Activation of leukocyte rolling by the cysteine-rich domain and the hyper-variable region of HF3, a snake venom hemorrhagic metalloproteinase (original) (raw)
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Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain
Biochemical and Biophysical Research Communications, 2019
Snake venom metalloproteinases (SVMPs) are key toxins involved in local inflammatory reactions after snakebites. This study aimed to investigate the effect of SVMP domains on the alterations in leukocyteendothelium interactions in the microcirculation of mouse cremaster muscle. We studied three toxins: BnP1, a PI-toxin isolated from Bothrops neuwiedi venom, which only bears a catalytic domain; Jararhagin (Jar), a PIII-toxin isolated from Bothrops jararaca venom with a catalytic domain, as well as ECDdisintegrin and cysteine-rich domains; and Jar-C, which is produced from the autolysis of Jar and devoid of a catalytic domain. All these toxins induced an increase in the adhesion and migration of leukocytes. By inhibiting the catalytic activity of Jar and BnP1 with 1.10-phenanthroline (oPhe), leukocytes were no longer recruited. Circular dichroism analysis showed structural changes in oPhe-treated Jar, but these changes were not enough to prevent the binding of Jar to collagen, which occurred through the ECD-disintegrin domain. The results showed that the catalytic domain of SVMPs is the principal domain responsible for the induction of leukocyte recruitment and suggest that the other domains could also present inflammatory potential only when devoid of the catalytic domain, as with Jar-C.
Collagen binding is a key factor for the hemorrhagic activity of snake venom metalloproteinases
Biochimie, 2008
Snake venom metalloproteinases (SVMPs) are multifunctional enzymes involved in several symptoms following snakebite, such as severe local hemorrhage. Multidomain P-III SVMPs are strongly hemorrhagic, whereas single domain P-I SVMPs are not. This indicates that disintegrin-like and cysteine-rich domains allocate motifs that enable catalytic degradation of ECM components leading to disruption of capillary vessels. Interestingly, some P-III SVMPs are completely devoid of hemorrhagic activity despite their highly conserved disintegrin-like and cysteine-rich domains. This observation was approached in the present study by comparing the effects of jararhagin, a hemorrhagic P-III SVMP, and berythractivase, a procoagulant and non-hemorrhagic P-III SVMP. Both toxins inhibited collagen-induced platelet aggregation, but only jararhagin was able to bind to collagen I with high affinity. The monoclonal antibody MAJar 3, that neutralizes the hemorrhagic effect of Bothrops venoms and jararhagin binding to collagen, did not react with berythractivase. The three-dimensional structures of jararhagin and berythractivase were compared to explain the differential binding to collagen and MAJar 3. Thereby, we pinpointed a motif within the Da disintegrin subdomain located opposite to the catalytic domain. Jararhagin binds to both collagen I and IV in a triple helix-dependent manner and inhibited in vitro fibrillogenesis. The jararhaginecollagen complex retained the catalytic activity of the toxin as observed by hydrolysis of fibrin. Thus, we suggest that binding of hemorrhagic SVMPs to collagens I and IV occurs through a motif located in the Da subdomain. This allows accumulation of toxin molecules at the site of injection, close to capillary vessels, where their catalytic activity leads to a local hemorrhage. Toxins devoid of this motif would be more available for vascular internalization leading to systemic pro-coagulant effects. This reveals a novel function of the disintegrin domain in hemorrhage formation. Abbreviations: ADAM, a disintegrin and metalloproteinase; ECM, extracellular matrix; MMP, matrix metalloproteinase; SVMP, snake venom metalloproteinase; VAP-1, vascular apoptosis-inducing protein-1. * Corresponding author. Tel.: þ55 11 3726 7222x2131; fax: þ55 11 3726 1505. E-mail address: anamoura@butantan.gov.br (A.M. Moura-da-Silva).
FEBS Letters, 2003
Atrolysin A and jararhagin are class P-III snake venom metalloproteinases (SVMPs) with three distinct domains: a metalloproteinase, a disintegrin-like and a cysteinerich. The metalloproteinase and the disintegrin-like domains of atrolysin A and jararhagin contain peptide sequences that interact with K K2L L1 integrin and inhibit the platelet responses to collagen. Recently, the recombinant cysteine-rich domain of atrolysin A was shown to have similar e¡ects, but the sequence(s) responsible for this is unknown. In this report, we demonstrate two complete peptide sequences from the homologous cysteine-rich domains of atrolysin A and jararhagin that inhibit both platelet aggregation by collagen and adhesion of K K2-expressing K562 cells to this protein. In addition, the peptide e¡ects on platelets do not seem to involve an inhibition of GPVI. These results identify, for the ¢rst time, sites in the cysteine-rich domain of SVMPs that inhibit cell responses to collagen and reveal the complexity of the potential biological e¡ects of these enzymes with multifunctional domains.
Toxins, 2021
Snake venom metalloproteinases (SVMP) are involved in local inflammatory reactions observed after snakebites. Based on domain composition, they are classified as PI (pro-domain + proteolytic domain), PII (PI + disintegrin-like domains), or PIII (PII + cysteine-rich domains). Here, we studied the role of different SVMPs domains in inducing the expression of adhesion molecules at the microcirculation of the cremaster muscle of mice. We used Jararhagin (Jar)—a PIII SVMP with intense hemorrhagic activity, and Jar-C—a Jar devoid of the catalytic domain, with no hemorrhagic activity, both isolated from B. jararaca venom and BnP-1—a weakly hemorrhagic P1 SVMP from B. neuwiedi venom. Toxins (0.5 µg) or PBS (100 µL) were injected into the scrotum of mice, and 2, 4, or 24 h later, the protein and gene expression of CD54 and CD31 in the endothelium, and integrins (CD11a and CD11b), expressed in leukocytes were evaluated. Toxins induced significant increases in CD54, CD11a, and CD11b at the ini...
Archives of Biochemistry and Biophysics, 2000
The P-III class of venom metalloproteinases has, in addition to the proteinase domain, a disintegrin-like domain and a cysteine-rich domain. Recent evidence has shown that the nonproteinase domains of the P-III class of hemorrhagic metalloproteinases function in the inhibition of platelet aggregation by blocking essential procoagulant integrins on platelets. A specific role for the highly conserved cysteine-rich domain has yet to be described. In this study, we expressed the cysteine-rich domain from the hemorrhagic metalloproteinase atrolysin A and demonstrated its ability to inhibit collagen-stimulated platelet aggregation. Additionally, the cysteine-rich domain was shown to interact with MG-63 cells to inhibit adhesion to collagen I. These data suggest a functional role for the cysteinerich domain of the P-III toxins in the observed coagulopathy by targeting the toxin to platelets and inhibiting collagen-stimulated platelet aggregation. These characteristics may function to synergistically increase the hemorrhagic effect of the toxins. . 2 Abbreviations used: ABTS, 2,2Ј-azino-di(3-ethylbenzthiozolinesulfonic acid); AcNMPV, Autographa californica nuclear polyhedrosis virus; MALD-TOF, matrix-assisted laser desorption time of flight; PBS, phosphate-buffered saline, pH 7.4; PMSF, phenylmethylsulfonyl fluoride; PRP, platelet-rich plasma; rp-HPLC, reverse-phase high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SVMP, snake venom metalloproteinase; TFA, trifluoroacetic acid; DC-protein, disintegrinlike/cysteine-rich domain protein; A/DC-protein, recombinant DCprotein of atrolysin; PCR, polymerase chain reaction.
Thrombosis and Haemostasis, 2010
SummaryHaemorrhage 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 coll...
Journal of Biological …, 2002
The integrins ␣ 2  1 and ␣ 1  1 have been shown to modulate cellular activities of fibroblasts on contact with fibrillar collagen. Previously it has been shown that collagen binding to ␣ 2  1 regulates matrix metalloproteinase MMP-1 and membrane-type MT1-MMP expression. Jararhagin is a snake venom metalloproteinase of the Reprolysin family of zinc metalloproteinases, containing a metalloproteinase domain followed by disintegrinlike and cysteine-rich domains. Jararhagin blocks type I collagen-induced platelet aggregation by binding to the ␣ 2  1 integrin and inhibiting collagen-mediated intracellular signaling events. Here we present evidence that, in contrast to the observations in platelets, jararhagin binding to the integrin receptor ␣ 2  1 in fibroblasts produces collagen-like cell signaling events such as up-regulation of MMP-1 and MT1-MMP. Inactivation of the metalloproteinase domain had no effect on these properties of jararhagin. Thus, in fibroblasts the snake venom metalloproteinase jararhagin functions as a collagen-mimetic substrate that binds to and activates integrins. Given the homology between the metalloproteinase, disintegrin-like and cysteine-rich domains of jararhagin and those of the members of the ADAMs (a disintegrin-like and metalloproteinase) family of proteins, this work demonstrates the potential of the disintegrin-like/cysteine-rich domains in the ADAMs as cellular signaling agents to elicit responses relevant to the biological function of these proteins. The abbreviations used are: MMP-1, matrix metalloproteinase-1; MMP-2, matrix metalloproteinase-2; MT1-MMP, membrane-type matrix metalloproteinase-1; PBS, phosphate-buffered saline; SVMP, snake venom metalloproteinases; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; BSA, bovine serum albumin.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2004
Metalloproteinases (MPs) are Zn + -dependent endoproteolytic enzymes, abundant in crotalid and viperid snake venoms. Most snake venom metalloproteinases (svMPs) are active on extracellular matrix components and this effect is thought to result in bleeding as a consequence of the basement membrane disruption in capillaries. Jararhagin and ACLH are hemorrhagic svMPs from Bothrops jararaca and Agkistrodon contortrix laticinctus venom, respectively. Both enzymes demonstrate proteolytic activity on fibrinogen and fibronectin and jararhagin inhibits collagen-induced platelet aggregation in vitro. This work describes the expression, purification and successful refolding of the recombinant ACLH zymogen (rPRO-ACLH) as well as the catalytic domain of jararhagin (rCDJARA). The heterologous proteins were produced in E. coli, an in vivo expression system that does not make post-translational modifications. The recombinant refolded proteins did not show any hemorrhagic activity in mice skin, as well as the native deglycosylated jararhagin and ACLH. However, they preserved their proteolytic activity on fibrinogen and fibronectin. It seems that the hemorrhagic properties of these hemorrhagins are dependent on posttranslational modifications, whereas their proteolytic activity is not dependent on such modifications. D