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Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

Adade CM, Carvalho AL, Tomaz MA, Costa TF, Godinho JL, Melo PA, Lima AP, Rodrigues JC, Zingali RB, Souto-Padrón T - PLoS Negl Trop Dis (2014)

Bottom Line: We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin.Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml).This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil.

ABSTRACT

Background: The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania.

Methodology/principal findings: Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells.

Conclusions: This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

No MeSH data available.


Related in: MedlinePlus

(A) Crovirin purification from Cvv venom using a reverse phase analytical C8 column, where the protein was eluted as peak 3. (B) SDS-PAGE analysis of peak 3 (lane B) containing the purified crovirin protein under reducing conditions. The gel was stained with Coomassie blue. Lane A, molecular weight markers. (C) MALDI-TOF mass spectrometry analyses of the intact protein yielded a molecular mass of 24,893.64 Da. The peaks of 12,424.36 and 12,477.62 Da correspond to doubly-charged (z = 2) cationic forms of crovirin.
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pntd-0003252-g001: (A) Crovirin purification from Cvv venom using a reverse phase analytical C8 column, where the protein was eluted as peak 3. (B) SDS-PAGE analysis of peak 3 (lane B) containing the purified crovirin protein under reducing conditions. The gel was stained with Coomassie blue. Lane A, molecular weight markers. (C) MALDI-TOF mass spectrometry analyses of the intact protein yielded a molecular mass of 24,893.64 Da. The peaks of 12,424.36 and 12,477.62 Da correspond to doubly-charged (z = 2) cationic forms of crovirin.

Mentions: In a previous study, we showed that the Cvv venom had anti-parasitic activity against T. cruzi[46]. Preliminary analysis of Cvv venom fractions by reverse-phase chromatography (not shown) indicated that the activity eluted with fractions containing peak 3 of the chromatographic profile (Fig. 1A). Thus, we analyzed the main chromatographic fraction corresponding to peak 3 by SDS-PAGE and MALDI-TOF mass spectrometry (Fig. 1B–C). SDS-PAGE analysis of peak 3 showed a single polypeptide, with a relative molecular mass of 24 kDa (Fig. 1B) and 28 kDa (data not shown), under reducing and non-reducing conditions, respectively. We will refer to this protein henceforth as crovirin. MALDI-TOF analysis of the intact protein showed a molecular mass of 24,893.64 Da (Fig. 1C). The peaks of 12,424.36 and 12,477.62 Da in the MS profile correspond to doubly-charged (z = 2) cationic forms of the protein. The amino acid sequence of tryptic crovirin peptides (produced by Nano LC-MS/MS mass spectrometry analysis) is nearly identical to a partial sequence of a Cvv CRISP (GenBank gi:190195319) (Fig. 2). The MS/MS-derived sequences are also nearly identical to those of a CRISP protein from Calloselasma rhodostoma (GenBank gi:190195317) and have high degree of sequence similarity to several other snake venom CRISPs, including ablomin (Fig. 2). The MS/MS spectrum of the fragmented peptide ions was matched by MASCOT displayed a coverage of 48% of identical peptides, with a p≥355 indicating extensive homology to the CRISP from C. rhodostoma. The MS results strongly suggested that a CRISP from Cvv snake venom had been purified, and corresponded to crovirin.


Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

Adade CM, Carvalho AL, Tomaz MA, Costa TF, Godinho JL, Melo PA, Lima AP, Rodrigues JC, Zingali RB, Souto-Padrón T - PLoS Negl Trop Dis (2014)

(A) Crovirin purification from Cvv venom using a reverse phase analytical C8 column, where the protein was eluted as peak 3. (B) SDS-PAGE analysis of peak 3 (lane B) containing the purified crovirin protein under reducing conditions. The gel was stained with Coomassie blue. Lane A, molecular weight markers. (C) MALDI-TOF mass spectrometry analyses of the intact protein yielded a molecular mass of 24,893.64 Da. The peaks of 12,424.36 and 12,477.62 Da correspond to doubly-charged (z = 2) cationic forms of crovirin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199522&req=5

pntd-0003252-g001: (A) Crovirin purification from Cvv venom using a reverse phase analytical C8 column, where the protein was eluted as peak 3. (B) SDS-PAGE analysis of peak 3 (lane B) containing the purified crovirin protein under reducing conditions. The gel was stained with Coomassie blue. Lane A, molecular weight markers. (C) MALDI-TOF mass spectrometry analyses of the intact protein yielded a molecular mass of 24,893.64 Da. The peaks of 12,424.36 and 12,477.62 Da correspond to doubly-charged (z = 2) cationic forms of crovirin.
Mentions: In a previous study, we showed that the Cvv venom had anti-parasitic activity against T. cruzi[46]. Preliminary analysis of Cvv venom fractions by reverse-phase chromatography (not shown) indicated that the activity eluted with fractions containing peak 3 of the chromatographic profile (Fig. 1A). Thus, we analyzed the main chromatographic fraction corresponding to peak 3 by SDS-PAGE and MALDI-TOF mass spectrometry (Fig. 1B–C). SDS-PAGE analysis of peak 3 showed a single polypeptide, with a relative molecular mass of 24 kDa (Fig. 1B) and 28 kDa (data not shown), under reducing and non-reducing conditions, respectively. We will refer to this protein henceforth as crovirin. MALDI-TOF analysis of the intact protein showed a molecular mass of 24,893.64 Da (Fig. 1C). The peaks of 12,424.36 and 12,477.62 Da in the MS profile correspond to doubly-charged (z = 2) cationic forms of the protein. The amino acid sequence of tryptic crovirin peptides (produced by Nano LC-MS/MS mass spectrometry analysis) is nearly identical to a partial sequence of a Cvv CRISP (GenBank gi:190195319) (Fig. 2). The MS/MS-derived sequences are also nearly identical to those of a CRISP protein from Calloselasma rhodostoma (GenBank gi:190195317) and have high degree of sequence similarity to several other snake venom CRISPs, including ablomin (Fig. 2). The MS/MS spectrum of the fragmented peptide ions was matched by MASCOT displayed a coverage of 48% of identical peptides, with a p≥355 indicating extensive homology to the CRISP from C. rhodostoma. The MS results strongly suggested that a CRISP from Cvv snake venom had been purified, and corresponded to crovirin.

Bottom Line: We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin.Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml).This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil.

ABSTRACT

Background: The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania.

Methodology/principal findings: Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells.

Conclusions: This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.

No MeSH data available.


Related in: MedlinePlus