Protein decomplexation and proteomics: A complementary assessment method of the physicochemical purity of antivenom.

The quality of antivenom is governed by its safety and efficacy profiles. These quality characteristics are much influenced by the purity of antivenom content. Rigorous assessment and meticulous monitoring of antivenom purity at the preclinical setting is hence crucial. This study aimed to explore an integrative proteomic method to assess the physicochemical purity of four commercially available antivenoms in the region. The antivenoms were subjected to Superdex 200 HR 10/30 size-exclusion fast-protein liquid chromatography (SE-FPLC). The proteins in each fraction were trypsin-digested and analyzed by nano-ESI-liquid chromatography-tandem mass spectrometry (LC-MS/MS). SE-FPLC resolved the antivenom proteins into three major protein components of very high (>200 kDa), high (100-120 kDa) and medium (<60 kDa) molecular weights. The major components (80-95% of total proteins) in the antivenoms were proteins of 100-120 kDa consisting of mainly the light and partially digested heavy immunoglobulin chains, consistent with F(ab')2 as the active principle of the antivenoms. However, LC-MS/MS also detected substantial quantity of large proteins (e.g. alpha-2-macroglobulins), immunoglobulin aggregates and impurities e.g. albumins in some products. The method is practical and able to unveil the quantitative and qualitative aspects of antivenom protein compositions. It is therefore a potentially useful preclinical assessment tool of antivenom purity.

Nutritional evaluation on Lignosus cameronensis C. S. Tan, a medicinal Polyporaceae.

Sclerotial powder of a cultivated species of the Tiger Milk Mushroom, Lignosus cameronensis was analysed for its nutritional components and compared against species of the same genus, Lignosus rhinocerus and Lignosus tigris. All three species have been used by indigenous tribes in Peninsular Malaysia as medicinal mushrooms. Content of carbohydrate, fibre, mineral, amino acid, palatable index, fat, ash and moisture were determined. L. cameronensis sclerotial material consists of carbohydrate (79.7%), protein (12.4%) and dietary fibre (5.4%) with low fat (1.7%) and no free sugar. It has the highest content of total carbohydrate (791 g kg-1 ), energy value (3,700 kcal kg-1 ) and calcium (0.85 g kg-1 ). The crude protein content (123 g kg-1 ) is comparable to that of L. rhinocerus with its main amino acids consisting of glutamic acid, aspartic acid and leucine. The umami index is determined to be 0.27. The total essential amino acid (45 g kg-1 ) is comparable to that of L. tigris. The main mineral is potassium (1.51 g kg-1 ) and the Na/K ratio was <0.6. Heavy metals such as mercury, cadmium, lead and arsenic were absent. L. cameronensis has the highest amount of food energy, total carbohydrate and calcium compared to those of both L. rhinocerus and L. tigris. The essential amino acids comprised almost 40% of the total amino acid content, slightly more than that reported from sclerotial powder of the L. tigris. © 2019 IUBMB Life, 9999(9999):1-6, 2019.

Heterologous expression of cytotoxic sesquiterpenoids from the medicinal mushroom Lignosus rhinocerotis in yeast.

BACKGROUND: Genome mining facilitated by heterologous systems is an emerging approach to access the chemical diversity encoded in basidiomycete genomes. In this study, three sesquiterpene synthase genes, GME3634, GME3638, and GME9210, which were highly expressed in the sclerotium of the medicinal mushroom Lignosus rhinocerotis, were cloned and heterologously expressed in a yeast system. RESULTS: Metabolite profile analysis of the yeast culture extracts by GC-MS showed the production of several sesquiterpene alcohols (C15H26O), including cadinols and germacrene D-4-ol as major products. Other detected sesquiterpenes include selina-6-en-4-ol, ß-elemene, ß-cubebene, and cedrene. Two purified major compounds namely (+)-torreyol and α-cadinol synthesised by GME3638 and GME3634 respectively, are stereoisomers and their chemical structures were confirmed by 1H and 13C NMR. Phylogenetic analysis revealed that GME3638 and GME3634 are a pair of orthologues, and are grouped together with terpene synthases that synthesise cadinenes and related sesquiterpenes. (+)-Torreyol and α-cadinol were tested against a panel of human cancer cell lines and the latter was found to exhibit selective potent cytotoxicity in breast adenocarcinoma cells (MCF7) with IC50 value of 3.5 ± 0.58 µg/ml while α-cadinol is less active (IC50 = 18.0 ± 3.27 µg/ml). CONCLUSIONS: This demonstrates that yeast-based genome mining, guided by transcriptomics, is a promising approach for uncovering bioactive compounds from medicinal mushrooms.

Venom-gland transcriptome and venom proteome of the Malaysian king cobra (Ophiophagus hannah).

BACKGROUND: The king cobra (Ophiophagus hannah) is widely distributed throughout many parts of Asia. This study aims to investigate the complexity of Malaysian Ophiophagus hannah (MOh) 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. RESULTS: Transcriptomic results reveal high redundancy of toxin transcripts (3357.36 FPKM/transcript) despite small cluster numbers, implying gene duplication and diversification within restricted protein families. Among the 23 toxin families identified, three-finger toxins (3FTxs) and snake-venom metalloproteases (SVMPs) have the most diverse isoforms. These 2 toxin families are also the most abundantly transcribed, followed in descending order by phospholipases A2 (PLA2s), cysteine-rich secretory proteins (CRISPs), Kunitz-type inhibitors (KUNs), and L-amino acid oxidases (LAAOs). 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 (43 % toxin sbundance). Within this toxin family, there are 6 long-chain, 5 short-chain and 2 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 PLA2 would explain tissue inflammation and necrotising destruction in local envenoming. Dissimilarities in the subtypes and sequences between the neurotoxins of MOh and Naja kaouthia (monocled cobra) 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. CONCLUSION: This study reports the diversity and abundance of toxins in the venom of the Malaysian king cobra (MOh). 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.

Genome-based proteomic analysis of Lignosus rhinocerotis (Cooke) Ryvarden sclerotium.

Lignosus rhinocerotis (Cooke) Ryvarden (Polyporales, Basidiomycota), also known as the tiger milk mushroom, has received much interest in recent years owing to its wide-range ethnobotanical uses and the recent success in its domestication. The sclerotium is the part with medicinal value. Using two-dimensional gel electrophoresis coupled with mass spectrometry analysis, a total of 16 non-redundant, major proteins were identified with high confidence level in L. rhinocerotis sclerotium based on its genome as custom mapping database. Some of these proteins, such as the putative lectins, immunomodulatory proteins, superoxide dismutase, and aegerolysin may have pharmaceutical potential; while others are involved in nutrient mobilization and the protective antioxidant mechanism in the sclerotium. The findings from this study provide a molecular basis for future research on potential pharmacologically active proteins of L. rhinocerotis.

The genome of the Tiger Milk mushroom, Lignosus rhinocerotis, provides insights into the genetic basis of its medicinal properties.

BACKGROUND: The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden or Tiger milk mushroom (Polyporales, Basidiomycota) is a valuable folk medicine for indigenous peoples in Southeast Asia. Despite the increasing interest in this ethnobotanical mushroom, very little is known about the molecular and genetic basis of its medicinal and nutraceutical properties. RESULTS: The de novo assembled 34.3 Mb L. rhinocerotis genome encodes 10,742 putative genes with 84.30% of them having detectable sequence similarities to others available in public databases. Phylogenetic analysis revealed a close evolutionary relationship of L. rhinocerotis to Ganoderma lucidum, Dichomitus squalens, and Trametes versicolor in the core polyporoid clade. The L. rhinocerotis genome encodes a repertoire of enzymes engaged in carbohydrate and glycoconjugate metabolism, along with cytochrome P450s, putative bioactive proteins (lectins and fungal immunomodulatory proteins) and laccases. Other genes annotated include those encoding key enzymes for secondary metabolite biosynthesis, including those from polyketide, nonribosomal peptide, and triterpenoid pathways. Among them, the L. rhinocerotis genome is particularly enriched with sesquiterpenoid biosynthesis genes. CONCLUSIONS: The genome content of L. rhinocerotis provides insights into the genetic basis of its reported medicinal properties as well as serving as a platform to further characterize putative bioactive proteins and secondary metabolite pathway enzymes and as a reference for comparative genomics of polyporoid fungi.

King cobra (Ophiophagus hannah) venom L-amino acid oxidase induces apoptosis in PC-3 cells and suppresses PC-3 solid tumor growth in a tumor xenograft mouse model.

King cobra (Ophiophagus hannah) venom L-amino acid oxidase (OH-LAAO), a heat stable enzyme, has been shown to exhibit very potent anti-proliferative activity against human breast and lung tumorigenic cells but not in their non-tumorigenic counterparts. We further examine its in vitro and in vivo anti-tumor activity in a human prostate adenocarcinoma (PC-3) model. OH-LAAO demonstrated potent cytotoxicity against PC-3 cells with IC50 of 0.05 µg/mL after 72 h incubation in vitro. It induced apoptosis as evidenced with an increase in caspase-3/7 cleavages and an increase in annexin V-stained cells. To examine its in vivo anti-tumor activity, we treated PC-3 tumor xenograft implanted subcutaneously in immunodeficient NU/NU (nude) mice with 1 µg/g OH-LAAO given intraperitoneally (i.p.). After 8 weeks of treatment, OH-LAAO treated PC-3 tumors were markedly inhibited, when compared to the control group (P <0.05). TUNEL staining analysis on the tumor sections showed a significantly increase of apoptotic cells in the LAAO-treated animals. Histological examinations of the vital organs in these two groups showed no significant differences with normal tissues, indicating no obvious tissue damage. The treatment also did not cause any significant changes on the body weight of the mice during the duration of the study. These observations suggest that OH-LAAO cytotoxic effects may be specific to tumor xenografts and less to normal organs. Given its potent anti-tumor activities shown in vitro as well as in vivo, the king cobra venom LAAO can potentially be developed to treat prostate cancer and other solid tumors.

Energy and nutritional composition of Tiger milk mushroom (Lignosus tigris Chon S. Tan) sclerotia and the antioxidant activity of its extracts.

The Lignosus is a genus of fungi that have useful medicinal properties. In Southeast Asia, three species of Lignosus (locally known collectively as Tiger milk mushrooms) have been reported including L. tigris, L. rhinocerotis, and L. cameronensis. All three have been used as important medicinal mushrooms by the natives of Peninsular Malaysia. In this work, the nutritional composition and antioxidant activities of the wild type and a cultivated strain of L. tigris sclerotial extracts were investigated. The sclerotia are rich in carbohydrates with moderate amount of protein and low fat content. Free radical scavenging activities of L. tigris sclerotial extracts correlate with their phenolic content, which ranges from 6.25 to 45.42 mg GAE/g extract. The FRAP values ranged from 0.002 to 0.041 mmol/min/g extract, while the DPPH(•), ABTS(•+), and superoxide anion (SOA) scavenging activities ranged from 0.18 to 2.53, 0.01 to 0.36, and -4.53 to 10.05 mmol Trolox equivalents/g extract, respectively. L. tigris cultivar shows good prospect to be developed into functional food due to its good nutritional value and potent SOA scavenging activity.

Anti-inflammatory effect of the sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden, the Tiger Milk mushroom.

BACKGROUND: The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) is used as a traditional medicine to relieve cough, asthma and chronic hepatitis. The traditional uses of the sclerotium are presumably related to its anti-inflammatory effect. The present study was carried out to evaluate the anti-inflammatory activity of the sclerotial powder of L. rhinocerotis (Cooke) Ryvarden (Tiger Milk mushroom) cultivar TM02. METHODS: The anti-acute inflammatory activity of the sclerotial powder of L. rhinocerotis cultivar TM02 was investigated using carrageenan-induced paw edema test while the inhibition of transudative and proliferative phases of chronic inflammation were studied by cotton pellet induced granuloma model. Sprague Dawley rats were used in both studies. The anti-inflammatory activity was also measured by inhibition of lipopolysaccharide induced TNF-alpha production in RAW 264.7 macrophage cells. RESULTS: Cold water extract (CWE), hot water extract (HWE) and methanol extract (ME) of the sclerotial powder of L. rhinocerotis cultivar TM02 possessed anti-acute inflammatory activity as was measured by carrageenan-induced paw edema test, with CWE being the most potent. The acute anti-inflammatory activity of the cold water extract (CWE) was mainly contributed by its high molecular weight (HMW) fraction isolated by Sephadex G50 gel filtration chromatography. Its protein component was very potent in the inhibition of TNF-alpha production with an IC50 of 0.76 µg/ml. CWE at 200 mg/kg did not inhibit transudative and proliferative phase of chronic inflammation as shown by using the cotton pellet induced granuloma model. CONCLUSIONS: Our results suggested that most of the bioactive substance(s) contributed to the acute-inflammatory activity of the sclerotial powder of L. rhinocerotis cultivar TM02 appear to be in the CWE, particularly its HMW fraction. The anti-inflammatory activity of CWE was mainly contributed by the protein component of the high molecular weight (HMW) fraction and it exhibited strong inhibitory effect on TNF-alpha production but the possibility of synergistic effect between HMW, MMW and LMW fractions cannot be excluded. Future studies will provide new insights into the anti-inflammatory activity of the sclerotial powder.

Prophylactic effect of Mucuna pruriens Linn (velvet bean) seed extract against experimental Naja sputatrix envenomation: gene expression studies.

Mucuna pruriens is widely used in traditional medicine for treatments of various diseases. In certain region of Nigeria, the seed is used as oral prophylactics for snakebite. Rats pretreated with the aqueous extract from M. pruriens seed (MPE) were protected against the lethal effects of Naja sputatrix (Javan spitting cobra) venom [Tan et al., J Ethnopharmacol, 123 (2009) 356]. The pretreatment also protected against venom-induced histopathological changes in rat heart. To contribute to the understanding of the mechanism of cardio-protective action, the present study examined the effects of MPE-pretreatment on gene expression profile of rat heart as well as effect of MPE-pretreatment on N. sputatrix venom-induced gene expression alterations in rat heart. The gene expression profiles were examined by microarray analysis and verified by real time PCR. The results showed that pretreatment with MPE caused 50 genes in the rat heart substantially up-regulated of which 19 were related to immune responses, 7 were related to energy production and metabolism. The up-regulation of genes related to energy metabolism probably plays a role in maintaining the viability of the heart. Four other genes that were up-regulated (alpha synuclein, natriuretic peptide precursor, calsequestrin and triadin) were involved in the maintenance of homeostasis of the heart or maintaining its viability, thereby contributing to the direct protective action. The results demonstrated that protective effect of MPE pretreatment against snake venom poisoning may involve a direct action on the heart.

Isolation, characterization and antigenic cross-reactivities of the major hemorrhagin from Cryptelytrops purpureomaculatus venom.

The major hemorrhagin from C. purpureomaculatus (mangrove pit viper) venom was purified to homogeneity and termed Maculatoxin. Maculatoxin has a molecular weight of 38 kDa as determined by SDS-PAGE. It is an acidic protein (pI= 4.2) and exhibited proteolytic and hemorrhagic activities (MHD10 = 0.84 microg in mice) but was not lethal to mice at a dose of 1 microg/g. The hemorrhagic activity of Maculatoxin was completely inactivated by EDTA and partially inhibited by ATP and citrate. The N-terminal sequence of Maculatoxin (TPEQQRFPPTYIDLGIFVDHGMYAT) shares a significant degree of homology with the metalloprotease domain of other venom hemorrhagins. Indirect ELISA showed anti-Maculatoxin cross reacted with protein components of many snake venoms. In the double-sandwich ELISA, however, anti-Maculatoxin cross-reacted only with venoms of certain species of the Trimeresurus (Asia lance-head viper) complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.

De Novo Assembly of Venom Gland Transcriptome of Tropidolaemus wagleri (Temple Pit Viper, Malaysia) and Insights into the Origin of Its Major Toxin, Waglerin.

The venom proteome of Temple Pit Viper (Tropidolaemus wagleri) is unique among pit vipers, characterized by a high abundance of a neurotoxic peptide, waglerin. To further explore the genetic diversity of its toxins, the present study de novo assembled the venom gland transcriptome of T. wagleri from west Malaysia. Among the 15 toxin gene families discovered, gene annotation and expression analysis reveal the dominating trend of bradykinin-potentiating peptide/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (BPP/ACEI-CNP, 76.19% of all-toxin transcription) in the transcriptome, followed by P-III snake venom metalloproteases (13.91%) and other toxins. The transcript TwBNP01 of BPP/ACEI-CNP represents a large precursor gene (209 amino acid residues) containing the coding region for waglerin (24 residues). TwBNP01 shows substantial sequence variations from the corresponding genes of its sister species, Tropidolaemus subannulatus of northern Philippines, and other viperid species which diversely code for proline-rich small peptides such as bradykinin-potentiating peptides (BPPs). The waglerin/waglerin-like peptides, BPPs and azemiopsin are proline-rich, evolving de novo from multiple highly diverged propeptide regions within the orthologous BPP/ACEI-CNP genes. Neofunctionalization of the peptides results in phylogenetic constraints consistent with a phenotypic dichotomy, where Tropidolaemus spp. and Azemiops feae convergently evolve a neurotoxic trait while vasoactive BPPs evolve only in other species.

De Novo Venom Gland Transcriptome Assembly and Characterization for Calloselasma rhodostoma (Kuhl, 1824), the Malayan Pit Viper from Malaysia: Unravelling Toxin Gene Diversity in a Medically Important Basal Crotaline.

In Southeast Asia, the Malayan Pit Viper (Calloselasma rhodostoma) is a venomous snake species of medical importance and bioprospecting potential. To unveil the diversity of its toxin genes, this study de novo assembled and analyzed the venom gland transcriptome of C. rhodostoma from Malaysia. The expression of toxin genes dominates the gland transcriptome by 53.78% of total transcript abundance (based on overall FPKM, Fragments Per Kilobase Million), in which 92 non-redundant transcripts belonging to 16 toxin families were identified. Snake venom metalloproteinase (SVMP, PI > PII > PIII) is the most dominant family (37.84% of all toxin FPKM), followed by phospholipase A2 (29.02%), bradykinin/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (16.30%), C-type lectin (CTL, 10.01%), snake venom serine protease (SVSP, 2.81%), L-amino acid oxidase (2.25%), and others (1.78%). The expressions of SVMP, CTL, and SVSP correlate with hemorrhagic, anti-platelet, and coagulopathic effects in envenoming. The SVMP metalloproteinase domains encode hemorrhagins (kistomin and rhodostoxin), while disintegrin (rhodostomin from P-II) acts by inhibiting platelet aggregation. CTL gene homologues uncovered include rhodocytin (platelet aggregators) and rhodocetin (platelet inhibitors), which contribute to thrombocytopenia and platelet dysfunction. The major SVSP is a thrombin-like enzyme (an ancrod homolog) responsible for defibrination in consumptive coagulopathy. The findings provide insight into the venom complexity of C. rhodostoma and the pathophysiology of envenoming.

Effect of Mucuna pruriens Seed Extract Pretreatment on the Responses of Spontaneously Beating Rat Atria and Aortic Ring to Naja sputatrix (Javan Spitting Cobra) Venom.

Mucuna pruriens Linn. (velvet bean) has been used by native Nigerians as a prophylactic for snakebite. Rats pretreated with M. pruriens seed extract (MPE) have been shown to protect against the lethal and cardiovascular depressant effects of Naja sputatrix (Javan spitting cobra) venoms, and the protective effect involved immunological neutralization of the venom toxins. To investigate further the mechanism of the protective effect of MPE pretreatment against cobra venom toxicity, the actions of Naja sputatrix venom on spontaneously beating rat atria and aortic rings isolated from both MPE pretreated and untreated rats were studied. Our results showed that the MPE pretreatment conferred protection against cobra venom-induced depression of atrial contractility and atrial rate in the isolated atrial preparations, but it had no effect on the venom-induced contractile response of aortic ring preparation. These observations suggested that the protective effect of MPE pretreatment against cobra venom toxicity involves a direct protective action of MPE on the heart function, in addition to the known immunological neutralization mechanism, and that the protective effect does not involve action on blood vessel contraction. The results also suggest that M. pruriens seed may contain novel cardioprotective agent with potential therapeutic value.

Equatorial Spitting Cobra (Naja sumatrana) from Malaysia (Negeri Sembilan and Penang), Southern Thailand, and Sumatra: Comparative Venom Proteomics, Immunoreactivity and Cross-Neutralization by Antivenom.

The Equatorial Spitting Cobra (Naja sumatrana) is a medically important venomous snake species in Southeast Asia. Its wide geographical distribution implies potential intra-specific venom variation, while there is no species-specific antivenom available to treat its envenoming. Applying a protein-decomplexing proteomic approach, the study showed that three-finger toxins (3FTX), followed by phospholipases A2 (PLA2), were the major proteins well-conserved across N. sumatrana venoms of different locales. Variations were noted in the subtypes and relative abundances of venom proteins. Of note, alpha-neurotoxins (belonging to 3FTX) are the least in the Penang specimen (Ns-PG, 5.41% of total venom proteins), compared with geographical specimens from Negeri Sembilan (Ns-NS, 14.84%), southern Thailand (Ns-TH, 16.05%) and Sumatra (Ns-SU, 10.81%). The alpha-neurotoxin abundance, in general, correlates with the venom's lethal potency. The Thai Naja kaouthia Monovalent Antivenom (NkMAV) was found to be immunoreactive toward the N. sumatrana venoms and is capable of cross-neutralizing N. sumatrana venom lethality to varying degrees (potency = 0.49-0.92 mg/mL, interpreted as the amount of venom completely neutralized per milliliter of antivenom). The potency was lowest against NS-SU venom, implying variable antigenicity of its lethal alpha-neurotoxins. Together, the findings suggest the para-specific and geographical utility of NkMAV as treatment for N. sumatrana envenoming in Southeast Asia.

Snake Venomics and Antivenomics of Cape Cobra (Naja nivea) from South Africa: Insights into Venom Toxicity and Cross-Neutralization Activity.

Naja nivea (Cape Cobra) is endemic to southern Africa. Envenoming by N. nivea is neurotoxic, resulting in fatal paralysis. Its venom composition, however, has not been studied in depth, and specific antivenoms against it remain limited in supply. Applying a protein decomplexation approach, this study unveiled the venom proteome of N. nivea from South Africa. The major components in the venom are cytotoxins/cardiotoxins (~75.6% of total venom proteins) and alpha-neurotoxins (~7.4%), which belong to the three-finger toxin family. Intriguingly, phospholipase A2 (PLA2) was undetected-this is a unique venom phenotype increasingly recognized in the African cobras of the Uraeus subgenus. The work further showed that VINS African Polyvalent Antivenom (VAPAV) exhibited cross-reactivity toward the venom and immunorecognized its toxin fractions. In mice, VAPAV was moderately efficacious in cross-neutralizing the venom lethality with a potency of 0.51 mg/mL (amount of venom completely neutralized per milliliter of antivenom). In the challenge-rescue model, VAPAV prevented death in 75% of experimentally envenomed mice, with slow recovery from neurotoxicity up to 24 h. The finding suggests the potential para-specific utility of VAPAV for N. nivea envenoming, although a higher dose or repeated administration of the antivenom may be required to fully reverse the neurotoxic effect of the venom.

On characterizing the Red-headed Krait (Bungarus flaviceps) venom: Decomplexation proteomics, immunoreactivity and toxicity cross-neutralization by hetero-specific antivenoms.

The Red-headed Krait (Bungarus flaviceps) is a medically important venomous snake species in Southeast Asia, while there is no specific antivenom available for its envenoming. This study investigated the venom composition through a decomplexation proteomic approach, and examined the immunoreactivity as well as cross-neutralization efficacy of two hetero-specific krait antivenoms, Bungarus candidus Monovalent Antivenom (BcMAV) and Bungarus fasciatus Monovalent Antivenom (BfMAV), against the venom of B. flaviceps from Peninsular Malaysia. A total of 43 non-redundant proteoforms belonging to 10 toxin families were identified in the venom proteome, which is dominated by phospholipases A2 including beta-bungarotoxin lethal subunit (56.20 % of total venom proteins), Kunitz-type serine protease inhibitors (19.40 %), metalloproteinases (12.85 %) and three-finger toxins (7.73 %). The proteome varied in quantitative aspect from the earlier reported Indonesian (Sumatran) sample, suggesting geographical venom variation. BcMAV and BfMAV were immunoreactive toward the B. flaviceps venom, with BcMAV being more efficacious in immunological binding. Both antivenoms cross-neutralized the venom lethality with varying efficacy, where BcMAV was more potent than BfMAV by ~13 times (normalized potency: 38.04 mg/g vs. 2.73 mg/g, defined as the venom amount completely neutralized by one-gram antivenom protein), supporting the potential utility of BcMAV for para-specific neutralization against B. flaviceps venom.

Mucuna pruriens Linn. seed extract pretreatment protects against cardiorespiratory and neuromuscular depressant effects of Naja sputatrix (Javan spitting cobra) venom in rats.

Mucuna pruriens has been used by native Nigerians as a prophylactic for snakebite. The protective effects of M. pruriens seed extract (MPE) were investigated against the pharmacological actions of N. sputatrix (Javan spitting cobra) venom in rats. The results showed that MPE-pretreatment protected against cardiorespiratory and, to a lesser extent, neuromuscular depressant effects of N. sputatrix venom. These may be explained at least in part by the neutralisation of the cobra venom toxins by anti-MPE antibodies elicited by the MPE pretreatment.

A Neurotoxic Snake Venom without Phospholipase A2: Proteomics and Cross-Neutralization of the Venom from Senegalese Cobra, Naja senegalensis (Subgenus: Uraeus).

The Senegalese cobra, Naja senegalensis, is a non-spitting cobra species newly erected from the Naja haje complex. Naja senegalensis causes neurotoxic envenomation in Western Africa but its venom properties remain underexplored. Applying a protein decomplexation proteomic approach, this study unveiled the unique complexity of the venom composition. Three-finger toxins constituted the major component, accounting for 75.91% of total venom proteins. Of these, cardiotoxin/cytotoxin (~53%) and alpha-neurotoxins (~23%) predominated in the venom proteome. Phospholipase A2, however, was not present in the venom, suggesting a unique snake venom phenotype found in this species. The venom, despite the absence of PLA2, is highly lethal with an intravenous LD50 of 0.39 µg/g in mice, consistent with the high abundance of alpha-neurotoxins (predominating long neurotoxins) in the venom. The hetero-specific VINS African Polyvalent Antivenom (VAPAV) was immunoreactive to the venom, implying conserved protein antigenicity in the venoms of N. senegalensis and N. haje. Furthermore, VAPAV was able to cross-neutralize the lethal effect of N. senegalensis venom but the potency was limited (0.59 mg venom completely neutralized per mL antivenom, or ~82 LD50 per ml of antivenom). The efficacy of antivenom should be further improved to optimize the treatment of cobra bite envenomation in Africa.

Proteomics and neutralization of Bungarus multicinctus (Many-banded Krait) venom: Intra-specific comparisons between specimens from China and Taiwan.

The Many-banded Krait (Bungarus multicinctus) is a medically important venomous snake in East Asia. This study investigated the venom proteomes of B. multicinctus from Guangdong, southern China (BM-China) and insular Taiwan (BM-Taiwan), and the neutralization activities of two antivenom products (produced separately in China and Taiwan) against the lethal effect of the venoms. The venom proteomes of both specimens contained similar toxin families, notwithstanding small variations in the subtypes and abundances of minor components. More than 90% of the total venom proteins belong to three-finger toxins (3FTx, including alpha-neurotoxins) and phospholipases A2 (PLA2, including beta-bungarotoxins), supporting their key involvement in the pathophysiology of krait envenomation which manifests as pre- and post-synaptic neurotoxicity. The venoms exhibited potent neurotoxic and lethal effects with extremely low i.v. LD50 of 0.027 µg/g (Bm-China) and 0.087 µg/g (Bm-Taiwan), respectively, in mice. Bungarus multicinctus monovalent antivenom (BMMAV) produced in China and Neuro bivalent antivenom (NBAV) produced in Taiwan were immunoreactive toward both venoms and their toxin fractions. The antivenoms neutralized the venom lethality variably, with BMMAV being more efficacious than NBAV by approximately two-fold. Findings suggest that the monovalent antivenom has a higher potency presumably due to its species-specificity toward the krait venom.