Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans

You, Weon Kyoo; Sohn, Young Doug; Kim, Ki-Yong; Park, Doo Hong; Jang, Yangsoo; Chung, Kyeong Woo

doi:10.1016/j.ibmb.2003.10.003

Cited by 42 publications

(25 citation statements)

References 36 publications

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“…Over the last 10 years, several effective thrombolytic agents have been identified and characterized from microorganisms [4][5][6], earthworms [7], snake venoms [8,9], centipede venoms [10], insects [11], and leeches [12]. The agents are of interest as useful tools for understanding fibrinolytic mechanism and as potential therapeutic drugs.…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Wang

Лі

et al. 2006

J IND MICROBIOL BIOTECHNOL

150

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Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55°C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS-PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), b-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-PhepNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bb-chains and Aa-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.

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Section: Introductionmentioning

confidence: 99%

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Wang

Лі

et al. 2006

J IND MICROBIOL BIOTECHNOL

150

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“…However, while TNGYT prolongs whole blood clotting time and bleeding time in mice, its potency is well below the range that would make it a useful therapeutic lead. The venom of this centipede also contains a serine protease with fibrinolytic activity [95].…”

Section: Expect the Unexpected: Centipede-venom Compounds With Anti-tmentioning

confidence: 99%

Centipede venoms as a source of drug leads

Undheim

Jenner

King

2016

Expert Opinion on Drug Discovery

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Introduction: Centipedes are one of the oldest and most successful lineages of venomous terrestrial predators. Despite their use for centuries in traditional medicine, centipede venoms remain poorly studied. However, recent work indicates that centipede venoms are highly complex chemical arsenals that are rich in disulfide-constrained peptides that have novel pharmacology and three-dimensional structure. Areas covered:This review summarizes what is currently know about centipede venom proteins, with a focus on disulfide-rich peptides that have novel or unexpected pharmacology that might be useful from a therapeutic perspective. We also highlight the remarkable diversity of constrained threedimensional peptide scaffolds present in these venoms that might be useful for bioengineering of drug leads. Expert opinion:The resurgence of interest in peptide drugs has stimulated interest in venoms as a source of highly stable, disulfide-constrained peptides with potential as therapeutics. Well-studied venomous taxa such as cone snails and snakes have yielded FDA-approved drugs, while ancient invertebrate predators such as spiders and sea anemones have yielded molecules that are currently in preclinical studies and clinical trials, respectively. However, until recently, the paucity of research on centipede venoms made it easy to discount them as a potential source of peptides with therapeutically useful properties. Seminal studies over the past five years have revealed that centipede venoms have exceedingly complex proteomes that are perhaps richer than any other venom in unique disulfide-rich peptide scaffolds. Like most arthropod predators, centipede venoms are rich in peptides that target neuronal ion channels and receptors, but it is also becoming increasingly apparent that many of these peptides have novel or unexpected pharmacological properties with potential applications in drug discovery and development.

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“…1a) by RT-PCR using specific primers ( Table 1) that were designed according to the conserved catalytic amino acids of serine protease: histidine (primer H) and serine (primer S) [11]. The expected PCR product (443 bp) showed a single sharp band for the partial CVSP sequence (Fig.…”

Section: Full-length Cdna Determinationmentioning

confidence: 99%

“…These primers were designed according to the conserved catalytic amino acids for serine proteases: histidine (named primerH) and serine (primerS) [11]. The gene was amplified using a PCR master mix reagent kit (Thermo Scientific, USA).…”

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confidence: 99%

Molecular cloning and sequence analysis of serine protease cDNA from the venom of the centipede Scolopendra subspinipes dehaani

Meunchan¹,

Thammasirirak²,

Daduang³

et al. 2015

Turk J Bioch

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Molecular cloning and sequence analysis of serine protease cDNA from the venom of the centipede Scolopendra subspinipes dehaani [Scolopendra subspinipes dehaani kırkayak zehrinden elde edilen serin proteaz cDNA'sının moleküler klonlaması ve dizi analizi] ABSTRACT Objective: Proteolytic enzymes are one of the well-known components of animal venom. The aim of this study was to identify protease from the venom gland of Scolopendra subspinipes dehaani. Methods: After total RNA extraction, a cDNA encoding a serine protease was identified by RT-PCR and was subsequently sequenced. The sequence was analysed by multiple alignment and homology modelling. Results: A cDNA encoding the precursor of a centipede venom serine protease was identified. The full-length cDNA was 1,014 nucleotides with 780 nucleotides of open reading frame. The precursor nucleotide sequence encoded a signal peptide of 19 residues and a mature protein of 260 residues. It belonged to clan PA of serine proteases in the MEROPS database classification. Catalytic residues were identified by multiple alignments, including an unusual hydrogen bond network in the catalytic site. Secondary structure prediction and homology modelling revealed a chymotrypsin-like fold, which is characteristic of PA clan proteases. Conclusion:The sequence of the identified serine protease showed unusual features, making this centipede venom serine protease an interesting candidate to investigate. A better understanding of the molecular mechanism of venom components will be useful for further applications and help to improve envenomation treatment. Key Words: Scolopendra subspinipes dehaani, cDNA, serine protease Conflict of Interest: The authors have no conflict of interest. ÖZETAmaç: Proteolitik enzimler hayvan zehirlerinin en çok bilinen bileşenlerinden biridir. Bu ça-lışmanın amacı Scolopendra subspinipes dehaani zehir salgı bezinden proteaz enziminin tanımlanmasıdır. Metod: Toplam RNA eldesinin ardından, RT-PCR tekniği kullanılarak serin proteaz proteinini kodlayan gene ait cDNA tanımlanmış ve dizi analizine tabi tutulmuştur. Elde edilen dizi çoklu dizi hizalama ve homoloji modelleme ile analiz edilmiştir. Bulgular: Kırkayak zehirinde bulunan serin proteaz öncülünü kodlayan bir cDNA tanımlan-mıştır. cDNA, 780 nükleotit açık okuma çerçevesinde olmak üzere, toplam 1.014 nükleotit uzunluğundadır. Öncül nükleotit dizisi 19 amino asitlik bir sinyal peptidini ve 260 amino asitlik olgun proteini kodlamaktadır. Bu protein MEROPS veritabanı sınıflandırmasına göre serin proteazlardan PA grubuna aittir. Çoklu dizi hizalama kullanılarak alışılmadık bir hidrojen bağı örgüsüne sahip katalitik bölge ve bu bölgedeki katalitik aminoasitler tanımlanmıştır. İkincil yapı tahmin ve homoloji modelleme çalışmaları PA grup proteazlara özgün olan kimotiripsin benzeri katlanmayı göstermiştir. Sonuç: Tanımlanan serin proteaz dizisi alışılmadık özellikler göstermesi sebebiyle kırkayak zehrindeki serin proteaz enzimini ileri çalışmalar için aday yapmıştır. Venom bileşenlerinin moleküler mekanizmalarının...

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Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans

Cited by 42 publications

References 36 publications

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Centipede venoms as a source of drug leads

Molecular cloning and sequence analysis of serine protease cDNA from the venom of the centipede Scolopendra subspinipes dehaani

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