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Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain Bianca Cestari Zychar a, Patrícia Bianca Clissa b, Eneas Carvalho c, Cristiani Baldo d, Luis Roberto C. Gonçalves a, * ~o Paulo, 05503-900, Brazil Laboratory of Pathophysiology, Butantan Institute, Sa ~o Paulo, 05503-900, Brazil Laboratory of Immunopathology, Butantan Institute, Sa c ~o Paulo, 05503-900, Brazil Laboratory of Bacteriology, Butantan Institute, Sa d , 86057-970, Brazil Department of Biochemistry and Biotechnology, State University of Londrina, Parana a
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a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 September 2019 Accepted 20 October 2019 Available online xxx
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. © 2019 Elsevier Inc. All rights reserved.
Keywords: Snake venom metalloproteinases Leukocyte recruitment Zinc-binding domain Microcirculation Bothrops
1. Introduction Bothrops snakebites are characterized by severe local effects, such as inflammatory edema, which can affect the whole bitten limb, and cause necrosis of skin and muscles, which can lead to significant tissue loss and debilitating morbidity [1,2]. The local inflammation observed in this envenomation is mainly mediated by eicosanoids [3e5]. Because of the involvement of endogenous mediators, antivenom is not sufficient to neutralize the massive local inflammation in the envenomation by Bothrops snakebites [6e8]. Transcriptomic and proteomic studies have shown that metalloproteases, serine proteases, and phospholipase A2 are the main components of Bothrops venoms [9e11], and an earlier study
* Corresponding author. Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil, 1500, 05503-900, S~ ao Paulo, SP, Brazil. E-mail address: [email protected] (L.R.C. Gonçalves).
showed that metalloproteases are the key toxins responsible for the local inflammatory response induced by Bothrops jararaca venom (BjV) [11]. Snake venom metalloproteases (SVMPs) are classified as PI through PIII, according to their domain composition. PI-SVMP toxins have only a catalytic domain and require a zinc molecule to confer enzymatic action. SVMPs with catalytic and disintegrinlike domains are classified as PII, and PIII-SVMPs have the domains present in PII toxins and a cysteine-rich domain [12]. Among the SVMPs isolated from BjV, Jararhagin (Jar), a PIII-SVMP with intense hemorrhagic action, is one of the most studied [13,14]. Jar can cleave recombinant pro-TNF-a in its active form [15,16] and can induce an acute inflammatory response, inducing paw edema formation in mice [16e18]. A degraded form of Jar named Jararhagin-C (Jar-C) can also induce changes in leukocyte-endothelial interactions in the microcirculation of mice [19], although it does not cause hemorrhage because it lacks a catalytic domain. The different domains of Jar play roles in the complex
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Please cite this article as: B.C. Zychar et al., Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.144
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pathogenesis of the local hemorrhage induced by BjV [20]. Catalytic activity is essential for the hemorrhagic action of SVMPs [5,21,22], for the degradation of extracellular matrix proteins [23], and for the induction of the release of neurogenic mediators [4]. However, for hemorrhage to occur more efficiently, PIII-SVMPs need to adhere to perivascular collagen fibers through the disintegrin-like domain and to other substrates through the cysteine-rich domain [20,24]. The roles of the domains of SVMPs in acute inflammatory response induction are not known, and the aim of this study was to investigate these roles. Here, we measured the changes in leukocyte-endothelial interactions in the microcirculation of the mouse cremaster muscle using three toxins: Jar and Jar-C, both isolated from BjV, and BnP1, a PI-SVMP with weak hemorrhagic action isolated from Bothrops neuwiedi venom (BnV).
2. Material and methods 2.1. Ethical statements All experimental procedures followed the ethical parameters proposed by the International Society of Toxinology and by the Brazilian College of Experimental Animals and were approved by the Ethical Committee for the Use of Animals at Butantan Institute (protocol nº466/08).
2.2. Animals Male Swiss mice weighing 20e25 g were used. Animals were maintained for two days in the laboratory with free access to pelleted food and water in a temperature-controlled environment and a 12/12 h light-dark circle before beginning experiments.
2.3. Toxins Venoms of B. jararaca and B. neuwiedi were collected from adult snakes kept under confinement at Butantan Institute serpentarium rio de Herpetologia). Jar and Jar-C were isolated from BjV (Laborato as previously described [13,25]. BnP1 was isolated from BnV, as previously described by Baldo et al. [26]. The purity of toxins was evaluated by 12.5% SDS-PAGE under reducing conditions [27]; Jar, Jar-C, and BnP1 had molecular masses of 52, 28 and 24 kDa, respectively. All the toxins used in the intravital microscopy experiments were LPS-free.
2.5. Intravital microscopy of murine cremaster venules Leukocyte-endothelial interactions in mouse cremaster venules were assessed by intravital microscopy. PBS (100 mL, control) or 0.5 mg/100 mL of either Jar or BnP1, treated or not with oPhe, or Jar-C was injected into the subcutaneous tissue of the scrotal bag of mice (n ¼ 5/group). The doses of Jar or BnP1 used were sub hemorrhagic. After 2 or 24 h, animals were anesthetized with an intraperitoneal dose of ketamine (100 mg/kg) and xylazine (10 mg/ kg), and the cremaster muscle was exteriorized for microscopic examination in situ, as previously described by Baez [31]. The microcirculation of the cremaster muscle was visualized in a transparent window of a board heated at 37 C, on which the anesthetized mouse was maintained. Leukocyte responses on cremaster muscle microcirculation were evaluated by light microscopy (Axioplan II, Carl Zeiss, Germany, equipped with Achroplan objectives with 10.0 longitudinal distance/0.25 numeric apertures and 1.60 optovar). Images were captured and digitalized with a computer for further analysis using image analyzer software (KS 300, Kontron, Carl Zeiss, Germany). One post capillary venule (30e40 mm diameter) was randomly selected. After the stabilization period (10 min), the adhered leukocytes were counted over 5 min in a 100-mm vascular section. Firmly adherent leukocytes were considered to be the cells that remained immobile for at least 30 s within a given 100-mm vessel segment. Transmigrated leukocytes were quantified as those in the extravascular tissue within 50 mm of each side of the 100-mm vessel segments studied. The results obtained in the groups analyzed two hours after the injection of the toxin were used to compare adhered leukocytes, while migrated leukocytes were evaluated 24 h after venom injection. 2.6. Circular dichroism The ordered secondary structure composition of the Jar toxin after treatment with oPhe was evaluated by circular dichroism (CD). Dialyzed samples of Jar treated with oPhe or untreated Jar were subjected to a CD study using a Jasco J-810 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan) equipped with a Peltier unit for temperature control. Measurements were performed at 20, 37, 60, and 75 C in a 0.1 mm path length cell. The CD spectra were recorded at each 0.1 nm from 190 to 260 nm. The spectra presented are the average of five scans, and the data obtained were reported as molar ellipticity (deg x cm2 x dmol1). A baseline assessment with sodium phosphate buffer was subtracted from each obtained spectra. The spectra deconvolution analyses were performed with Dichroweb software [32] using the SELCON3 algorithm [33]. 2.7. Collagen binding assay
2.4. Inhibition of proteolytic activity In some experiments, Jar and BnP1 were treated with the Zn2þchelating metalloproteinase inhibitor 1,10-phenanthroline monohydrate (oPhe - Sigma Chemical Co., St. Louis, MO) as described by Borkow et al. [28]. Samples of 0.5 mg/mL of Jar or BnP1 were incubated with oPhe (20 mM) for 30 min at 37 C, followed by dialysis against phosphate-buffered saline (PBS) for 12 h. Control samples of Jar or BnP1 were submitted to incubation and dialysis, but no inhibitor was used. After this treatment, samples were kept at 20 C until use. The efficacy of this treatment on the proteolytic activity of Jar and BnP1 was evaluated by comparing the hemorrhagic and fibrinolytic activities of toxins treated with or without oPhe. Hemorrhagic activity was evaluated in mice [29], while fibrinolytic activity was evaluated in fibrin-agarose plates, as described by Jespersen and Astrup [30].
The interaction of Jar and oPhe-treated Jar with collagen was evaluated by a solid-phase binding assay as described by Moura da Silva et al. [34], with some modifications. For this assay, 10 mg of type I collagen diluted in 0.01 N acetic acid was coated on microtiter plates and incubated for 18 h at 4 C. The plates were then blocked with Tris-buffered saline containing 2% nonfat milk. After blocking, the plates were incubated for 1 h at 37 C with samples containing Jar or oPhe-treated Jar in decreasing concentrations in serial dilutions (1:1), and 25 nM was the initial concentration. Collagenadhered Jar was detected using an anti-Jar polyclonal serum raised in rabbits (1:100) followed by incubation with goat antirabbit IgG labeled with horseradish peroxidase (1:2000). Each step was followed by washing with PBS/Tween 0.05%. After the final incubation, orthophenylene diamine/H2O2 was added, and the reaction was developed for 10 min. The optical density was read at 492 nm.
Please cite this article as: B.C. Zychar et al., Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.144
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2.8. Statistical analysis The results of leukocyte adherence and migration were expressed as the mean ± standard error of the mean of five animals per group. The values were compared by one-way ANOVA followed by Bonferroni’s posttest, and differences were considered significant when p < 0.05. 3. Results All the SVMPs used were able to induce changes in leukocyteendothelial cell interactions, significantly increasing adhered and migrated cells at the 2nd and the 24th hour after injection, respectively (Fig. 1). A similar inflammatory reaction was induced when using both Jar and BnP1 toxins, whereas a lower, but still significant, was observed when using Jar-C toxin. To evaluate the role of the catalytic domain in this inflammatory effect, Jar and BnP1 were treated with oPhe. This treatment completely inhibited the hemorrhagic and fibrinolytic activities of these toxins (data not shown) and completely abrogated the alterations of leukocyte-endothelial cell interactions induced by both toxins, such as adhered and migrated cells (Fig. 2). To assess a possible structural change in Jar induced by the treatment with oPhe, we submitted oPhe-treated Jar to a CD analysis. Samples of Jar treated with oPhe showed remarkable differences in their CD spectra and in their calculated secondary structure composition compared with the untreated samples of this toxin (Fig. 3A). The alpha-helix and beta-sheet content were reduced by 37.5 and 15.4%, respectively, while the proportion of unstructured regions increased by 40.5% (Fig. 3A). The exposure of
Fig. 2. Analysis of leukocyte-endothelium interactions in the microcirculation of the mouse cremaster muscle at 2 h (A) or 24 h (B) after the injection of PBS (control) or toxins treated with oPhe or untreated toxins. Parameters such as adhered (A) or migrated (B) leukocytes were evaluated. The results are expressed as the mean ± s.e.m. (n ¼ 5). *p < 0.05, significantly different from the group treated with PBS.
untreated samples to denaturing temperatures led to a loss of regular secondary structures (Fig. 3B and C). High temperatures reduced the alpha-helix and beta-sheet content of untreated Jar to levels similar to those observed in samples treated with oPhe (Fig. 3C). Additionally, samples of oPhe-treated Jar did not undergo a noticeable change in CD spectra after exposure to denaturing temperatures (Fig. 3B). Therefore, high temperatures were not able to produce an additional effect on the CD spectra of oPhe-treated Jar, probably because these samples had already lost their secondary structure during the chemical treatment (Fig. 3B and C). Temperature denaturation was also evaluated by following the molar ellipticity at 222 nm, from 20 to 75 C, at intervals of 0.1 C. Similar to the results that we obtained for CD spectra, the molar ellipticity at 222 nm showed that the Jar samples treated with oPhe presented fewer structured regions and were not affected by an increase or decrease in temperature (Fig. 3C). On the other hand, untreated Jar lost its structure during temperature denaturation, leading us to determine the protein melting temperature. The melting temperature was found to be 59.43 ± 0.27 C. To determine whether this structural modification of Jar induced by oPhe treatment interfered with the binding of the toxin to components of the extracellular matrix, we performed a binding assay of oPhe-treated and untreated Jar with collagen. The results showed that despite the structural modifications revealed by CD, oPhe-treated Jar was still able to bind to collagen type I (Kd ¼ 90.58 nM) but with a lower affinity than that of untreated Jar (Kd ¼ 40.27 nM) (Fig. 4). Fig. 1. Analysis of leukocyte-endothelium interactions in the microcirculation of the mouse cremaster muscle at 2 h (A) or 24 h (B) after the injection of toxins or PBS (control). Parameters such as adhered (A) or migrated (B) leukocytes were evaluated. The results are expressed as the mean ± s.e.m. (n ¼ 5). *p < 0.05, significantly different from the group treated with PBS. #p < 0.05, significantly different from other groups.
4. Discussion Snake venom metalloproteinases (SVMPs) play a pivotal role in the inflammatory processes observed in patients bitten by viperid
Please cite this article as: B.C. Zychar et al., Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.144
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Fig. 3. Secondary structure of Jararhagin (Jar) as determined by circular dichroism spectroscopy. CD spectra of Jar and Jar-oPhe samples are shown in (A) with deconvolution analysis (insert in A). The molar ellipticity of Jar and Jar-oPhe at 222 nm was recorded from 20 C to 75 C (B), and the percentage of secondary structures in Jar and Jar-oPhe determined during temperature increase is shown in (C).
snakes. In BjV, SVMPs are the main class of toxins that induce inflammatory reactions [11]. The hemorrhagic activity of these toxins is related to their catalytic domain, but their efficacy as a hemorrhagic factor is also related to their disintegrin-like and cysteinerich domains. These domains contribute to the hemorrhagic effect by binding to perivascular components of the basal membrane of blood vessels [20,22,34,35], but the role played by the different domains of SVMPs in the inflammatory response is still unknown. Here, we have shown that all SVMPs used in this study were able to induce alterations in the leukocyte-endothelial interactions of the cremaster microcirculation when injected into the scrotal bag of mice, even considering the significant difference in the molarities of the injected toxins. The magnitude of this inflammatory effect
Fig. 4. Interaction of Jararhagin with collagen I: Collagen I was immobilized to a microtiter plate. After blocking nonspecific protein binding sites, Jar or Jar-oPhe in decreasing concentrations (with an initial concentration of 25 109 M) were added to the plate. The bound toxins were detected using rabbit anti-jararhagin polyclonal serum followed by incubation with goat IgG anti-rabbit IgG labeled with horseradish peroxidase and orthophenylene diamine/H2O2 as enzyme substrates. The optical density was read at 492 nm. The mean values of triplicate measurements of one of several representative experiments are shown.
did not depend on the domain composition of the toxins, since BnP1, a P1 toxin, containing only the catalytic domain, Jar, a P3 toxin, contain the catalytic domain and the ECD-disintegrin and cysteine-rich domains, and Jar-C, a toxin containing only ECDdisintegrin and cysteine-rich domains were all able to induce a significant increase in the adherence and migration of leukocytes. Nevertheless, when Jar and BnP1 were treated with oPhe, the inflammatory effect was completely abolished. These data corroborate the importance of the catalytic domain in the inflammatory reaction induced by Bothrops snake venoms [11,21,36,37], but in the case of Jar, a residual inflammatory effect should be expected, since this toxin also has ECD-disintegrin and cysteine-rich domains, and Jar-C was effective in inducing the adherence and migration of leukocytes. Disintegrin-like/cysteine-rich toxins are the result of the auto degradation of PIII-SVMPs and are present in minor quantities in crude venom [38,39]. Studies have shown that a recombinant protein containing only the disintegrin-like and cysteine-rich domain derived from HF3, a hemorrhagic PIII-SVMP from BjV, is able to significantly increase leukocyte rolling in the microcirculation [40,41], confirming a previous study [19] and our observation with Jar-C in the present study. To evaluate whether the treatment with oPhe altered the conformation of Jar, we analyzed oPhe-treated Jar by circular dichroism and compared the structures of oPhe-treated Jar with that of the untreated toxin. The results showed a significant alteration in the toxin structure, similar to that induced by irreversible denaturation due to high temperatures. The alpha-helix regions were the structures most affected by oPhe treatment, suggesting that they may play a major role in Jar activities, either directly, as an integral part of the active sites or indirectly, by maintaining the major structural motifs that are essential for biological activities. This finding also confirms the importance of Zn2þ ions in the active site of SVMP toxins, as demonstrated in three-dimensional modeling structures of SVMP toxins [42].
Please cite this article as: B.C. Zychar et al., Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.144
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Despite this structural modification, oPhe treatment did not prevent the binding of Jar to collagen. This result corroborates studies with oPhe-treated Jar suggesting that the catalytic domain is not involved in the binding of Jar to a2-b1 integrin or to collagen [34,43]. It is known that binding of Jar to a2-b1 integrin and collagen occurs through the cysteine-rich and disintegrin-like domains, respectively [44,45]. The results obtained in the microcirculation with Jar-C demonstrates that Jar inhibited with oPhe maintains its capacity to bind collagen, suggesting that, in addition to the catalytic domain of SVMPs, other domains also have inflammatory potential. Some studies indicated that the cysteine-rich domain of PIII-SVMPs has a pivotal role in the interaction with extracellular matrix proteins [34,46] and that this domain contributes to the activation of leukocyte rolling [40]. Our results corroborate these observations. Treatment with oPhe did not abolish the ability of Jar to adhere to collagen, suggesting that the structural changes induced by oPhe did not significantly affect the disintegrin-like domain structure, which is related to this activity. This result may indicate that in oPhe-treated Jar, the disintegrin-like domain is not sufficient to promote leukocyte-endothelial cell interactions and that the catalytic domain of SVMPs is the main factor responsible for the changes in leukocyte-endothelial cell interactions. However, since Jar-C, which does not have the catalytic domain, had an effect on leukocyte-endothelial cell interactions, it seems that the effect of the disintegrin-like domain on the inflammatory response may be dependent on the absence of the catalytic domain or may be dependent on the cysteine-rich domain. In conclusion, our results show that the catalytic domain of Jar and BnP1 is essential for inducing the inflammatory response caused by these SVMPs and, in particular, for promoting the alterations in leukocyte-endothelial cell interactions induced by those toxins in the microcirculation.
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Declaration of competing interest
[21]
The authors declare that there are no conflicts of interest regarding this work.
[22]
Acknowledgments ~o Paulo Research Foundation (FAPESP), grants Supported by Sa n 2008/00108-8 and 2010/09002-8. Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.144. [26]
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Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain Bianca Cestari Zychar a, Patrícia Bianca Clissa b, Eneas Carvalho c, Cristiani Baldo d, Luis Roberto C. Gonçalves a, * ~o Paulo, 05503-900, Brazil Laboratory of Pathophysiology, Butantan Institute, Sa ~o Paulo, 05503-900, Brazil Laboratory of Immunopathology, Butantan Institute, Sa c ~o Paulo, 05503-900, Brazil Laboratory of Bacteriology, Butantan Institute, Sa d , 86057-970, Brazil Department of Biochemistry and Biotechnology, State University of Londrina, Parana a
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a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 September 2019 Accepted 20 October 2019 Available online xxx
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. © 2019 Elsevier Inc. All rights reserved.
Keywords: Snake venom metalloproteinases Leukocyte recruitment Zinc-binding domain Microcirculation Bothrops
1. Introduction Bothrops snakebites are characterized by severe local effects, such as inflammatory edema, which can affect the whole bitten limb, and cause necrosis of skin and muscles, which can lead to significant tissue loss and debilitating morbidity [1,2]. The local inflammation observed in this envenomation is mainly mediated by eicosanoids [3e5]. Because of the involvement of endogenous mediators, antivenom is not sufficient to neutralize the massive local inflammation in the envenomation by Bothrops snakebites [6e8]. Transcriptomic and proteomic studies have shown that metalloproteases, serine proteases, and phospholipase A2 are the main components of Bothrops venoms [9e11], and an earlier study
* Corresponding author. Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil, 1500, 05503-900, S~ ao Paulo, SP, Brazil. E-mail address: [email protected] (L.R.C. Gonçalves).
showed that metalloproteases are the key toxins responsible for the local inflammatory response induced by Bothrops jararaca venom (BjV) [11]. Snake venom metalloproteases (SVMPs) are classified as PI through PIII, according to their domain composition. PI-SVMP toxins have only a catalytic domain and require a zinc molecule to confer enzymatic action. SVMPs with catalytic and disintegrinlike domains are classified as PII, and PIII-SVMPs have the domains present in PII toxins and a cysteine-rich domain [12]. Among the SVMPs isolated from BjV, Jararhagin (Jar), a PIII-SVMP with intense hemorrhagic action, is one of the most studied [13,14]. Jar can cleave recombinant pro-TNF-a in its active form [15,16] and can induce an acute inflammatory response, inducing paw edema formation in mice [16e18]. A degraded form of Jar named Jararhagin-C (Jar-C) can also induce changes in leukocyte-endothelial interactions in the microcirculation of mice [19], although it does not cause hemorrhage because it lacks a catalytic domain. The different domains of Jar play roles in the complex
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Please cite this article as: B.C. Zychar et al., Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.144
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pathogenesis of the local hemorrhage induced by BjV [20]. Catalytic activity is essential for the hemorrhagic action of SVMPs [5,21,22], for the degradation of extracellular matrix proteins [23], and for the induction of the release of neurogenic mediators [4]. However, for hemorrhage to occur more efficiently, PIII-SVMPs need to adhere to perivascular collagen fibers through the disintegrin-like domain and to other substrates through the cysteine-rich domain [20,24]. The roles of the domains of SVMPs in acute inflammatory response induction are not known, and the aim of this study was to investigate these roles. Here, we measured the changes in leukocyte-endothelial interactions in the microcirculation of the mouse cremaster muscle using three toxins: Jar and Jar-C, both isolated from BjV, and BnP1, a PI-SVMP with weak hemorrhagic action isolated from Bothrops neuwiedi venom (BnV).
2. Material and methods 2.1. Ethical statements All experimental procedures followed the ethical parameters proposed by the International Society of Toxinology and by the Brazilian College of Experimental Animals and were approved by the Ethical Committee for the Use of Animals at Butantan Institute (protocol nº466/08).
2.2. Animals Male Swiss mice weighing 20e25 g were used. Animals were maintained for two days in the laboratory with free access to pelleted food and water in a temperature-controlled environment and a 12/12 h light-dark circle before beginning experiments.
2.3. Toxins Venoms of B. jararaca and B. neuwiedi were collected from adult snakes kept under confinement at Butantan Institute serpentarium rio de Herpetologia). Jar and Jar-C were isolated from BjV (Laborato as previously described [13,25]. BnP1 was isolated from BnV, as previously described by Baldo et al. [26]. The purity of toxins was evaluated by 12.5% SDS-PAGE under reducing conditions [27]; Jar, Jar-C, and BnP1 had molecular masses of 52, 28 and 24 kDa, respectively. All the toxins used in the intravital microscopy experiments were LPS-free.
2.5. Intravital microscopy of murine cremaster venules Leukocyte-endothelial interactions in mouse cremaster venules were assessed by intravital microscopy. PBS (100 mL, control) or 0.5 mg/100 mL of either Jar or BnP1, treated or not with oPhe, or Jar-C was injected into the subcutaneous tissue of the scrotal bag of mice (n ¼ 5/group). The doses of Jar or BnP1 used were sub hemorrhagic. After 2 or 24 h, animals were anesthetized with an intraperitoneal dose of ketamine (100 mg/kg) and xylazine (10 mg/ kg), and the cremaster muscle was exteriorized for microscopic examination in situ, as previously described by Baez [31]. The microcirculation of the cremaster muscle was visualized in a transparent window of a board heated at 37 C, on which the anesthetized mouse was maintained. Leukocyte responses on cremaster muscle microcirculation were evaluated by light microscopy (Axioplan II, Carl Zeiss, Germany, equipped with Achroplan objectives with 10.0 longitudinal distance/0.25 numeric apertures and 1.60 optovar). Images were captured and digitalized with a computer for further analysis using image analyzer software (KS 300, Kontron, Carl Zeiss, Germany). One post capillary venule (30e40 mm diameter) was randomly selected. After the stabilization period (10 min), the adhered leukocytes were counted over 5 min in a 100-mm vascular section. Firmly adherent leukocytes were considered to be the cells that remained immobile for at least 30 s within a given 100-mm vessel segment. Transmigrated leukocytes were quantified as those in the extravascular tissue within 50 mm of each side of the 100-mm vessel segments studied. The results obtained in the groups analyzed two hours after the injection of the toxin were used to compare adhered leukocytes, while migrated leukocytes were evaluated 24 h after venom injection. 2.6. Circular dichroism The ordered secondary structure composition of the Jar toxin after treatment with oPhe was evaluated by circular dichroism (CD). Dialyzed samples of Jar treated with oPhe or untreated Jar were subjected to a CD study using a Jasco J-810 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan) equipped with a Peltier unit for temperature control. Measurements were performed at 20, 37, 60, and 75 C in a 0.1 mm path length cell. The CD spectra were recorded at each 0.1 nm from 190 to 260 nm. The spectra presented are the average of five scans, and the data obtained were reported as molar ellipticity (deg x cm2 x dmol1). A baseline assessment with sodium phosphate buffer was subtracted from each obtained spectra. The spectra deconvolution analyses were performed with Dichroweb software [32] using the SELCON3 algorithm [33]. 2.7. Collagen binding assay
2.4. Inhibition of proteolytic activity In some experiments, Jar and BnP1 were treated with the Zn2þchelating metalloproteinase inhibitor 1,10-phenanthroline monohydrate (oPhe - Sigma Chemical Co., St. Louis, MO) as described by Borkow et al. [28]. Samples of 0.5 mg/mL of Jar or BnP1 were incubated with oPhe (20 mM) for 30 min at 37 C, followed by dialysis against phosphate-buffered saline (PBS) for 12 h. Control samples of Jar or BnP1 were submitted to incubation and dialysis, but no inhibitor was used. After this treatment, samples were kept at 20 C until use. The efficacy of this treatment on the proteolytic activity of Jar and BnP1 was evaluated by comparing the hemorrhagic and fibrinolytic activities of toxins treated with or without oPhe. Hemorrhagic activity was evaluated in mice [29], while fibrinolytic activity was evaluated in fibrin-agarose plates, as described by Jespersen and Astrup [30].
The interaction of Jar and oPhe-treated Jar with collagen was evaluated by a solid-phase binding assay as described by Moura da Silva et al. [34], with some modifications. For this assay, 10 mg of type I collagen diluted in 0.01 N acetic acid was coated on microtiter plates and incubated for 18 h at 4 C. The plates were then blocked with Tris-buffered saline containing 2% nonfat milk. After blocking, the plates were incubated for 1 h at 37 C with samples containing Jar or oPhe-treated Jar in decreasing concentrations in serial dilutions (1:1), and 25 nM was the initial concentration. Collagenadhered Jar was detected using an anti-Jar polyclonal serum raised in rabbits (1:100) followed by incubation with goat antirabbit IgG labeled with horseradish peroxidase (1:2000). Each step was followed by washing with PBS/Tween 0.05%. After the final incubation, orthophenylene diamine/H2O2 was added, and the reaction was developed for 10 min. The optical density was read at 492 nm.
Please cite this article as: B.C. Zychar et al., Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.144
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2.8. Statistical analysis The results of leukocyte adherence and migration were expressed as the mean ± standard error of the mean of five animals per group. The values were compared by one-way ANOVA followed by Bonferroni’s posttest, and differences were considered significant when p < 0.05. 3. Results All the SVMPs used were able to induce changes in leukocyteendothelial cell interactions, significantly increasing adhered and migrated cells at the 2nd and the 24th hour after injection, respectively (Fig. 1). A similar inflammatory reaction was induced when using both Jar and BnP1 toxins, whereas a lower, but still significant, was observed when using Jar-C toxin. To evaluate the role of the catalytic domain in this inflammatory effect, Jar and BnP1 were treated with oPhe. This treatment completely inhibited the hemorrhagic and fibrinolytic activities of these toxins (data not shown) and completely abrogated the alterations of leukocyte-endothelial cell interactions induced by both toxins, such as adhered and migrated cells (Fig. 2). To assess a possible structural change in Jar induced by the treatment with oPhe, we submitted oPhe-treated Jar to a CD analysis. Samples of Jar treated with oPhe showed remarkable differences in their CD spectra and in their calculated secondary structure composition compared with the untreated samples of this toxin (Fig. 3A). The alpha-helix and beta-sheet content were reduced by 37.5 and 15.4%, respectively, while the proportion of unstructured regions increased by 40.5% (Fig. 3A). The exposure of
Fig. 2. Analysis of leukocyte-endothelium interactions in the microcirculation of the mouse cremaster muscle at 2 h (A) or 24 h (B) after the injection of PBS (control) or toxins treated with oPhe or untreated toxins. Parameters such as adhered (A) or migrated (B) leukocytes were evaluated. The results are expressed as the mean ± s.e.m. (n ¼ 5). *p < 0.05, significantly different from the group treated with PBS.
untreated samples to denaturing temperatures led to a loss of regular secondary structures (Fig. 3B and C). High temperatures reduced the alpha-helix and beta-sheet content of untreated Jar to levels similar to those observed in samples treated with oPhe (Fig. 3C). Additionally, samples of oPhe-treated Jar did not undergo a noticeable change in CD spectra after exposure to denaturing temperatures (Fig. 3B). Therefore, high temperatures were not able to produce an additional effect on the CD spectra of oPhe-treated Jar, probably because these samples had already lost their secondary structure during the chemical treatment (Fig. 3B and C). Temperature denaturation was also evaluated by following the molar ellipticity at 222 nm, from 20 to 75 C, at intervals of 0.1 C. Similar to the results that we obtained for CD spectra, the molar ellipticity at 222 nm showed that the Jar samples treated with oPhe presented fewer structured regions and were not affected by an increase or decrease in temperature (Fig. 3C). On the other hand, untreated Jar lost its structure during temperature denaturation, leading us to determine the protein melting temperature. The melting temperature was found to be 59.43 ± 0.27 C. To determine whether this structural modification of Jar induced by oPhe treatment interfered with the binding of the toxin to components of the extracellular matrix, we performed a binding assay of oPhe-treated and untreated Jar with collagen. The results showed that despite the structural modifications revealed by CD, oPhe-treated Jar was still able to bind to collagen type I (Kd ¼ 90.58 nM) but with a lower affinity than that of untreated Jar (Kd ¼ 40.27 nM) (Fig. 4). Fig. 1. Analysis of leukocyte-endothelium interactions in the microcirculation of the mouse cremaster muscle at 2 h (A) or 24 h (B) after the injection of toxins or PBS (control). Parameters such as adhered (A) or migrated (B) leukocytes were evaluated. The results are expressed as the mean ± s.e.m. (n ¼ 5). *p < 0.05, significantly different from the group treated with PBS. #p < 0.05, significantly different from other groups.
4. Discussion Snake venom metalloproteinases (SVMPs) play a pivotal role in the inflammatory processes observed in patients bitten by viperid
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Fig. 3. Secondary structure of Jararhagin (Jar) as determined by circular dichroism spectroscopy. CD spectra of Jar and Jar-oPhe samples are shown in (A) with deconvolution analysis (insert in A). The molar ellipticity of Jar and Jar-oPhe at 222 nm was recorded from 20 C to 75 C (B), and the percentage of secondary structures in Jar and Jar-oPhe determined during temperature increase is shown in (C).
snakes. In BjV, SVMPs are the main class of toxins that induce inflammatory reactions [11]. The hemorrhagic activity of these toxins is related to their catalytic domain, but their efficacy as a hemorrhagic factor is also related to their disintegrin-like and cysteinerich domains. These domains contribute to the hemorrhagic effect by binding to perivascular components of the basal membrane of blood vessels [20,22,34,35], but the role played by the different domains of SVMPs in the inflammatory response is still unknown. Here, we have shown that all SVMPs used in this study were able to induce alterations in the leukocyte-endothelial interactions of the cremaster microcirculation when injected into the scrotal bag of mice, even considering the significant difference in the molarities of the injected toxins. The magnitude of this inflammatory effect
Fig. 4. Interaction of Jararhagin with collagen I: Collagen I was immobilized to a microtiter plate. After blocking nonspecific protein binding sites, Jar or Jar-oPhe in decreasing concentrations (with an initial concentration of 25 109 M) were added to the plate. The bound toxins were detected using rabbit anti-jararhagin polyclonal serum followed by incubation with goat IgG anti-rabbit IgG labeled with horseradish peroxidase and orthophenylene diamine/H2O2 as enzyme substrates. The optical density was read at 492 nm. The mean values of triplicate measurements of one of several representative experiments are shown.
did not depend on the domain composition of the toxins, since BnP1, a P1 toxin, containing only the catalytic domain, Jar, a P3 toxin, contain the catalytic domain and the ECD-disintegrin and cysteine-rich domains, and Jar-C, a toxin containing only ECDdisintegrin and cysteine-rich domains were all able to induce a significant increase in the adherence and migration of leukocytes. Nevertheless, when Jar and BnP1 were treated with oPhe, the inflammatory effect was completely abolished. These data corroborate the importance of the catalytic domain in the inflammatory reaction induced by Bothrops snake venoms [11,21,36,37], but in the case of Jar, a residual inflammatory effect should be expected, since this toxin also has ECD-disintegrin and cysteine-rich domains, and Jar-C was effective in inducing the adherence and migration of leukocytes. Disintegrin-like/cysteine-rich toxins are the result of the auto degradation of PIII-SVMPs and are present in minor quantities in crude venom [38,39]. Studies have shown that a recombinant protein containing only the disintegrin-like and cysteine-rich domain derived from HF3, a hemorrhagic PIII-SVMP from BjV, is able to significantly increase leukocyte rolling in the microcirculation [40,41], confirming a previous study [19] and our observation with Jar-C in the present study. To evaluate whether the treatment with oPhe altered the conformation of Jar, we analyzed oPhe-treated Jar by circular dichroism and compared the structures of oPhe-treated Jar with that of the untreated toxin. The results showed a significant alteration in the toxin structure, similar to that induced by irreversible denaturation due to high temperatures. The alpha-helix regions were the structures most affected by oPhe treatment, suggesting that they may play a major role in Jar activities, either directly, as an integral part of the active sites or indirectly, by maintaining the major structural motifs that are essential for biological activities. This finding also confirms the importance of Zn2þ ions in the active site of SVMP toxins, as demonstrated in three-dimensional modeling structures of SVMP toxins [42].
Please cite this article as: B.C. Zychar et al., Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.144
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Despite this structural modification, oPhe treatment did not prevent the binding of Jar to collagen. This result corroborates studies with oPhe-treated Jar suggesting that the catalytic domain is not involved in the binding of Jar to a2-b1 integrin or to collagen [34,43]. It is known that binding of Jar to a2-b1 integrin and collagen occurs through the cysteine-rich and disintegrin-like domains, respectively [44,45]. The results obtained in the microcirculation with Jar-C demonstrates that Jar inhibited with oPhe maintains its capacity to bind collagen, suggesting that, in addition to the catalytic domain of SVMPs, other domains also have inflammatory potential. Some studies indicated that the cysteine-rich domain of PIII-SVMPs has a pivotal role in the interaction with extracellular matrix proteins [34,46] and that this domain contributes to the activation of leukocyte rolling [40]. Our results corroborate these observations. Treatment with oPhe did not abolish the ability of Jar to adhere to collagen, suggesting that the structural changes induced by oPhe did not significantly affect the disintegrin-like domain structure, which is related to this activity. This result may indicate that in oPhe-treated Jar, the disintegrin-like domain is not sufficient to promote leukocyte-endothelial cell interactions and that the catalytic domain of SVMPs is the main factor responsible for the changes in leukocyte-endothelial cell interactions. However, since Jar-C, which does not have the catalytic domain, had an effect on leukocyte-endothelial cell interactions, it seems that the effect of the disintegrin-like domain on the inflammatory response may be dependent on the absence of the catalytic domain or may be dependent on the cysteine-rich domain. In conclusion, our results show that the catalytic domain of Jar and BnP1 is essential for inducing the inflammatory response caused by these SVMPs and, in particular, for promoting the alterations in leukocyte-endothelial cell interactions induced by those toxins in the microcirculation.
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Declaration of competing interest
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The authors declare that there are no conflicts of interest regarding this work.
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Acknowledgments ~o Paulo Research Foundation (FAPESP), grants Supported by Sa n 2008/00108-8 and 2010/09002-8. Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.144. [26]
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Please cite this article as: B.C. Zychar et al., Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.144