfv against the lethal effect of king cobra  in mice.
however, matching of the said interactive amino acids of q2vbp <dig> to the major ntxs  in moh venom  yielded limited similarity .
this suggests that nkln-huscfv may not be effective in neutralising the king cobra venom from malaysia.
from the practical standpoint, leong et al.
<cit>  confirmed in a mouse model that thai naja kaouthia monovalent antivenom  has low neutralization capacity against moh venom, indicating the antigenic dissimilarities of both venoms despite the conserved protein scaffolds between neurotoxins of moh and n. kaouthia.
this is not surprising considering the fact that the antivenom manufacturer has to combine venom immunogens from both king cobra and n. kaouthia  in order to produce the effective neuro polyvalent antivenom .
clinically, an attempt to treat malaysian o. hannah envenomation in kuantan, malaysia with nkmav has proven in vain as the antivenom against n. kaouthia failed to cross-neutralize both the systemic and local effects from the bite  <cit> .
in the moh envenoming case, the systemic effects were only arrested much later with the arrival and administration of the king cobra-specific antivenom.
we postulate that the neurotoxins of moh are highly distinct to those of naja cobras’ as a result of adaptive evolution and positive selection to ensure that the moh neurotoxins are sufficiently effective to bind naja’s nicotinic receptor since naja cobra is a prey top in king cobra’s food list – a known predator–prey phenomenon in palm oil estates in malaysia.
generally, naja cobra is resistant to ntx as their receptors have been mutated with a sugar chain that masks the binding by ntx  <cit> .
in addition to distinct neurotoxins, the moh venom proteome also reveals abundant high molecular mass proteins  not commonly present in large amount in naja cobra venoms, implying their potential roles in killing and digestion of other neurotoxic snakes by king cobra.
the higher share of non-3ftx components  in the moh venom may be a reason for the venom’s higher ld <dig>  in mice compared to that of naja venom  that is predominated with alpha-neurotoxins  <cit> .
this is supported by an earlier report  <cit>  where the major neurotoxic fraction of moh venom, purified by ion-exchange chromatography and accounted for ~ <dig> % venom weight, has an i.v.
ld <dig> of  <dig>  μg/g mice, a value disproportionally lower than that of the crude venom .
snake venom metalloproteinases
from the moh venom-gland transcriptome, the svmp transcripts encode at least  <dig> unique svmps, with sequence similarities matched to svmp-ohanin, svmp-cobrin, svmp-bmmp, svmp-kaouthiagin, svmp-mocarhagin- <dig>  svmp-atrase-a, chain-a-svmp, asrin and carinatease- <dig> from different elapid snakes .
the sequence divergence is evident, supporting the view of extensive diversification of svmp functions under accelerated evolution  <cit> .
all the identified svmp transcripts belong to the p-iii class  domains), the svmp type that has a rich array of biological activities, including potent haemorrhagic activity, inflammation, apoptosis, prothrombin activation, and inhibition of platelet aggregation  <cit> .
from the svmp transcript sequences of moh, we identified the absence of the seventh cysteine residue in domain m that takes part in the disulfide bond exchange for autolysis during secretion or formation of the biologically active dc domain, typical for viperidae svmp-piiis  <cit> .
figure  <dig> shows that the moh and annotated svmp-piii  m domains from several elapids have only six cysteine residues, consistent with previous studies  <cit>  including that for balinese king cobra  <cit> .
the effect of structural difference as such has been shown by guan et al.
<cit>  that no autolytic activity was observed during the preparations of two p-iii svmps sourced from the taiwanese naja atra.
thus, the svmp-piiis of malaysian o. hannah and most elapids likely exhibit the similar structure-function properties distinct from those of viperids.fig.
<dig> sequence alignments of snake venom metalloproteinase  transcripts of malaysian o. hannah venom-gland transcriptome in comparison with svmp sequences of representative venomous snakes.
some proteomic studies indicated that svmps are expressed mainly in the viperidae and colubridae venoms, and in a relatively smaller amount in elapidae venoms  <cit> .
our findings revealed that svmps are present in a substantial amount in the moh venom, with a total of  <dig> proteins detected.
our finding of diverse and abundant svmps in moh venom is in agreement with vonk et al.
<cit>  on the view that svmps are important pathogenic toxins besides 3ftxs in the balinese king cobra venom.
this is also supported by chang et al.
<cit>  who reported high metalloproteolytic enzyme activity in king cobra venoms, although the authors did not determine the subtypes and composition of svmps in the venoms.
on the other hand, svmp was not identified at all in the previous 2de proteomic study of malaysian king cobra  <cit> , a rather peculiar finding that raises a conflict in local venom database, possibly due to inadequacy of the methodology.
earlier studies indicated that at least five svmp isoforms with proinflammatory and oedema-inducing activities were present in king cobra venoms, two of which were proven to be hemorrhagic proteases  <cit> .
the major hemorrhagic protease, termed hannahtoxin, is a 66-kda zinc-dependent metalloprotease that exhibited species-specific lethality, where it is highly lethal to rabbit but not to mice at comparable doses  <cit> .
although local hemorrhage is not a well-recognized clinical feature of king cobra envenoming, the proteolytic and proinflammatory activities of svmp may be involved in cytotoxicity leading to necrosis and tissue destruction.
cysteine-rich secretory proteins
multiple sequence alignment  of the  <dig> crisp transcripts shows that the sequences are highly conserved across different snake species and the complete sequence of moh_crp <dig> obtained is almost identical to ophanin sequence derived from uniprotkb database, including its functional region known for the inhibition of smooth muscle contraction  <cit> .
this is in agreement with the transcriptomic finding reported for the balinese king cobra, where crisp is found with limited diversity and little evidence of gene duplication  <cit> .
this gene family likely has conserved functional activities across most lineages, and does not participate in the evolutionary arms race seen in the more pathogenic toxin families such as 3ftx.fig.
<dig> sequence alignments of cysteine-rich secretory protein  transcripts of malaysian o. hannah venom-gland transcriptome in comparison with crisp sequences of representative venomous snakes.
the presence of crisps in king cobra venom proteome varies between studies.
crisp is not reported in two previous studies on the venom proteome of malaysian king cobra  <cit> .
in the present study on moh venom, one crisp isoform was identified, consistent with the finding from the thai specimen  <cit>  but varied from the balinese which recorded a total of  <dig> crisp isoforms  <cit> .
in moh venom, opharin precusor is the identified crisp isoform.
this toxin has the putative function of reducing smooth muscle contraction that is elicited by high potassium-induced depolarization.
the degree of inhibition by opharin is however lower than most other crisps  <cit> , possibly due to the critical amino acid difference essential for channel activity inhibition: as with ophanin, the translated moh crisp possesses y <dig> in substitution of f <dig>  and y <dig> instead of e <dig>  .
although the pharmacological activity of ophanin as a weak antagonist of smooth muscle contraction has been characterized, its exact role in envenomation remains elusive especially in moh which venom contains a considerable amount of this toxin.
l-amino acid oxidases
the laao transcripts revealed by the moh venom-gland transcriptome consist of the three main functional domains  <cit>  and share high sequence similarity with sequences of other king cobras  .
the major transcript sequence  in our study is highly homologous to the sequence of hypothetical protein l345_ <dig> from the balinese king cobra  <cit>  and king cobra oh-laao .
unlike 3ftxs , laaos appear rather well conserved within king cobras from different regions.fig.
<dig> sequence alignments of l-amino acid oxidase  transcripts of malaysian o. hannah venom-gland transcriptome in comparison with laao sequences of representative venomous snakes.
snake venom laaos are reported present natively as a homodimer with molecular mass around  <dig> kda, while under reducing conditions, present as subunit of around 50- <dig> kda  <cit> .
we showed in our proteomic study that the laaos of moh venom distributed mainly in gel section  <dig> corresponding to 55- <dig> kda .
despite the relatively lower mrna expression, the protein comprises close to  <dig> % of moh venom , comparable to the approximation of  <dig> % w/w in an earlier study of malaysian king cobra laao  <cit> .
the peptide sequences exhibit low variability with only two isoforms identified in the proteome, comparable with the thai and balinese samples where one laao subtype was identified  <cit> .
our finding is also in agreement with chang et al.
<cit>  who reported substantial laao activity in four king cobra venoms of different localities, although the paper reported neither the compositional subtype nor the abundance of this enzyme.
these studies however strongly disagree with another proteomic study on a malaysian king cobra sample where no laao was detected at all  <cit> .
the substantial amount of laao in the moh venom proteome  suggests that the toxin may serve beyond ancillary function.
its role in king cobra predation and envenomation may be related to its potent proinflammatory and cytotoxic activity  <cit>  that can induce extensive tissue destruction.
in addition, the functional uniqueness of king cobra laao has been shown on its substrate specificity compared to other snakes, where its heat-stable property contributes greatly to its potential for biomedical application  <cit> .
notably, laao from the malaysian king cobra has been shown to exhibit potent in vitro and in vivo selective cytotoxicity  <cit> .
further characterizations of king cobra laao is therefore a feasible and promising research direction towards cytotoxic drug discovery.
vespryn
the complete sequence of the only vespryn transcript in moh matches identically to the ohanin sequence .
the sequence, covering signal and mature chain, is also identical to the corresponding sequence regions reported for the balinese specimen  <cit>  .
the balinese king cobra vespryn sequence, however, contains additional pre-signal  <dig> amino acid residues , the significance of which is uncertain.
in contrast to other members of snake venom proteins, ohanin presents a unique single cysteine residue , and the protein sequence shares little similarity to the pry-spry domains  with relatively short n-terminal extension  <cit> .
although thaicobrin  isolated from the monocled cobra, naja kaouthia was deposited in the protein database much earlier than ohanin, there is no published literature and little characterization work has been done.
together with thaicobrin, the distinct sequence of ohanin has made them the first member of vespryns  family sourced from snake venom.fig.
<dig> sequence alignments of vespryn  transcripts of malayan o. hannah venom-gland transcriptome in comparison with vespryn sequences of representative venomous snakes.
pung et al.
<cit>  reported the presence of ohanin at a low abundance  in king cobra venom sourced from jakarta, indonesia.
in contrast, the moh venom proteomic study shows the venom contains a higher amount of ohanin   that were distributed mainly in gel sections  <dig> and  <dig> , corresponding to the known molecular mass of ohanin .
the difference in ohanin content in o. hannah venoms from malaysia  and indonesia  may seem to represent a remarkable geographical variation of the venoms.
phospholipases a <dig>
the two complete pla <dig> transcripts  of moh were annotated to group ia  and ib , respectively, similar to the pla <dig> enzymes in chinese o. hannah venoms  <cit> , and they share the characteristic asn <dig> n-terminus for venom pla <dig> of king cobra .
the major pla <dig>  expressed in the proteome of moh venom is the acidic pla2- <dig> , consistent with tan and saifuddin  <cit>  who reported that the major pla <dig> of malaysian king cobra was an acidic isoform constituting  <dig> % of the venom weight.
the enzyme was however non-lethal in mice at an intravenous dose of  <dig> μg/g but exhibited moderate anticoagulant and oedema-inducing  activities, in contrast to the fujian pla2- <dig>  that was mildly lethal and cardiotoxic in mice  <cit> .
remarkable variations are noted within the group i pla <dig> among king cobra venoms of different origin: while we identified group ia pla <dig> from moh, huang et al.
<cit>  isolated only group ib pla <dig> from a fujian chinese sample; vonk et al.
<cit>  identified two each for group ia and group ib from a balinese sample; vejayan et al.
<cit>  identified one each of group ia and group ib from a malaysian sample; danpaiboon et al.
<cit>  identified  <dig> group ia and  <dig> group ib pla <dig> from a thai sample.
with the inconsistency of king cobra pla <dig> subtypes identified, the comparative analysis is further compounded by undetermined toxin abundance in each study.
chang et al.
<cit>  showed with quantitation in another malaysian sample the presence of  <dig> group ia and  <dig> group ib pla <dig> subtypes, however each with exceptionally low abundance ranging from  <dig> - <dig>  % only, indicating that the total pla <dig> content in the venom was far less than  <dig> %  <cit> .
the intraspecific pla <dig> variations are therefore vast and may be related to not only geographical distribution but also ontogenic factors.
the current proteomic study, nonetheless, shows that the moh major pla <dig> is the acidic, proinflammatory but non-lethal variant of group ia pla <dig>  it constitutes a small portion of the malaysian king cobra  venom, approximately  <dig> times less than pla2s found in the regional naja cobra venoms   <cit> .
in view of the negligible cytotoxin content and small amount of pla <dig>  local tissue necrosis that occurs in moh envenoming is possibly induced by other cytotoxic components in the venom, for instances, svmps and laaos that exist at a high abundance.fig.
<dig> sequence alignments of phospholipase a <dig>  transcripts of malaysian o. hannah venom-gland transcriptome in comparison with pla <dig> sequences of representative venomous snakes.
cobra venom factors
the trasncripts of cobra venom factors from the moh transcriptome are highly homologous to ovf, ophiophagus venom factor, the cvf first purified from the venom of a chinese  king cobra  <cit> .
interestingly, the ovf transcripts in moh transcriptome reveal  <dig> amino acid residues  absent in the reported sequence for guangxi ovf, but present identically in that of the balinese specimen  <cit> .
this suggests that the structures of ovf from king cobras of the two southeast asian regions share high similarity and are sufficiently distinct from that of the china specimen.
in addition, two novel regions  not reported previously in cvf/ovf have been identified in the ovf transcript of moh .
blast analysis of these peptides retrieved no identifiable protein or peptide region.
the role of the additional amino acid sequences in the structure-function relationship of malaysian cvf/ovf remains to be investigated.fig.
<dig> sequence alignments of cobra venom factor  transcripts of malaysian o. hannah venom gland transcriptome in comparison with cvf sequences of representative venomous snakes.
the number of cvf subtypes expressed in the moh venom  is comparable to that reported in the balinese specimen   <cit> , but more diverse than the thai specimen and another malaysian sample  <cit> .
cvf is a protein with high molecular mass  and it dissociates into α, β and γ chains under reducing condition  <cit> , consistent with our findings where the gel sections  <dig>   <dig>   <dig> and  <dig>  corresponded to the reported sizes of α , β  and γ chain .
cvf is not directly lethal; it facilitates the absorption and spread of venom from the bite site, as cvf-activated local complement system releases anaphylatoxins c3a and c5a that increase the regional vascular permeability and blood flow  <cit> .
low-abundance transcripts and toxins
toxins constituents expressed in both venom-gland transcriptome and venom proteome
kunitz/bpti serine protease inhibitors  transcripts consitutute  <dig>  % of toxin fpkm, while one kun was expressed into the venom proteome at  <dig>  % of toxin abundance.
the expressed kun protein is similar to oh-tci, the first reported kun from snake venom that exhibited equivalent trypsin and chymotrypsin inhibitory activities  <cit> .
in addition, cystatin, an inhibitor of various c <dig> cysteine proteases that may play a role in preventing toxin auto-digestion  <cit> , was detected at a low amount in the transcriptome  and proteome .
in comparison, the balinese and thai king cobra venoms contain, respectively,  <dig> and  <dig> kun subtypes but cystatin has not been reported  <cit> .
the exact function and action of kuns remain to be elucidated.
from the moh venom-gland transcriptome, we have identified  <dig> transcripts  that encoded neprilysin, a metalloendopeptidase with affinity for a vast range of physiological targets, including natriuretic and vasodilatory neuropeptides for their regulation  <cit> .
novel metallopeptidases exhibiting similarity to neprilysin was first demonstrated in the venom-gland transcriptomic study of echis sp.
<cit> , and recently the neprilysin transcript was revealed too from the venom gland of the balinese king cobra, but its expression in the king cobra venom proteome was not reported  <cit> .
in this study, we showed that neprilysin is present in the venom proteome of malaysian king cobra, although its identity and function as a secretory toxin in the venom are still elusive at this stage of venom study.
from the venom gland transcriptome, three transcripts were identified for encoding snake venom serine proteases  at rather low fpkm .
through lcms/ms and homology search, the expressed peptides were noted similar to alpha- and beta-fibrinogenase ohs <dig> .
in comparison,  <dig> svsps and  <dig> kallikrein were identified from the venom proteome of balinese and thai specimens, respectively  <cit>  but not reported in the other two malaysian samples  <cit> .
the serine fibrinogenase in moh venom has a putative role of a potent fibrinogenolytic and amidolytic agent without haemorrhagic effect  <cit> .
serine proteases at 5- <dig> % in viperid venom is able to elicit significant coagulopathic effect in vivo and in vitro  <cit> ; this effect however has not been a well-recognized clinical syndrome in king cobra envenomation, and laboratory test  using human plasma for the venom’s procoagulant effect has not been positive  <cit> .
in addition to the fibrinogenolytic toxins, phospholipase b  with haemolytic activity from drysdalia coronoides  <cit>  was detected in a minute amount in the moh transcriptome and the venom proteome.
this enzyme was also identified in the venom proteome of the thai specimen  <cit>  but not reported from the other sources  <cit> .
acetylcholinesterases  are usually expressed at low level in snake venoms; the highest abundance ever reported was  <dig>  %  from the genus bungarus   <cit> .
the major isoform of ache transcript from moh exhibits extremely high homology to ache full sequence of bungarus fasciatus , supporting the antigenic cross-reactivity observed between king cobra ache and the monoclonal antibodies of krait ache  <cit> .
one ache  was detected in the moh venom proteome at a relatively low abundance .
the presence of this enzyme in king cobra venom was previously identified only through functional study.
chang et al.
<cit>  showed that the ache activities of malaysian and thai king cobra venoms were several-fold higher than the indonesian and chinese samples, and described an inverse relationship between the ache activity and the neurotoxic fatality of the venom.
however, the postulation of the ‘inverse relationship’ seems contradictory as it was suggested that the immediate acetylcholine hydrolysis at synapses might reduce the competition between the neurotransmitter  and the curare-like neurotoxins, thus potentiating the neurotoxicity.
besides, although venom aches may interrupt musculatory control through rapid degradation of ach, their effect on cholinergic transmission in the central nervous system and autonomic ganglia remains unclear  <cit> .
systemic hypotension may be a possible effect associated with the interruption of cholinergic transmission at the autonomic ganglia, thus favouring prey immobilization.
the transcription of nerve growth factor  genes in the moh venom glands is consistent with the findings for the balinese king cobra  <cit> .
a small amount  of ngf was also detected in the moh venom proteome.
of note, the proteomic presence of this minor toxin in king cobra venoms has not been reported nor quantitated previously  <cit> .
it has been suggested that ngf can exhibit arginine esterase activity that contributes to hypotensive effect through nitric oxide liberation and histamine release  <cit> .
interestingly, this is a group of snake venom constituents, after the widely-studied anticoagulant, antiproliferative and antimicrobial toxins, shown to have therapeutic potential for neurodegenerative brain disorders, e.g., alzheimer’s disease  <cit> .
phosphodiesterase  and 5’-nucleotidase were two other minor toxins detected in both the venom-gland trasncriptome and venom proteome of moh.
both the enzymes were detected in the venom proteome of thai king cobra  <cit> , while only pde was identified from the balinese specimen  <cit> .
these enzymes primarily act to liberate nucleosides which may help in prey immobilization .
venom phosphodiesterases are known to cause locomotor depression and reduction of mean arterial blood pressure  <cit> .
together with 5’nucs and toxins with hypotensive and immobilising potentials such as ovf, ache and ngf, these components may act synergistically to enhance prey subduing.
in comparison with the study on balinese king cobra  <cit> , we noticed some variations in the “–omic” findings between king cobras from the two geographical regions .
besides the variabilities of sequence and abundance described, we also found the inconsistent presence of a number of toxins between the two specimens.
for instance, ngf, ache, neprilysin, plb, cystatin and 5’-nucleotidase present in the moh proteome were not reported in the balinese specimen proteome  <cit> .
conversely, natriuretic peptide  and vascular endothelial growth factor  were detected in the venom proteome of the balinese specimen  <cit>  but absent in the moh although the expression was detected at low mrna level.
one interesting, consistent feature shared by both king cobras from the malayan peninsula and bali island is the absence of lectin in the venom proteome in spite of their gene expression in the venom glands.
this observation supports that lectins do not contribute to king cobra envenoming  <cit>  which is in contrast to many other venomous snakes particularly from the viperidae family  <cit> .
in addition, although the two specimens did not differ remarkably in the composition of major pathogenic toxin components e.g., 3ftxs and svmps, variations are noted in the gene sequences  and possibly proteomic abundances , indicating intra-specific venom diversity that may lead to variations in immunological profile and antivenom effectiveness.
toxins not translated from venom-gland transcriptome
twenty seven toxin transcripts were identified only at the transcriptional level of moh venom glands.
these transcripts encoded protein genes for insulin growth factors , natriuretic peptides , hyaluronidase, dipeptidylpeptidase iv , vascular endothelial growth factors , waprins and c-type lectins , totalled to  <dig>  % of all toxin fpkm.
the activities of these toxins have not been previously investigated in king cobra venom.
it is an assumption that their occurence in the venom is extremely low or negligible since the peptides of these proteins were not detected by the high resolution tandem mass spectrometry.
transcripts of igfs have been previously categorized into secretory toxin transcripts by vonk et al.
<cit> , although its toxic role is uncertain to date.
at the proteomic level, this toxin was also not reported in both moh and the balinese king cobra venoms, but its presence was detected along with a hepatocyte growth factor in the thai specimen   <cit> .
natriuretic peptide, a hormone that controls over natriuresis, diuresis, blood pressure, homeostasis and inhibition of aldosterone secretion in all vertebrates was not identified in the proteomes of both moh and thai  <cit>  specimen, but reported in the balinese specimen proteome  <cit> .
this is another physiological protein which gene was likely recruited earlier into the toxin arsenal; the expression however has been low among venomous snakes.
besides, vegfs, potential vasodilators, were expressed in the venom proteome of the balinese specimen  <cit>  but never reported in moh and the other king cobra venoms studied  <cit> .
hyaluronidase, dpp-iv, waprin and ctl were consistently not reported in the venom proteomes of king cobras  although the genes were transcribed in the venom glands  <cit> .
interestingly, the enzymatic activity of hyaluronidase has been reported previously in king cobra venom  <cit>  but at a level much lower than that of naja sp., bungarus sp.
and viperid venoms.
in this study, a full sequence of hyaluronidase from moh venom gland transcriptome was identified.
its high homology to sequences of viperid snakes suggests that the gene and protein function appear to be conserved across many venomous lineages despite its trivial expression in the venom.
dpp-iv, waprin and ctl have not been reported thus far in king cobra venom proteomes; but it remains interesting for further study as complete sequences of these toxins have been made available from the moh venom-gland transcriptome .
the expression of these genes without translation into proteins indicate their early recruitment into the venom-gland transcriptome, however, adaptation and selection have favored the expression of other toxins to suit the predatory arm-race and ecological niche changes for the king cobras.
an alternative possible explanation for this is the convergence of some genes which begin to emerge as potential contributor of toxins in adaptation to the changing niche.
data of these sequences may serve useful purposes for biodiversity study and drug discovery in the future.
CONCLUSIONS
this study demonstrates complex and diverse composition of malaysian king cobra venom at both the transcriptomic and proteomic levels.
unlike venoms of southeast asian naja sp.
that mainly comprise neurotoxins, pla2s and cytotoxins, moh venom contains a higher degree of toxin complexity predominated with neurotoxins and svmps, in addition to appreciable amounts of crisp, laao, cvfs, vespryn, pla <dig> and ngf.
additionally, the moh venom contains small amounts of neprilysins and ache which may have synergistic action on the neurotoxicity of the venom.
the findings also suggest the presence of toxins that may induce hypolocomotive and hypotensive effects in the venom, although clinically humans are susceptible more rapidly to neuromuscular paralysis due to the faster absorption of neurotoxins and their intrinsic toxicity with a rapid onset  <cit> .
the unique toxin composition of king cobra venom may serve to aid this ophiophagic species in preying on other snakes especially naja cobras which are known to be neurotoxin-resistant  <cit> .
as anticipated, intra-species variation of venom is not uncommon, and this is exemplified by the king cobras among the peninsular malaysian , the thai and the insular balinese specimens.
we propose the need to address the medical concerns of such phenomenon, where comparative studies in the future should aim to provide variation details in terms of the sequence diversification and the relative abundance of toxins between venoms of different geographical sources.
the integrated transcriptomic-proteomic knowledge as such is fundamental for deeper insights into the mechanisms of snake venom toxicity and the natural history of venom evolution.
the knowledge may provide crucial information for optimizing the production of an effective pan-regional antivenom for related species in the future.

 toxin proteins appeared more than million years ago in the common ancestor of venomous reptiles such as snakes and their saurian cousins. the prevailing thoughts on venom evolution agree that toxin genes were initially recruited from genes of proteins with normal physiological functions, followed by repeated gene duplication that creates redundancy, allowing a gene copy to be selectively expressed in the venom gland. the ‘free’ copies subsequently underwent neofunctionalization through positive selection and molecular adaptation at accelerated rates, driven primarily by changes in the ecological niche, diet and predator–prey arms race. besides gene duplications, alternative splicing and alterations of domain sturctures are other mechanisms capable of generating novel toxin genes. the emergence of paralogous groups of multigene families across taxonomic lineages is accompanied by the occurrence of multiple isoforms within each major toxin family, resulting in diverse functional variations of venom proteins that have great impacts on medical management and toxinological research. the immerse variety of toxins also serves as a source of leads for drug discovery. from a clinical standpoint, the phenomenon gives rise to diverse snakebite syndromes and potentially suboptimal antivenom effectiveness. the phenomenon complicates snakebite management that relies heavily on the understanding of venom complexity and the availability of effective antivenom. in most developing and under-developed countries, snake envenomation remains a neglected tropical disease and a disease of poverty. malaysia, as a tropical country that is home to many venomous snake species, shares similar public health concerns. the problem affects not only the rural populations but also the suburban regions due to rapid urbanization as the human populations increasingly encroach upon snake habitat. among the native snakes in malaysia, the king cobra is a known cause of envenomation, partly due to its popularity among snake hobbyists. the king cobra is the world’s largest venomous snake, capable of delivering huge amounts of venom per bite. clinically king cobra bites cause neurotoxicty, paralysing respiratory muscles and leading to rapid death if untreated. in surviving victims, the bites can result in extensive tissue necrosis and crippling deformity, adding to the heavy toll of human suffering in socioeconomically deprived countries. ophiophagus hannah is widely distributed across the indian subcontinent, southeastern asia and the southern part of china. as the generic name ophiophagus suggests, its diet consists primarily of snakes, although small mammals are also eaten. the ophiophagus is a monotypic genus with only one species recognized currently. however, its vast geographical distribution accompanied by morphological differences suggests potential taxonomic divergence and variation in venom composition. in fact, the venom of king cobra has been studied since the early 1970’s. various components have been isolated and characterized, including l-amino acid oxidases, metalloproteinases, three finger toxins, phospholipases a, ohanin, kunitz-type protease inhibitors and factor x activator. nonetheless, the significance of the origin and intraspecific variation of king cobra venom has only been recently documented. in a study with venom samples sourced from a few countries, differences in venom properties were noted between chinese and southeast asian king cobras. a genomic study of an indonesian king cobra by vonk et al. indicated great diversity in the pla and 3ftx gene families in a single specimen. indeed, intraspecific variations of snake venom are attributed to multiple factors such as diet, geographical distribution, ontogeny, season/climate changes etc., and the phenomenon is hence highly relevant to the king cobra in view of its extremely vast distribution. producing regional or local antivenom would be ideal; unfortunately the production of an affordable and effective antivenom tailored to different areas by local manufacturers would be hampered by financial and technical constraints. thus, a new approach has been recently proposed by the toxinological community to focus on the investigation of specific venom-gland transcriptomes and venom proteomes for the development of pan-regional antivenoms. vonk et al. reported the venom-gland transcriptome and venom proteome of a specimen from the indonesian island of bali. however, the possibility of intraspecific venom variations from king cobra in the distant malayan peninsula cannot be excluded. in addition, the paper reported the numbers of toxin subtypes identified in the venom proteome without the relative toxin abundances. earlier, vejayan et al. reported 2-dimensional gel electrophoresis findings of the malaysian king cobra venom. unfortunately, their mass spectrometric analysis yielded a large number of unidentifiable proteins, whilst only five toxin protein families were determined as well as one non-toxin protein, thioredoxin. the study verified the presence of some previously isolated toxins but shed little light on the venom proteome. another recent report on king cobra venom of probable thai origin profiled the venom as well, detecting protein types/families, but again without the estimation of relative toxin abundance. the 1d electrophoretic gel findings of the thai king cobra venom appeared similar to that reported by chang et al., suggesting that 3ftxs are the major component of king cobra venoms of different localities. however, the diversity in toxin sequences and isoforms as well as variations in toxin abundance could not be ascertained. to remedy the current lack of venom-gland transcriptomic and venom proteomic data for the malaysian king cobra, the current study employed next-generation sequencing technology and nano-liquid chromatography-tandem mass spectrometry for a deeper insight into the toxin diversity of king cobra venom.  sequencing and transcriptome assembly from the primary output statistics of transcript sequencing, a total of, pairs of clean reads passed the illumina quality filter for further de novo assembly using the short reads assembling program, trinity. trinity created contigs, connected to form unigenes in total as shown in fig.  blastx alignment between the unigenes and sequences in the ncbi non-redundant protein database yielded annotated unigenes. after filtering low-frequency transcripts, the assemblies were reduced from to transcripts and categorized into unidentified, non-toxin and toxin groups. of these, toxin transcripts encode full-length toxin sequences. although the toxin group accounted for transcripts only, it is abundantly expressed as much as % of the total expression, whereas the unidentified and non-toxin groups were % and % respectively. toxin transcripts were expressed at extremely higher redundancies compared to non-toxin transcripts.fig. de novo transcriptome assembly with short reads assembling program, trinity. a length distribution of contigs from reads assembly. b length distribution of unigenes generated from connected contigs fig. the malaysian ophiophagus hannah venom gland mrna expression profile. pie charts mainly display the percentage abundance of toxin transcripts. toxins transcripts accounted for % of the total fpkm. three-finger toxins are the most abundant toxin family in the venom gland. 3ftx, three-finger toxin; svmp, snake venom metalloproteinase; pla phospholipase a2; crisp, cysteine-rich secretory protein; kun, kunitz-type proteinase inhibitor; laao, l-amino acid oxidase; cvf, cobra venom factor; igf, insulin-like growth factor; np, natriuretic peptide; svsp, snake venom serine protease; pde, phosphodiesterase; ngf, nerve growth factor; ache, acetylcholinesterase; plb, phospholipase-b; dpp iv, dipeptidylpeptidase iv; ctl, c-type lectin; vegf, vascular endothelial growth factor venom-gland transcriptome the identifiable toxin transcripts represent protein families related to venom toxic functions. the toxin constituents, as indicated by fpkm, are dominated by 3ftxs. the toxin transcript abundances are followed by snake venom metalloproteases, phospholipases a, cysteine-rich secretory proteins, kunitz-type proteinase inhibitors and l-amino acid oxidases. a variety of toxin or protein mrnas is expressed in low abundances, including cobra venom factors, vespryn, natriuretic peptides, snake venom serine proteases, phosphodiesterases, nerve growth factors, acetylcholinesterases, neprilysins, hyaluronidase, phopholipases b, cystatins, dipeptidylpeptidase iv, 5’nucleotidases, waprins, c-type lectins, insulin-like growth factors, and vascular endothelial growth factors. the sequences and parameter details of the transcripts were sorted according to gene families and compiled in additional file venom proteome when examined under reducing % sds-page, moh venom showed three major protein bands each with a distinct molecular mass range: high, intermediate, and low. the gel as shown in fig.  was divided into sections of equal sizes in preparation for in-gel digestion and nano-lcms/ms, and the protein abundances from each section were estimated by gel densitometry for in-gel protein amount correlation. proteins were detected by nano-lcms/ms from all gel sections including the pale-staining ones notwithstanding their low abundances. the moh venom proteins mainly migrated to section and accounting for approximately % of the total venom protein.fig. densitometry quantitative measurement of sds-page gel using myimageanalysis software. a sds-page gel sections cutting scale with protein marker range from ~10kda to 170kda. b gel densitometry section to displayed in percentages a customized reference database was used in data mining to identify peptides sequenced by tandem mass spectrometry. a total of toxins were identified from the moh venom; of these were matched to the transcripts. all toxins were assigned to protein families. 3ftxs and svmps were the most abundantly expressed toxins in the proteome, accounting for % and %, respectively, of the venom. this was followed by crisp, laaos, vespryn, pla and cvfs. protein families with an abundance of less than % each were categorized into “others” and considered as low-abundance proteins. the sequences and parameter details of the proteins identified were sorted according to gel sections and protein families as compiled in additional file fig. protein expression profiles of malaysian o. hannah venom with a total of unique toxins peptides identified encoded for protein families. three-finger toxins are the most abundant toxin family in the moh venom. 3ftx, three-finger toxin; laao, l-amino acid oxidase; svmp, snake venom metalloproteinase; crisp, cysteine-rich secretory protein; cvf, cobra venom factor; pla phospholipase a2; 5’nuc, 5’nucleotidase; kun, kunitz-type protease inhibitor; svsp, snake venom serine protease; pde, phosphodiesterase; ngf, nerve growth factor; plb, phospholipase-b; ache, acetylcholinesterase based on the proteomic data, transcript sequences of translated major toxins were selected for sequence comparison with related toxins. the sequence alignments of the major transcripts were illustrated in the following figures according to their respective toxin families: 3ftxs, svmps, crisps, laaos, vespryns, pla2s and cvfs. figure  shows the phylogenetic tree of the major 3ftxs of moh.fig. sequence alignments of three-finger toxin transcripts of malaysian o. hannah venom-gland transcriptome. a malaysian o. hannah 3ftxs were aligned to show disulfide bonding. b l-3ftxs were aligned and compared to sequences from balinese king cobra and public database. c s-3ftxs were aligned and compared to sequences from balinese king cobra and and public database fig. phylogenetic tree of selected 3ftx transcripts of malaysian o. hannah constructed with mega using the maximum likelihood method and presented by figtree.. neurotoxin- androctonus australis used as outgroup comparison between transcriptome and proteome in this study, data from the venom-gland transcriptome and the venom proteome derived from the same specimen did not show significant correlation. table  summarized the quantitative results of the transcripts and proteins by toxin family for the malaysian king cobra studied. in addition, the “–omic” information available for the balinese king cobra was included along in table  for comparison purposes. of note, the transcripts by protein family for the balinese specimen were denoted as present or absent as quantitative data on transcript abundances were not available. in addition, the expression of the percentage of toxin family in the venom proteome is different between the two studies: this current study reports the relative abundance of protein in each toxin family; whereas the other indicated the relative number of toxin subtypes without estimation of protein abundance. sequences of toxin transcripts of the two king cobras were compared and compiled in additional file table comparison of the venom-gland transcriptomes and the venom proteomes of king cobra from malaysia and indonesia athe relative abundance of a particular transcript was expressed in percentage of total fpkm of all toxins. values shown are percentages sorted according to toxin gene families bthe detection of transcript sorted according to toxin gene family. exact quantitative values were not provided in the original report. √ indicates detection cthe relative abundance of a particular toxin was expressed in percentage of total mean spectral intensities of all toxins. values shown are percentages sorted according to toxin protein families. the numbers of subtype within each family are indicated in brackets dthe relative ‘presence’ of a particular toxin was expressed in percentage of the total number of all toxin subtypes. values shown are percentages sorted according to toxin protein families. the numbers of subtype within each family are indicated in brackets discussion venom-gland transcriptome high redundancy was observed overall in the toxin genes, indicating that multiple toxin isoforms are highly expressed in the venom gland in contrast to non-toxin genes. the phenomenon supports the hypothesis that the toxins were products of repeated gene duplications which underwent molecular adaptation for subsequent neofunctionalization: the evolutionary mechanism proposed to be behind the astonishing molecular diversity of snake venoms. sixty three of the toxin transcripts show high homology with sequences previously reported for ophiophagus hannah, while the other sequences are at variable degrees of similarity to toxins from other taxa, consistent with the view that toxin genes expanded from a limited pool of protein families among the advanced snakes. the moh venom-gland transcriptome reflects the use of venom as an innovated predatory strategy. the role of 3ftxs as the principal toxins in moh venom is supported by the fact that they are the most abundantly expressed and the most isomerically diverse toxin genes. the 3ftx mrna expression level in our study appears to be higher than that reported by vonk et al. in a balinese specimen. a total of unique 3ftx transcripts are identified and classified as short-chain, long-chain and weak neurotoxin isoforms, as well as neurotoxin-like hypothetical proteins. in king cobra envenomation, the neurotoxins are thought to be the major pathogenic cause of rapid death mediated through neuromuscular paralysis and respiratory failure, attributable to the curare-like neurotoxins of the 3ftx family. from the venom-gland trasncriptome, high expression levels were also noted for the genes of svmps and pla2s. svmps and pla are two enzymes commonly present in snake venoms across the elapidae and viperidae. the svmp genes of moh appear highly diverse; the multiple isoforms are suggestive of the vast biological activities of the enzyme. the pla transcripts, on the other hand, are comprised of only distinct sequences, representing a finding of low sequence diversification similar to that in laaos. the enzymatic products of these genes are not usually involved directly in lethality but play ancillary roles in causing tissue inflammation and destruction. meanwhile, approximately % of the non-toxin genes appear unrelated to envenomation. most of these are housekeeping genes associated with cellular metabolisms, and they are transcribed at very low fpkm levels, marked with low redundancies. venom proteome when used in combination, the next generation transcriptomic and proteomic analyses can provide a powerful approach to unveil the molecular complexity of venom toxins. this approach was adopted in the current study for a better correlation of the two processes of gene expression and protein synthesis in the venom glands of the malaysian king cobra. among the many proteomic methods, we employed a sequential method of 1d-sds-page followed by in-gel digestion and nano-esi-lcms/ms. a simpler method of whole venom in-solution lcms/ms is time-saving, cost-friendly, and has been tested out in several venomic studies; however, additional steps of venom protein separation such as sds-page prior to lcms/ms are known to be effective to “de-complex” the venom and thus greatly enhance the protein identification by lcms/ms. in this study, the 1d-sds-page pattern of moh venom is similar to the observation reported in chang et al.. the authors deduced that the prominent bands of king cobra venoms were represented by laaos and svmps, crisps, pla2s and 3ftxs, without reporting their relative abundances. our approach in examining the protein composition throughout the electrophoretic passage has made it possible to estimate the relative abundance of these toxins, in addition to the detection of several other proteins. while the genomic disclosure by vonk et al. has been an excellent reference for the evolution and toxin diversity of the balinese king cobra venom, the individual toxin abundance has not been reported. our current study that revealed the relative abundance of toxins in moh venom is thus hoped to provide further insights and correlation between the tissue transcriptome and toxin expression profiles in king cobra. it is however worth noting that toxin variations can still exist between venoms of different geographical origins. it is interesting to note that 3ftxs and pla in moh venom made up approximately % of the overall abundance, apparently less than in the venoms of local elapids such as naja cobras and kraits, where the total abundance of these two major toxins can be as high as 70- %. instead, a substantial amount of svmps, crisp and laaos were found expressed in the moh venom. a hypothesis for the difference could be that the non-neurotoxin components act synergistically in moh venom in order to subdue neurotoxic elapids such as cobras, which are known to be resistant to neurotoxins. comparison of transcriptome and proteome the expression of mrnas and protein abundance are not necessarily identical due to various cellular regulatory processes e.g., post-transcriptional, translational and protein degradation that control the steady-state protein abundances. moreover, the correlation between transcriptomic and proteomic abundances in snake venom has traditionally been poor. it should be noted that in the correlation study, the protein expression is ideally measured based on peptide unit, analogous to the analysis for rna expression described by mortazavi et al. in order to correct for the molecule size of messenger transcript and protein. using this approach, aird et al. reported a significant correlation of the data for two east asiatic crotalids, although some variances in the results remained unexplained possibly due to biological as well as analytical factors. our study on the moh specimen, however, showed no significant correlation between the venom-gland transciptome and the venom proteome. the correlation may be influenced by many factors, one of which is the time span between venom collection and gland tissue harvesting. in this study, the venom glands had been stimulated by venom milking several days prior to their harvesting as a step to maximise the mrna synthesis. admittedly, toxin components can be synthesized at different rates and hence the respective metrics of mrna and protein expressions taken at one particular time point may not be well correlated. in addition, extensive post-translational modifications occur in many complex toxins such as svmps; hence the mature proteins produced may not be necessarily congruent to the mrna expressed. these factors may explain some of the variances in the data presented here and elsewhere. in the following section, we further discuss the expression of moh toxins according to their protein families. major transcripts and venom constituents three-finger toxins three-finger toxins comprise a family of non-enzymatic polypeptides of 60- amino acid residues with four or five disulfide bridges. 3ftxs are usually the principal toxins found in the venoms of elapid snakes such as king cobra, naja cobras, coral snakes, kraits, mambas and sea snakes. the genes may have been recruited before the divergence of different venomous snakes, as low levels of 3ftx transcripts have been reported in the venom-gland transcriptomes of some viperids and colubrids. the classification of 3ftxs in general is made based on the number and position of disulfide bonds. short-chain 3ftxs possess only four disulfide bonds, while long-chain 3ftxs have five with an additional disulfide bond at the distal end of loop ii. “non-conventional 3ftxs” also have five disulfide bonds, with the fifth bond located within loop i of the molecule. the molecular diversity of 3ftx genes of moh is well reflected by the presence of multiple unique 3ftx transcripts. the two main 3ftxs of moh, moh_ftx and moh_ftx share sequence consensus to most curare-like α-neurotoxins as shown in fig. 5b. moh_ftx is similar to an uncharacterized 3ftxs in the database with an unknown mechanism of action. l-3ftxs constitute the majority of 3ftx transcripts in the venom gland, notably moh_ftx. interestingly, moh_ftx a long-chain neurotoxin, has an n-terminal peptide sequence not seen in other l-3ftxs but identical to that of a weak neurotoxin, wntx-. the sequence in the central core of moh_ftx is nevertheless similar to most other long-chain neurotoxins. it is known that the functional sites of long-chain neurotoxins e.g., α-cobrotoxin/elapitoxin from naja kaouthia reside mainly in the second loop of the toxin, with important residues of k d r and k while the first loop is not involved in the toxin function. moh_ftx and moh_ftx were found to contain all the critical residues except that the major l-3ftxs, moh_ftx showed substitution of k with glutamine and k with asparagine. the effect of variations on these critical residues were unknown. among the s-3ftx transcripts identified, the three most abundantly expressed isoforms are homologous to neurotoxin-like protein hypothetical protein l345_ and muscarinic toxin-like protein homolog, respectively. these transcripts belong to the 3ftx “orphan group”; with the exception of the muscarinic toxin-like protein, the functions of the other two neurotoxin-like proteins are not well understood. a similar type of transcript has also been reported in balinese o. hannah, suggesting that some atypical 3ftxs are conserved. site-directed mutagenesis and nicotinic cholinergic receptor binding studies using the short-chain α-neurotoxin, erabutoxin a, revealed that the functional sites of the molecule are distributed over the tips of all three loops in the toxin, including s and q in loop i and k w d and r that play important roles in short-chain neurotoxin selectivity and affinity to nachr. in contrast, the functional site of l-3ftxs lies mostly within the second loop of the toxin. the lack of the critical resides q in moh_ftx06; q and w in moh_ftx07; q and k in moh_ftx implies their potential structural-function differences in the moh venom. although they are categorized as s-3ftxs, the unusual n-terminal amino acid sequences presented in these transcripts may influence the toxin-receptor binding as the tip of the first loop of the typical short-chain neurotoxin, ea has been shown critical for its binding with the receptor. three other s-3ftx transcripts encode a typical short-chain neurotoxin sntx, a type a muscarinic toxin and a cytotoxin, cardiotoxin-, all at extremely low expression levels. their roles in envenomation have not been elucidated through functional studies. on phylogenetic analysis, s-3ftx, l-3ftx and nc-3ftx were grouped into separate clusters on the phylogenetic tree. genes for both the s-3ftxs identified in moh venom proteome are basal in the phylogenetic relationship. the finding supports that 3ftx genes of moh evolved from short-chain isoforms which later adapted and diverged into genes encoding non-conventional 3ftxs and long-chain 3ftxs. the phylogenetic analysis also showed that the main l-3ftx, moh_ftx is more related to the long-chain neurotoxin oh55/ of the balinese specimen, but distantly diverged from α-cobrotoxin/elapitoxin, indicating their distinctness from the long-chain neurotoxins of naja cobra. on the venom proteome, the number of expressed moh 3ftxs in this study is less than that reported for the balinese specimen. it is however comparable to the number of 3ftxs reported for the thai specimen and is more diverse than that of the other two malaysian king cobra samples studied using chromatography separation and 2de method. the discrepancies could be due to the use of the sensitive nano-lcms/ms in our study, or geographical variations of the venom within malaysia. the current study further showed that the l-3ftxs and s-3ftxs account for % and % of total 3ftx abundance in the proteome, while the non-conventional 3ftxs constitute the least. in term of all protein abundance, the amount of 3ftxs in moh venom is hence %, a finding comparable to chang et al., although the 3ftx subtypes varied between these two studies. while chang et al. concluded that s-3ftxs constituted % w/w of their malaysian venom sample with l-3ftxs constituting only % w/w, we demonstrated a lesser extreme distribution of s-3ftx:l-3ftx with a ratio of 1:. the major form of l-3ftx by toxin abundance is nonetheless same in both studies, viz. oh. similarly, the 3ftxs in our moh venom appear to be primarily neurotoxins of variable isoforms, with a very minimum content of cytotoxin or cardiotoxin, a finding comparable to chang et al.. interestingly, muscarinic toxin-like proteins and several nc-3ftxs were expressed in moh venom at trace amounts; these isoforms have not been reported in the previous studies of malaysian specimens. the cytotoxin and non-conventional 3ftxs of low abundance in moh venom likely play minimal role in the lethal action of the venom in view of their minute amount and the higher ld value compared to alpha-neurotoxins. the proteomic presence of long neurotoxin oh in the moh venom also contrasts with the previous conclusion which proposed this to be one of the unique 3ftx markers exclusive for chinese king cobra and not supposed to be present in the southeast asian specimens. our finding hence indicates that the 3ftx profile of king cobra may be more complex and diverse than previously thought. nonetheless, the presence of a large number of 3ftx variants in high abundance also implies the possibilities of variable protein structure folding, toxin-receptor interaction and potency. in addition, the immunogenicity of the venom is likely determined by the abundant 3ftxs; the small molecular sizes and diverse epitopes of 3ftxs can be a challenge to the production of a high efficacy antivenom. using a computerized model to analyse long ntx sequences retrieved from uniprotkb database, danpaiboon et al. predicted that a total of amino acids in a king cobra long ntx interacted with the human single-chain variable antibody fragments specific to n. kaouthia long ntx at the following amino acids: c w g r k in loop and r n in loop hence supporting the apparently weak cross-neutralization effect of nkln-huscfv against the lethal effect of king cobra in mice. however, matching of the said interactive amino acids of q2vbp to the major ntxs in moh venom yielded limited similarity. this suggests that nkln-huscfv may not be effective in neutralising the king cobra venom from malaysia. from the practical standpoint, leong et al. confirmed in a mouse model that thai naja kaouthia monovalent antivenom has low neutralization capacity against moh venom, indicating the antigenic dissimilarities of both venoms despite the conserved protein scaffolds between neurotoxins of moh and n. kaouthia. this is not surprising considering the fact that the antivenom manufacturer has to combine venom immunogens from both king cobra and n. kaouthia in order to produce the effective neuro polyvalent antivenom. clinically, an attempt to treat malaysian o. hannah envenomation in kuantan, malaysia with nkmav has proven in vain as the antivenom against n. kaouthia failed to cross-neutralize both the systemic and local effects from the bite. in the moh envenoming case, the systemic effects were only arrested much later with the arrival and administration of the king cobra-specific antivenom. we postulate that the neurotoxins of moh are highly distinct to those of naja cobras’ as a result of adaptive evolution and positive selection to ensure that the moh neurotoxins are sufficiently effective to bind naja’s nicotinic receptor since naja cobra is a prey top in king cobra’s food list – a known predator–prey phenomenon in palm oil estates in malaysia. generally, naja cobra is resistant to ntx as their receptors have been mutated with a sugar chain that masks the binding by ntx. in addition to distinct neurotoxins, the moh venom proteome also reveals abundant high molecular mass proteins not commonly present in large amount in naja cobra venoms, implying their potential roles in killing and digestion of other neurotoxic snakes by king cobra. the higher share of non-3ftx components in the moh venom may be a reason for the venom’s higher ld in mice compared to that of naja venom that is predominated with alpha-neurotoxins. this is supported by an earlier report where the major neurotoxic fraction of moh venom, purified by ion-exchange chromatography and accounted for ~ % venom weight, has an i.v. ld of μg/g mice, a value disproportionally lower than that of the crude venom. snake venom metalloproteinases from the moh venom-gland transcriptome, the svmp transcripts encode at least unique svmps, with sequence similarities matched to svmp-ohanin, svmp-cobrin, svmp-bmmp, svmp-kaouthiagin, svmp-mocarhagin- svmp-atrase-a, chain-a-svmp, asrin and carinatease- from different elapid snakes. the sequence divergence is evident, supporting the view of extensive diversification of svmp functions under accelerated evolution. all the identified svmp transcripts belong to the p-iii class domains), the svmp type that has a rich array of biological activities, including potent haemorrhagic activity, inflammation, apoptosis, prothrombin activation, and inhibition of platelet aggregation. from the svmp transcript sequences of moh, we identified the absence of the seventh cysteine residue in domain m that takes part in the disulfide bond exchange for autolysis during secretion or formation of the biologically active dc domain, typical for viperidae svmp-piiis. figure  shows that the moh and annotated svmp-piii m domains from several elapids have only six cysteine residues, consistent with previous studies including that for balinese king cobra. the effect of structural difference as such has been shown by guan et al. that no autolytic activity was observed during the preparations of two p-iii svmps sourced from the taiwanese naja atra. thus, the svmp-piiis of malaysian o. hannah and most elapids likely exhibit the similar structure-function properties distinct from those of viperids.fig. sequence alignments of snake venom metalloproteinase transcripts of malaysian o. hannah venom-gland transcriptome in comparison with svmp sequences of representative venomous snakes. some proteomic studies indicated that svmps are expressed mainly in the viperidae and colubridae venoms, and in a relatively smaller amount in elapidae venoms. our findings revealed that svmps are present in a substantial amount in the moh venom, with a total of proteins detected. our finding of diverse and abundant svmps in moh venom is in agreement with vonk et al. on the view that svmps are important pathogenic toxins besides 3ftxs in the balinese king cobra venom. this is also supported by chang et al. who reported high metalloproteolytic enzyme activity in king cobra venoms, although the authors did not determine the subtypes and composition of svmps in the venoms. on the other hand, svmp was not identified at all in the previous 2de proteomic study of malaysian king cobra, a rather peculiar finding that raises a conflict in local venom database, possibly due to inadequacy of the methodology. earlier studies indicated that at least five svmp isoforms with proinflammatory and oedema-inducing activities were present in king cobra venoms, two of which were proven to be hemorrhagic proteases. the major hemorrhagic protease, termed hannahtoxin, is a 66-kda zinc-dependent metalloprotease that exhibited species-specific lethality, where it is highly lethal to rabbit but not to mice at comparable doses. although local hemorrhage is not a well-recognized clinical feature of king cobra envenoming, the proteolytic and proinflammatory activities of svmp may be involved in cytotoxicity leading to necrosis and tissue destruction. cysteine-rich secretory proteins multiple sequence alignment of the crisp transcripts shows that the sequences are highly conserved across different snake species and the complete sequence of moh_crp obtained is almost identical to ophanin sequence derived from uniprotkb database, including its functional region known for the inhibition of smooth muscle contraction. this is in agreement with the transcriptomic finding reported for the balinese king cobra, where crisp is found with limited diversity and little evidence of gene duplication. this gene family likely has conserved functional activities across most lineages, and does not participate in the evolutionary arms race seen in the more pathogenic toxin families such as 3ftx.fig. sequence alignments of cysteine-rich secretory protein transcripts of malaysian o. hannah venom-gland transcriptome in comparison with crisp sequences of representative venomous snakes. the presence of crisps in king cobra venom proteome varies between studies. crisp is not reported in two previous studies on the venom proteome of malaysian king cobra. in the present study on moh venom, one crisp isoform was identified, consistent with the finding from the thai specimen but varied from the balinese which recorded a total of crisp isoforms. in moh venom, opharin precusor is the identified crisp isoform. this toxin has the putative function of reducing smooth muscle contraction that is elicited by high potassium-induced depolarization. the degree of inhibition by opharin is however lower than most other crisps, possibly due to the critical amino acid difference essential for channel activity inhibition: as with ophanin, the translated moh crisp possesses y in substitution of f and y instead of e. although the pharmacological activity of ophanin as a weak antagonist of smooth muscle contraction has been characterized, its exact role in envenomation remains elusive especially in moh which venom contains a considerable amount of this toxin. l-amino acid oxidases the laao transcripts revealed by the moh venom-gland transcriptome consist of the three main functional domains and share high sequence similarity with sequences of other king cobras. the major transcript sequence in our study is highly homologous to the sequence of hypothetical protein l345_ from the balinese king cobra and king cobra oh-laao. unlike 3ftxs, laaos appear rather well conserved within king cobras from different regions.fig. sequence alignments of l-amino acid oxidase transcripts of malaysian o. hannah venom-gland transcriptome in comparison with laao sequences of representative venomous snakes. snake venom laaos are reported present natively as a homodimer with molecular mass around kda, while under reducing conditions, present as subunit of around 50- kda. we showed in our proteomic study that the laaos of moh venom distributed mainly in gel section corresponding to 55- kda. despite the relatively lower mrna expression, the protein comprises close to % of moh venom, comparable to the approximation of % w/w in an earlier study of malaysian king cobra laao. the peptide sequences exhibit low variability with only two isoforms identified in the proteome, comparable with the thai and balinese samples where one laao subtype was identified. our finding is also in agreement with chang et al. who reported substantial laao activity in four king cobra venoms of different localities, although the paper reported neither the compositional subtype nor the abundance of this enzyme. these studies however strongly disagree with another proteomic study on a malaysian king cobra sample where no laao was detected at all. the substantial amount of laao in the moh venom proteome suggests that the toxin may serve beyond ancillary function. its role in king cobra predation and envenomation may be related to its potent proinflammatory and cytotoxic activity that can induce extensive tissue destruction. in addition, the functional uniqueness of king cobra laao has been shown on its substrate specificity compared to other snakes, where its heat-stable property contributes greatly to its potential for biomedical application. notably, laao from the malaysian king cobra has been shown to exhibit potent in vitro and in vivo selective cytotoxicity. further characterizations of king cobra laao is therefore a feasible and promising research direction towards cytotoxic drug discovery. vespryn the complete sequence of the only vespryn transcript in moh matches identically to the ohanin sequence. the sequence, covering signal and mature chain, is also identical to the corresponding sequence regions reported for the balinese specimen. the balinese king cobra vespryn sequence, however, contains additional pre-signal amino acid residues, the significance of which is uncertain. in contrast to other members of snake venom proteins, ohanin presents a unique single cysteine residue, and the protein sequence shares little similarity to the pry-spry domains with relatively short n-terminal extension. although thaicobrin isolated from the monocled cobra, naja kaouthia was deposited in the protein database much earlier than ohanin, there is no published literature and little characterization work has been done. together with thaicobrin, the distinct sequence of ohanin has made them the first member of vespryns family sourced from snake venom.fig. sequence alignments of vespryn transcripts of malayan o. hannah venom-gland transcriptome in comparison with vespryn sequences of representative venomous snakes. pung et al. reported the presence of ohanin at a low abundance in king cobra venom sourced from jakarta, indonesia. in contrast, the moh venom proteomic study shows the venom contains a higher amount of ohanin that were distributed mainly in gel sections and, corresponding to the known molecular mass of ohanin. the difference in ohanin content in o. hannah venoms from malaysia and indonesia may seem to represent a remarkable geographical variation of the venoms. phospholipases a the two complete pla transcripts of moh were annotated to group ia and ib, respectively, similar to the pla enzymes in chinese o. hannah venoms, and they share the characteristic asn n-terminus for venom pla of king cobra. the major pla expressed in the proteome of moh venom is the acidic pla2-, consistent with tan and saifuddin who reported that the major pla of malaysian king cobra was an acidic isoform constituting % of the venom weight. the enzyme was however non-lethal in mice at an intravenous dose of μg/g but exhibited moderate anticoagulant and oedema-inducing activities, in contrast to the fujian pla2- that was mildly lethal and cardiotoxic in mice. remarkable variations are noted within the group i pla among king cobra venoms of different origin: while we identified group ia pla from moh, huang et al. isolated only group ib pla from a fujian chinese sample; vonk et al. identified two each for group ia and group ib from a balinese sample; vejayan et al. identified one each of group ia and group ib from a malaysian sample; danpaiboon et al. identified group ia and group ib pla from a thai sample. with the inconsistency of king cobra pla subtypes identified, the comparative analysis is further compounded by undetermined toxin abundance in each study. chang et al. showed with quantitation in another malaysian sample the presence of group ia and group ib pla subtypes, however each with exceptionally low abundance ranging from - % only, indicating that the total pla content in the venom was far less than %. the intraspecific pla variations are therefore vast and may be related to not only geographical distribution but also ontogenic factors. the current proteomic study, nonetheless, shows that the moh major pla is the acidic, proinflammatory but non-lethal variant of group ia pla it constitutes a small portion of the malaysian king cobra venom, approximately times less than pla2s found in the regional naja cobra venoms. in view of the negligible cytotoxin content and small amount of pla local tissue necrosis that occurs in moh envenoming is possibly induced by other cytotoxic components in the venom, for instances, svmps and laaos that exist at a high abundance.fig. sequence alignments of phospholipase a transcripts of malaysian o. hannah venom-gland transcriptome in comparison with pla sequences of representative venomous snakes. cobra venom factors the trasncripts of cobra venom factors from the moh transcriptome are highly homologous to ovf, ophiophagus venom factor, the cvf first purified from the venom of a chinese king cobra. interestingly, the ovf transcripts in moh transcriptome reveal amino acid residues absent in the reported sequence for guangxi ovf, but present identically in that of the balinese specimen. this suggests that the structures of ovf from king cobras of the two southeast asian regions share high similarity and are sufficiently distinct from that of the china specimen. in addition, two novel regions not reported previously in cvf/ovf have been identified in the ovf transcript of moh. blast analysis of these peptides retrieved no identifiable protein or peptide region. the role of the additional amino acid sequences in the structure-function relationship of malaysian cvf/ovf remains to be investigated.fig. sequence alignments of cobra venom factor transcripts of malaysian o. hannah venom gland transcriptome in comparison with cvf sequences of representative venomous snakes. the number of cvf subtypes expressed in the moh venom is comparable to that reported in the balinese specimen, but more diverse than the thai specimen and another malaysian sample. cvf is a protein with high molecular mass and it dissociates into α, β and γ chains under reducing condition, consistent with our findings where the gel sections and corresponded to the reported sizes of α, β and γ chain. cvf is not directly lethal; it facilitates the absorption and spread of venom from the bite site, as cvf-activated local complement system releases anaphylatoxins c3a and c5a that increase the regional vascular permeability and blood flow. low-abundance transcripts and toxins toxins constituents expressed in both venom-gland transcriptome and venom proteome kunitz/bpti serine protease inhibitors transcripts consitutute % of toxin fpkm, while one kun was expressed into the venom proteome at % of toxin abundance. the expressed kun protein is similar to oh-tci, the first reported kun from snake venom that exhibited equivalent trypsin and chymotrypsin inhibitory activities. in addition, cystatin, an inhibitor of various c cysteine proteases that may play a role in preventing toxin auto-digestion, was detected at a low amount in the transcriptome and proteome. in comparison, the balinese and thai king cobra venoms contain, respectively, and kun subtypes but cystatin has not been reported. the exact function and action of kuns remain to be elucidated. from the moh venom-gland transcriptome, we have identified transcripts that encoded neprilysin, a metalloendopeptidase with affinity for a vast range of physiological targets, including natriuretic and vasodilatory neuropeptides for their regulation. novel metallopeptidases exhibiting similarity to neprilysin was first demonstrated in the venom-gland transcriptomic study of echis sp., and recently the neprilysin transcript was revealed too from the venom gland of the balinese king cobra, but its expression in the king cobra venom proteome was not reported. in this study, we showed that neprilysin is present in the venom proteome of malaysian king cobra, although its identity and function as a secretory toxin in the venom are still elusive at this stage of venom study. from the venom gland transcriptome, three transcripts were identified for encoding snake venom serine proteases at rather low fpkm. through lcms/ms and homology search, the expressed peptides were noted similar to alpha- and beta-fibrinogenase ohs. in comparison, svsps and kallikrein were identified from the venom proteome of balinese and thai specimens, respectively but not reported in the other two malaysian samples. the serine fibrinogenase in moh venom has a putative role of a potent fibrinogenolytic and amidolytic agent without haemorrhagic effect. serine proteases at 5- % in viperid venom is able to elicit significant coagulopathic effect in vivo and in vitro; this effect however has not been a well-recognized clinical syndrome in king cobra envenomation, and laboratory test using human plasma for the venom’s procoagulant effect has not been positive. in addition to the fibrinogenolytic toxins, phospholipase b with haemolytic activity from drysdalia coronoides was detected in a minute amount in the moh transcriptome and the venom proteome. this enzyme was also identified in the venom proteome of the thai specimen but not reported from the other sources. acetylcholinesterases are usually expressed at low level in snake venoms; the highest abundance ever reported was % from the genus bungarus. the major isoform of ache transcript from moh exhibits extremely high homology to ache full sequence of bungarus fasciatus, supporting the antigenic cross-reactivity observed between king cobra ache and the monoclonal antibodies of krait ache. one ache was detected in the moh venom proteome at a relatively low abundance. the presence of this enzyme in king cobra venom was previously identified only through functional study. chang et al. showed that the ache activities of malaysian and thai king cobra venoms were several-fold higher than the indonesian and chinese samples, and described an inverse relationship between the ache activity and the neurotoxic fatality of the venom. however, the postulation of the ‘inverse relationship’ seems contradictory as it was suggested that the immediate acetylcholine hydrolysis at synapses might reduce the competition between the neurotransmitter and the curare-like neurotoxins, thus potentiating the neurotoxicity. besides, although venom aches may interrupt musculatory control through rapid degradation of ach, their effect on cholinergic transmission in the central nervous system and autonomic ganglia remains unclear. systemic hypotension may be a possible effect associated with the interruption of cholinergic transmission at the autonomic ganglia, thus favouring prey immobilization. the transcription of nerve growth factor genes in the moh venom glands is consistent with the findings for the balinese king cobra. a small amount of ngf was also detected in the moh venom proteome. of note, the proteomic presence of this minor toxin in king cobra venoms has not been reported nor quantitated previously. it has been suggested that ngf can exhibit arginine esterase activity that contributes to hypotensive effect through nitric oxide liberation and histamine release. interestingly, this is a group of snake venom constituents, after the widely-studied anticoagulant, antiproliferative and antimicrobial toxins, shown to have therapeutic potential for neurodegenerative brain disorders, e.g., alzheimer’s disease. phosphodiesterase and 5’-nucleotidase were two other minor toxins detected in both the venom-gland trasncriptome and venom proteome of moh. both the enzymes were detected in the venom proteome of thai king cobra, while only pde was identified from the balinese specimen. these enzymes primarily act to liberate nucleosides which may help in prey immobilization. venom phosphodiesterases are known to cause locomotor depression and reduction of mean arterial blood pressure. together with 5’nucs and toxins with hypotensive and immobilising potentials such as ovf, ache and ngf, these components may act synergistically to enhance prey subduing. in comparison with the study on balinese king cobra, we noticed some variations in the “–omic” findings between king cobras from the two geographical regions. besides the variabilities of sequence and abundance described, we also found the inconsistent presence of a number of toxins between the two specimens. for instance, ngf, ache, neprilysin, plb, cystatin and 5’-nucleotidase present in the moh proteome were not reported in the balinese specimen proteome. conversely, natriuretic peptide and vascular endothelial growth factor were detected in the venom proteome of the balinese specimen but absent in the moh although the expression was detected at low mrna level. one interesting, consistent feature shared by both king cobras from the malayan peninsula and bali island is the absence of lectin in the venom proteome in spite of their gene expression in the venom glands. this observation supports that lectins do not contribute to king cobra envenoming which is in contrast to many other venomous snakes particularly from the viperidae family. in addition, although the two specimens did not differ remarkably in the composition of major pathogenic toxin components e.g., 3ftxs and svmps, variations are noted in the gene sequences and possibly proteomic abundances, indicating intra-specific venom diversity that may lead to variations in immunological profile and antivenom effectiveness. toxins not translated from venom-gland transcriptome twenty seven toxin transcripts were identified only at the transcriptional level of moh venom glands. these transcripts encoded protein genes for insulin growth factors, natriuretic peptides, hyaluronidase, dipeptidylpeptidase iv, vascular endothelial growth factors, waprins and c-type lectins, totalled to % of all toxin fpkm. the activities of these toxins have not been previously investigated in king cobra venom. it is an assumption that their occurence in the venom is extremely low or negligible since the peptides of these proteins were not detected by the high resolution tandem mass spectrometry. transcripts of igfs have been previously categorized into secretory toxin transcripts by vonk et al., although its toxic role is uncertain to date. at the proteomic level, this toxin was also not reported in both moh and the balinese king cobra venoms, but its presence was detected along with a hepatocyte growth factor in the thai specimen. natriuretic peptide, a hormone that controls over natriuresis, diuresis, blood pressure, homeostasis and inhibition of aldosterone secretion in all vertebrates was not identified in the proteomes of both moh and thai specimen, but reported in the balinese specimen proteome. this is another physiological protein which gene was likely recruited earlier into the toxin arsenal; the expression however has been low among venomous snakes. besides, vegfs, potential vasodilators, were expressed in the venom proteome of the balinese specimen but never reported in moh and the other king cobra venoms studied. hyaluronidase, dpp-iv, waprin and ctl were consistently not reported in the venom proteomes of king cobras although the genes were transcribed in the venom glands. interestingly, the enzymatic activity of hyaluronidase has been reported previously in king cobra venom but at a level much lower than that of naja sp., bungarus sp. and viperid venoms. in this study, a full sequence of hyaluronidase from moh venom gland transcriptome was identified. its high homology to sequences of viperid snakes suggests that the gene and protein function appear to be conserved across many venomous lineages despite its trivial expression in the venom. dpp-iv, waprin and ctl have not been reported thus far in king cobra venom proteomes; but it remains interesting for further study as complete sequences of these toxins have been made available from the moh venom-gland transcriptome. the expression of these genes without translation into proteins indicate their early recruitment into the venom-gland transcriptome, however, adaptation and selection have favored the expression of other toxins to suit the predatory arm-race and ecological niche changes for the king cobras. an alternative possible explanation for this is the convergence of some genes which begin to emerge as potential contributor of toxins in adaptation to the changing niche. data of these sequences may serve useful purposes for biodiversity study and drug discovery in the future.  this study demonstrates complex and diverse composition of malaysian king cobra venom at both the transcriptomic and proteomic levels. unlike venoms of southeast asian naja sp. that mainly comprise neurotoxins, pla2s and cytotoxins, moh venom contains a higher degree of toxin complexity predominated with neurotoxins and svmps, in addition to appreciable amounts of crisp, laao, cvfs, vespryn, pla and ngf. additionally, the moh venom contains small amounts of neprilysins and ache which may have synergistic action on the neurotoxicity of the venom. the findings also suggest the presence of toxins that may induce hypolocomotive and hypotensive effects in the venom, although clinically humans are susceptible more rapidly to neuromuscular paralysis due to the faster absorption of neurotoxins and their intrinsic toxicity with a rapid onset. the unique toxin composition of king cobra venom may serve to aid this ophiophagic species in preying on other snakes especially naja cobras which are known to be neurotoxin-resistant. as anticipated, intra-species variation of venom is not uncommon, and this is exemplified by the king cobras among the peninsular malaysian, the thai and the insular balinese specimens. we propose the need to address the medical concerns of such phenomenon, where comparative studies in the future should aim to provide variation details in terms of the sequence diversification and the relative abundance of toxins between venoms of different geographical sources. the integrated transcriptomic-proteomic knowledge as such is fundamental for deeper insights into the mechanisms of snake venom toxicity and the natural history of venom evolution. the knowledge may provide crucial information for optimizing the production of an effective pan-regional antivenom for related species in the future. 
the venom gland transcriptome was investigated using the illumina hiseq™ platform, while the venom proteome was profiled by 1d-sds-page-nano-esi-lcms/ms. besides, the presence of cobra venom factor, nerve growth factors, phosphodiesterase, 5’-nucleotidase, and dpp-iv in the venom proteome suggests its probable hypotensive action in subduing prey. ophiophagus hannahking cobravenom proteomevenom gland transcriptomenano-lcmsesi-lcms/msissue-copyright-statement© the author 2015 transcriptomic results reveal high redundancy of toxin transcripts despite small cluster numbers, implying gene duplication and diversification within restricted protein families. the results correlate with the pathophysiological actions of moh venom, and dispute the use of naja cobra antivenoms to treat moh envenomation. among the toxin families identified, three-finger toxins and snake-venom metalloproteases have the most diverse isoforms. these toxin families are also the most abundantly transcribed, followed in descending order by phospholipases a, cysteine-rich secretory proteins, kunitz-type inhibitors, and l-amino acid oxidases. the king cobra is widely distributed throughout many parts of asia. this study aims to investigate the complexity of malaysian ophiophagus hannah venom for a better understanding of king cobra venom variation and its envenoming pathophysiology. this study reports the diversity and abundance of toxins in the venom of the malaysian king cobra. on the other hand, the moh proteome includes 3ftxs, the most abundantly expressed proteins in the venom. neurotoxins comprise the major 3ftxs in the moh venom, consistent with rapid neuromuscular paralysis reported in systemic envenoming. dissimilarities in the subtypes and sequences between the neurotoxins of moh and naja kaouthia are in agreement with the poor cross-neutralization activity of n. kaouthia antivenom used against moh venom. within this toxin family, there are long-chain, short-chain and non-conventional 3ftx. the presence of toxic enzymes such as laaos, svmps and pla would explain tissue inflammation and necrotising destruction in local envenoming. seventeen toxin families exhibited low mrna expression, including hyaluronidase, dpp-iv and 5’-nucleotidase that were not previously reported in the venom-gland transcriptome of a balinese o. hannah.
 the king cobra is widely distributed throughout many parts of asia. this study aims to investigate the complexity of malaysian ophiophagus hannah venom for a better understanding of king cobra venom variation and its envenoming pathophysiology. the venom gland transcriptome was investigated using the illumina hiseq™ platform, while the venom proteome was profiled by 1d-sds-page-nano-esi-lcms/ms.  transcriptomic results reveal high redundancy of toxin transcripts despite small cluster numbers, implying gene duplication and diversification within restricted protein families. among the toxin families identified, three-finger toxins and snake-venom metalloproteases have the most diverse isoforms. these toxin families are also the most abundantly transcribed, followed in descending order by phospholipases a, cysteine-rich secretory proteins, kunitz-type inhibitors, and l-amino acid oxidases. seventeen toxin families exhibited low mrna expression, including hyaluronidase, dpp-iv and 5’-nucleotidase that were not previously reported in the venom-gland transcriptome of a balinese o. hannah. on the other hand, the moh proteome includes 3ftxs, the most abundantly expressed proteins in the venom. within this toxin family, there are long-chain, short-chain and non-conventional 3ftx. neurotoxins comprise the major 3ftxs in the moh venom, consistent with rapid neuromuscular paralysis reported in systemic envenoming. the presence of toxic enzymes such as laaos, svmps and pla would explain tissue inflammation and necrotising destruction in local envenoming. dissimilarities in the subtypes and sequences between the neurotoxins of moh and naja kaouthia are in agreement with the poor cross-neutralization activity of n. kaouthia antivenom used against moh venom. besides, the presence of cobra venom factor, nerve growth factors, phosphodiesterase, 5’-nucleotidase, and dpp-iv in the venom proteome suggests its probable hypotensive action in subduing prey.  this study reports the diversity and abundance of toxins in the venom of the malaysian king cobra. the results correlate with the pathophysiological actions of moh venom, and dispute the use of naja cobra antivenoms to treat moh envenomation. the findings also provide a deeper insight into venom variations due to geography, which is crucial for the development of a useful pan-regional antivenom. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords ophiophagus hannahking cobravenom proteomevenom gland transcriptomenano-lcmsesi-lcms/msissue-copyright-statement© the author 2015 