New insights into the structural elements involved in the skin haemorrhage induced by snake venom metalloproteinases

Abstract

Haemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon resulting in capillary disruption and extravasation. This study analysed structural elements important for the interaction of four Bothrops jararaca SVMPs of different domain organisation and glycosylation levels with plasma and extracellular matrix proteins: HF3 (P-III class) is highly glycosylated and ~80 times more haemorrhagic than bothropasin (P-III class), which has a minor carbohydrate moiety; BJ-PI (P-I class) is not haemorrhagic and the DC protein is composed of disintegrin-like/cysteine-rich domains of bothropasin. HF3, bothropasin and BJ-PI showed different degradation profiles of fibrinogen, fibronectin, vitronectin, von Willebrand factor, collagens IV and VI, laminin and Matrigel; however, only bothropasin degraded collagen I. In solid-phase binding assays HF3 and bothropasin interacted with fibrinogen, fibronectin, laminin, collagens I and VI; the DC protein bound only to collagens I and VI; however, no binding of BJ-PI to these proteins was detected. N-deglycosylation caused loss of structural stability of bothropasin and BJ-PI but HF3 remained intact, although its haemorrhagic and fibrinogenolytic activities were partially impaired. Nevertheless, N-deglycosylated HF3 bound with higher affinity to collagens I and VI, although its proteolytic activity upon these collagens was not enhanced. This study demonstrates that features of carbohydrate moieties of haemorrhagic SVMPs may play a role in their interaction with substrates of the extracellular matrix, and the ability of SVMPs to degrade proteins in vitro does not correlate to their ability to cause haemorrhage, suggesting that novel, systemic approaches are necessary for understanding the mechanism of haemorrhage generation by SVMPs.

Overlooked Issues of Snakebite Management: Time for Strategic Approach

“…The P-IV SVMPs are high molecular mass proteinases and have a similar domain structure to that of P-III class but with an additional C-type lectin domain. In contrast to the P-I class of SVMPs that are readily inhibited by the human 2-macroglobulin an endogenous protease inhibitor; the P-III class are not inhibited by 2-macroglobulin [57][58][59][60][61][62][63][64].…”

Identification of Novel Interaction between ADAM17 (a Disintegrin and Metalloprotease 17) and Thioredoxin-1

“…Solid Phase Binding Assay-One g of purified His-FLAGADAM17cyto or His-Trx-1-HA were immobilized into a 96-well polystyrene High Bind microtiter plate (Corning Glass) in a 0.05 M sodium carbonate buffer, pH 9.6, with gentle agitation overnight at 4°C, as described by Oliveira et al (29). The wells were washed three times with wash solution (PBS supplemented with 0.05% Tween 20) and then blocked with PBS containing 5% nonfat dry milk for 2 h at room temperature.…”

Análise proteômica da atividade proteolítica do HF3, uma metaloproteinase do veneno da Bothrops jararaca, no plasma humano e de serpente

“…No domínio tipo-disintegrina, a sequência RGD encontrada nos domínios disintegrina é substituída pela sequência SECD (Silva et al, 2004 (Assakura et al, 1986;Oliveira et al, 2009). O HF3 apresenta atividade proteolítica sobre a caseína e o fibrinogênio, com atividades específicas de 17±0.006 U/mg e 1.3±0.17 U/mg, respectivamente (Oliveira et al, 2009) e in vitro gera diferentes perfis de degradação de proteínas do plasma e proteínas da matriz extracelular, importantes para a manutenção da integridade dos vasos capilares e hemostasia, como fibrinogênio, fibronectina, vitronectina, fator de Willebrand, colágenos IV e VI, laminina e Matrigel™ (Oliveira et al, 2010 (Oliveira et al, 2010). O HF3 também é capaz de inibir a agregação plaquetária induzida por colágeno e tem efeito pró-inflamatório, estimulando a fagocitose por macrófagos mediada pela integrina αMβ2 (Silva et al, 2004).…”

“…Foram identificados 8 peptídeos da cadeia gama do fibrinogênio, incluindo os peptídeos localizados na região C-terminal, confirmando os dados obtidos pela incubação do HF3 com o plasma. Estes dados também corroboram com estudos anteriores que descrevem a clivagem mais extensiva da cadeia alfa, seguida por clivagem mais limitada da cadeia beta do fibrinogênio (Oliveira et al, 2010), pelo HF3. A cadeia gama do fibrinogênio não havia sido descrita como substrato do HF3.…”

“…Ainda que as cadeias alfa e beta fibrinogênio já tenham sido descritas como substratos do HF3 (Oliveira et al, 2010;Paes Leme et al, 2012), neste estudo a cadeia gama do fibrinogênio foi também detectada como um alvo desta metaloproteinase. Peptídeos oriundos da cadeia gama do fibrinogênio foram identificados como produto da atividade do HF3 no P(dep20) e P(enrq) (8)(9).…”