BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility

Silva, Floriano Paes; Guedes, Herbert L.M.; Garvey, Laura C.; Aguiar, Aniesse Silva; Bourguignon, Saulo Cabral; Di, Enrico; De-Simone, Salvatore Giovanni

doi:10.1016/j.toxicon.2007.02.018

Cited by 42 publications

(30 citation statements)

References 44 publications

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“…Further identification of the glycoside sequence of thrombin was not undertaken on account of the presence of hardly any data on its effect on the enzymatic and cellular activity of thrombin [12]. However, present-day investigations of thrombin-like proteases from snake venoms demonstrate the importance of the presence of the glycosidic chains in the structure of the enzyme during their thermal treatment [13]. Therefore the sequence and function of the oligosaccharide residue of thrombin can play an important role during the action of other factors, such as various types of radiation for example, on the enzyme.…”

Section: Resultsmentioning

confidence: 99%

Behavior of serine proteases thrombin, trypsin, and chymotrypsin under conditions of MALDI-TOF-Mass spectrometry

et al. 2008

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Features of ion formation under the influence of laser emission under the conditions of MALDI-TOF mass spectrometry in the glycosylated protein thrombin and the related proteases trypsin and chymotrypsin were studied for the first time. It was shown that trypsin and chymotrypsin are ionized in the form of molecular ions with masses 23800 and 25660 Da respectively. The molecular ion of thrombin was not detected; three main fragment ions with masses 9951, 10230, and 11982 Da were detected in the mass spectrum of thrombin. It was suggested that these fragments relate to peptide and glycopeptides fragment ions formed as a result of bond cleavage in the region of Asn60G during exposure to laser emission under the conditions of MALDI-TOF mass spectrometry.Today the methods of tandem mass spectrometry have aroused considerable interest among investigators throughout the world. They have been used not only to determine the molecular mass of an investigated sample but also to study the various mechanisms of intermolecular reactions.In spite of this on account of the increased complexity of the interpretation of the results due attention as not been paid to investigation of the ionization of high-molecular natural compounds.A special position among contemporary methods of mass-spectrometric analysis is occupied by matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI TOF MS). The method is maybe very young. It was proposed in 1988It was proposed in -1989, where UV-laser desorption was first used for the analysis of bioorganic compounds with molecular masses of more than 10 kDa.The main application of MALDI-TOF mass spectrometry is in the field of proteomics, the aim of which is the study of protein structure.The majority of the proteins taking part in particularly important biochemical processes are glycosylated, and analysis of their post-translational modification is also an extremely important aspect of proteomic investigations. Of particular interest are the proteases -enzymes that catalyze the selective cleavage of a protein chain and thereby regulate vitally important 0040-5760/08/4402-0075

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Section: Resultsmentioning

confidence: 99%

Behavior of serine proteases thrombin, trypsin, and chymotrypsin under conditions of MALDI-TOF-Mass spectrometry

et al. 2008

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“…The resistance of SVSPs to variations in temperature and pH is probably due to their three-dimensional structure, which is stabilized by six disulfide bonds, what increases the threshold energy required to generate significant conformational changes in the molecular scaffold. In addition, some SVSPs, such as BJ-48 from Bothrops jararacussu, protease A from Bothrops jararaca, and brevinase from Agkistrodon blomhoffi brevicaudus, completely or partially lost their thermal stability when deglycosylated, indicating that the carbohydrate motif play important role in their resistance to heat denaturation (Silva-Junior et al 2007;Paes-Leme et al 2008;Lee et al 1999).…”

Section: Discussionmentioning

confidence: 99%

Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme

Boldrini-França

Rodrigues

Santos-Silva

et al. 2015

Appl Microbiol Biotechnol

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Snake venom serine proteases (SVSPs) act primarily on plasma proteins related to blood clotting and are considered promising for the treatment of several hemostatic disorders. We report the heterologous expression of a serine protease from Crotalus durissus collilineatus, named collinein-1, in Pichia pastoris, as well as the enzymatic comparative characterization of the toxin in native and recombinant forms. The complementary DNA (cDNA) encoding collinein-1 was amplified from cDNA library of C. d. collilineatus venom gland and cloned into the pPICZαA vector. The recombinant plasmid was used to transform cells of KM71H P. pastoris. Heterologous expression was induced by methanol and yielded 56 mg of recombinant collinein-1 (rCollinein-1) per liter of culture. The native collinein-1 was purified from C. d. collilineatus venom, and its identity was confirmed by amino acid sequencing. The native and recombinant enzymes showed similar effects upon bovine fibrinogen by releasing preferentially fibrinopeptide A. Although both enzymes have induced plasma coagulation, native Colinein-1 has shown higher coagulant activity. The serine proteases were able to hydrolyze the chromogenic substrates S-2222, S-2238, and S2302. Both enzymes showed high stability on different pH and temperature, and their esterase activities were inhibited in the presence of Zn2+ and Cu2+. The serine proteases showed similar k cat/K m values in enzyme kinetics assays, suggesting no significant differences in efficiency of these proteins to hydrolyze the substrate. These results demonstrated that rCollinein-1 was expressed with functional integrity on the evaluated parameters. The success in producing a functionally active recombinant SVSP may generate perspectives to their future therapeutic applications.

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“…For example, metalloproteinase comprised of a series of zinc-containing protease, which cause bleeding, as well as muscle necrosis, skin lesions, edema and other inflammation [23,24] , is identified to be glycosylated in all the three venoms (Table S1). Another important protein in snake venom is snake venom serine proteinase (SVSP), which has a close relationship with coagulation system [25] and also has been detected to be glycoprotein in Vipera russelii siamensis Smith venoms. Since the majority of functional proteins in snake venoms are glycoproteins, the study of glycosylation in addition to functional activities is important for better understanding the complexity of the envenomation cases caused by different species as well as the variability of the venom itself, which will be helpful for the basic snake venom study and the management of snake envenomation.…”

Section: Discussionmentioning

confidence: 99%

Global insight into N-glycome and N-glycoproteome of three most abundant snake venoms in Asia

Cao

Huang

Cao

et al. 2014

Chem. Res. Chin. Univ.

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Snake venom is a complex cocktail including a variety of biological active proteins and proteinaceous components, which have considerable medical and pharmacological importance. N-Glycosylation is widely implicated as a common modification in numerous venom proteins and impacts the in vivo venomic functions. However, systematic survey of N-glycome and N-glycoproteome on snake venoms has not been undertaken. In this study, employing combination of N-glycomics and N-glycoproteomics strategies, we explored the N-glycosylation including both N-glycoproteins and N-glyco-chains in three venoms from Agkistrodon blomhoffii, Naja naja atra Cantor and Vipera russelii siamensis Smith, respectively, which are amongst the most abundant venomous snakes in Asia. As a result, numbers of N-glycoproteins and N-glycans were identified. However, the overlaps of N-glycoproteins and N-glycans of the three venoms were small. Thus, the exploration results of N-glycome and N-glycoproteome indicate that N-glycosylation increases the complexity and variety of the three venoms. Our research provided some new horizons for the comprehensive understanding of venoms variation, which is helpful for the basic venom research as well as the management of snake envenomation.

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BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility

Cited by 42 publications

References 44 publications

Behavior of serine proteases thrombin, trypsin, and chymotrypsin under conditions of MALDI-TOF-Mass spectrometry

Behavior of serine proteases thrombin, trypsin, and chymotrypsin under conditions of MALDI-TOF-Mass spectrometry

Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme

Global insight into N-glycome and N-glycoproteome of three most abundant snake venoms in Asia

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