Fluorometric assay using naphthylamide substrates for assessing novel venom peptidase activities

Gasparello-Clemente, Elaine; Silveira, Paulo Flávio

doi:10.1016/s0041-0101(02)00180-0

Cited by 28 publications

(16 citation statements)

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“…The presence of these peptidases could in fact contribute to the degradation/activation of many different pharmacological and physiopathological processes in the victims of spider bites. Regardless, the presence of some aminopeptidases has been previously reported in the venom of L. intermedia [27]. Whether these enzymes are either naturally from spider venom or secreted by bacteria which colonize the venom still remains to be determined.…”

Section: Resultsmentioning

confidence: 97%

Brown Recluse Spider Venom: Proteomic Analysis and Proposal of a Putative Mechanism of Action

Santos¹,

Dias²,

Roberto³

et al. 2009

PPL

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Section: Resultsmentioning

confidence: 97%

Brown Recluse Spider Venom: Proteomic Analysis and Proposal of a Putative Mechanism of Action

Santos¹,

Dias²,

Roberto³

et al. 2009

PPL

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“…APs are exo-metalloproteases that function in the physiological maintenance of the blood pressure (Vaiyapuri et al, 2010), while DPP-IV is a highly glycosylated serine protease that may counteract the hypertensive response in the envenomed prey by destroying hypertensive peptidyl hormones (Aird, 2008). The level of expression of these three putative toxins was very low (<0.03%) and the expressed proteins were not detectable even with the use of highly sensitive nano-LCMS/MS technique (Tan et al, 2015d), although some authors reported the presence of AP protein in certain snake venoms (Faiz et al, 1996; Gasparello-Clemente & Silveira, 2002). For N. kaouthia , full sequences of DPP-IV and IGF were available from the present study and these are the only ones reported from the venom gland transcriptomes of Naja species thus far.…”

Section: Resultsmentioning

confidence: 99%

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

Tan

Chanhome

et al. 2017

PeerJ

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BackgroundThe monocled cobra (Naja kaouthia) is a medically important venomous snake in Southeast Asia. Its venom has been shown to vary geographically in relation to venom composition and neurotoxic activity, indicating vast diversity of the toxin genes within the species. To investigate the polygenic trait of the venom and its locale-specific variation, we profiled and compared the venom gland transcriptomes of N. kaouthia from Malaysia (NK-M) and Thailand (NK-T) applying next-generation sequencing (NGS) technology.MethodsThe transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T.Results and discussionThe toxin transcripts showed high redundancy (41–82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin’s fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the exclusively and abundantly expressed cytotoxin CTX-3 in NK-T. The findings suggested correlation with the geographical variation in proteome and toxicity of the venom, and support the call for optimising antivenom production and use in the region. Besides, the current study uncovered full and partial sequences of numerous toxin genes from N. kaouthia which have not been reported hitherto; these include N. kaouthia-specific l-amino acid oxidase (LAAO), snake venom serine protease (SVSP), cystatin, acetylcholinesterase (AChE), hyaluronidase (HYA), waprin, phospholipase B (PLB), aminopeptidase (AP), neprilysin, etc. Taken together, the findings further enrich the snake toxin database and provide deeper insights into the genetic diversity of cobra venom toxins.

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“…The aminopeptidase activity of the B. g. rhinoceros venom as a whole is different from that of the purified protein; the detection of APL and APB activity even in the presence of calcium suggests the presence of further aminopeptidases in this venom. APL and APB activities have been reported in the venoms of several other snakes [4], [6], [7], [8] but to date no-one has identified the specific enzymes responsible.…”

Section: Discussionmentioning

confidence: 99%

“…For example aminopeptidase L (APL) removes an N-terminal leucine residue, aminopeptidase A (APA) removes an acidic N-terminal residue, aminopeptidase B (APB) removes a basic N-terminal residue, and aminopeptidase N (APN) removes a neutral N-terminal residue, typically alanine. There have been several reports of aminopeptidase activities present in venoms from elapids and vipers [3], [4], [5], [6], [7], [8], and a fraction exhibiting aminopeptidase A activity has been separated from the venom of Gloydius blomhoffi brevicaudus , a member of the Crotalinae (pit viper) subfamily of vipers [7]. A cDNA sequence from this snake represents the only determined sequence to date of a reptile venom aminopeptidase A. Interestingly, none of the complete snake venom proteomic studies done thus far has identified aminopeptidases [9].…”

Section: Introductionmentioning

confidence: 99%

Purification and Functional Characterisation of Rhiminopeptidase A, a Novel Aminopeptidase from the Venom of Bitis gabonica rhinoceros

et al. 2010

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BackgroundSnake bite is a major neglected public health issue within poor communities living in the rural areas of several countries throughout the world. An estimated 2.5 million people are bitten by snakes each year and the cost and lack of efficacy of current anti-venom therapy, together with the lack of detailed knowledge about toxic components of venom and their modes of action, and the unavailability of treatments in rural areas mean that annually there are around 125,000 deaths worldwide. In order to develop cheaper and more effective therapeutics, the toxic components of snake venom and their modes of action need to be clearly understood. One particularly poorly understood component of snake venom is aminopeptidases. These are exo-metalloproteases, which, in mammals, are involved in important physiological functions such as the maintenance of blood pressure and brain function. Although aminopeptidase activities have been reported in some snake venoms, no detailed analysis of any individual snake venom aminopeptidases has been performed so far. As is the case for mammals, snake venom aminopeptidases may also play important roles in altering the physiological functions of victims during envenomation. In order to further understand this important group of snake venom enzymes we have isolated, functionally characterised and analysed the sequence-structure relationships of an aminopeptidase from the venom of the large, highly venomous West African gaboon viper, Bitis gabonica rhinoceros. Methodology and Principal Findings The venom of B. g. rhinoceros was fractionated by size exclusion chromatography and fractions with aminopeptidase activities were isolated. Fractions with aminopeptidase activities showed a pure protein with a molecular weight of 150 kDa on SDS-PAGE. In the absence of calcium, this purified protein had broad aminopeptidase activities against acidic, basic and neutral amino acids but in the presence of calcium, it had only acidic aminopeptidase activity (APA). Together with the functional data, mass spectrometry analysis of the purified protein confirmed this as an aminopeptidase A and thus this has been named as rhiminopeptidase A. The complete gene sequence of rhiminopeptidase A was obtained by sequencing the PCR amplified aminopeptidase A gene from the venom gland cDNA of B. g. rhinoceros. The gene codes for a predicted protein of 955 amino acids (110 kDa), which contains the key amino acids necessary for functioning as an aminopeptidase A. A structural model of rhiminopeptidase A shows the structure to consist of 4 domains: an N-terminal saddle-shaped β domain, a mixed α and β catalytic domain, a β-sandwich domain and a C-terminal α helical domain.ConclusionsThis study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, ...

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Fluorometric assay using naphthylamide substrates for assessing novel venom peptidase activities

Cited by 28 publications

References 96 publications

Brown Recluse Spider Venom: Proteomic Analysis and Proposal of a Putative Mechanism of Action

Brown Recluse Spider Venom: Proteomic Analysis and Proposal of a Putative Mechanism of Action

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

Purification and Functional Characterisation of Rhiminopeptidase A, a Novel Aminopeptidase from the Venom of Bitis gabonica rhinoceros

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