Habutobin Splits the Arg16-Gly17 Bond in the Aa Chain of Rabbit Fibrinogen

Kinjoh, Kiyohiko; Kosugi, Tadayoshi; Nakamura, Mariko; Hanashiro, Kazuhiko; Sunagawa, Makoto; Tokeshi, Yoshihiro; Eguchi, Yukinori

doi:10.1055/s-0038-1656124

Cited by 12 publications

(5 citation statements)

References 5 publications

(6 reference statements)

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“…Protobothrops flavoviridis SP2, a complete transcript, is an isoform of a serine protease variously known as habutobin or flavoxobin. It is a strongly anticoagulant thrombin-like enzyme that releases only fibrinopeptide A [ 71 , 72 ], inhibits collagen-induced platelet aggregation [ 73 ], and releases urokinase-tissue plasminogen activator from bovine arterial endothelial cells [ 74 ]. In addition, it functions as a C3-convertase that activates the complement cascade [ 75 ].…”

Section: Resultsmentioning

confidence: 99%

Snake venoms are integrated systems, but abundant venom proteins evolve more rapidly

et al. 2015

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BackgroundWhile many studies have shown that extracellular proteins evolve rapidly, how selection acts on them remains poorly understood. We used snake venoms to understand the interaction between ecology, expression level, and evolutionary rate in secreted protein systems. Venomous snakes employ well-integrated systems of proteins and organic constituents to immobilize prey. Venoms are generally optimized to subdue preferred prey more effectively than non-prey, and many venom protein families manifest positive selection and rapid gene family diversification. Although previous studies have illuminated how individual venom protein families evolve, how selection acts on venoms as integrated systems, is unknown.ResultsUsing next-generation transcriptome sequencing and mass spectrometry, we examined microevolution in two pitvipers, allopatrically separated for at least 1.6 million years, and their hybrids. Transcriptomes of parental species had generally similar compositions in regard to protein families, but for a given protein family, the homologs present and concentrations thereof sometimes differed dramatically. For instance, a phospholipase A2 transcript comprising 73.4 % of the Protobothrops elegans transcriptome, was barely present in the P. flavoviridis transcriptome (<0.05 %). Hybrids produced most proteins found in both parental venoms. Protein evolutionary rates were positively correlated with transcriptomic and proteomic abundances, and the most abundant proteins showed positive selection. This pattern holds with the addition of four other published crotaline transcriptomes, from two more genera, and also for the recently published king cobra genome, suggesting that rapid evolution of abundant proteins may be generally true for snake venoms. Looking more broadly at Protobothrops, we show that rapid evolution of the most abundant components is due to positive selection, suggesting an interplay between abundance and adaptation.ConclusionsGiven log-scale differences in toxin abundance, which are likely correlated with biosynthetic costs, we hypothesize that as a result of natural selection, snakes optimize return on energetic investment by producing more of venom proteins that increase their fitness. Natural selection then acts on the additive genetic variance of these components, in proportion to their contributions to overall fitness. Adaptive evolution of venoms may occur most rapidly through changes in expression levels that alter fitness contributions, and thus the strength of selection acting on specific secretome components.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1832-6) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Snake venoms are integrated systems, but abundant venom proteins evolve more rapidly

et al. 2015

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“…In the rabbit fibrinogen-fibrin conversion induced by them, only fibrinopeptide A is preferentially released, revealing their SVTLE-A profile. Habutobin also does not recognize monkey, dog, rat, or guinea pig fibrinogens [88]. It is interesting to note, and perhaps not surprising, which SVTLE did not clot its own nor any other snake plasma in contrast to human thrombin.…”

Section: Biological Properties Of Svtlementioning

confidence: 96%

“…3). The species specificity of SVTLE clotting activity is not well characterized, with the exception of habutobin from T. flavoviridis and elegaxobin from T. elegans [88,89]. Both enzymes act only upon rabbit fibrinogen, whereas they show no effect on human and bovine fibrinogens.…”

Section: Biological Properties Of Svtlementioning

confidence: 99%

Snake venom thrombin-like enzymes: from reptilase to now

Castro

Zingali

Albuquerque

et al. 2004

Cellular and Molecular Life Sciences (CMLS)

170

123

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The snake venom thrombin-like enzymes (SVTLEs) comprise a number of serine proteases functionally and structurally related to thrombin. Until recently, only nine complete sequences of this subgroup of the serine protease family were known. Over the past 5 years, the primary structure of several SVTLEs has been characterized, and now this family includes several members. Of particular interest is their possible use in pathologies such as thrombosis. The aim of the present review is to summarize the state of the art concerning the evolutionary, structural and biological features of the SVTLEs.

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“…Electroblotting was performed by the method of Hirano and Watanabe [17], as described previously [18]. The protein-blotted membranes were blocked with 1% milk (dried nonfat milk dissolved in D-PBS) overnight at 4°C, and then reacted with rat antirabbit fibrinogen and rat antibovine thrombin (500 Ìg/ml) for 24 h at 4°C.…”

Section: Analysis Of Bound Thrombin By Sds-page and Western Blottingmentioning

confidence: 99%

“…Rabbit fibrinogen (2 mg) was also dissolved in 100 Ìl of the electrophoresis buffer and subjected to SDS-PAGE using a 12% polyacrylamide gel, and then electroblotted onto a PVDF membrane. The blotted proteins on PVDF membranes were subjected to N-terminal sequence analysis, using a Shimadzu PSQ-1 gas-phase sequencer system (Shimadzu Co., Kyoto, Japan), as described previously [18,20].…”

Section: N-terminal Sequence Analysis Of Bound Thrombinmentioning

confidence: 99%

Bound Thrombin from Crushed Clots Is Composed of α-Thrombin and the N-Terminal Regions of α- and γ-Chains of Fibrinogen

Kinjoh

Nakamura²,

Zeng³

et al. 2002

Pathophysiol Haemos Thromb

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We aimed at clarifying the structural characteristics of the bound thrombin that is liberated by mechanical breakdown of fibrin clots. Fibrin clots were prepared with bovine thrombin and rabbit fibrinogen, and were crushed mechanically with a glass rod. The supernatant of the crushed clots was subjected to immunoaffinity chromatography to isolate the bound thrombin. Western blotting analysis revealed that the bound thrombin could be reacted with both antithrombin and antifibrinogen under unreduced conditions. SDS-PAGE under reduced conditions revealed that there were three bands, two of which were found to be the N-terminal fragments of the α- and γ-chains of fibrinogen. The bound thrombin could be dissociated into three distinct fibrin fragments and bovine α-thrombin when denatured by 8 M urea. Thus, the bound thrombin liberated from crushed clots is a stable complex between bovine α-thrombin and fibrin fragments of the N-terminal regions of rabbit α- and γ-chains.

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Habutobin Splits the Arg16-Gly17 Bond in the Aa Chain of Rabbit Fibrinogen

Cited by 12 publications

References 5 publications

Snake venoms are integrated systems, but abundant venom proteins evolve more rapidly

Snake venoms are integrated systems, but abundant venom proteins evolve more rapidly

Snake venom thrombin-like enzymes: from reptilase to now

Bound Thrombin from Crushed Clots Is Composed of α-Thrombin and the N-Terminal Regions of α- and γ-Chains of Fibrinogen

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