Identification of sulfenylation patterns in trophozoite stage Plasmodium falciparum using a non-dimedone based probe

Abstract

Plasmodium falciparum causes the deadliest form of malaria. Adequate redox control is crucial for this protozoan parasite to overcome oxidative and nitrosative challenges, thus enabling its survival. Sulfenylation is an oxidative post-translational modification, which acts as a molecular on/off switch, regulating protein activity. To obtain a better understanding of which proteins are redox regulated in malaria parasites, we established an optimized affinity capture protocol coupled with mass spectrometry analysis for identification of in vivo sulfenylated proteins. The non-dimedone based probe BCN-Bio1 shows reaction rates over 100-times that of commonly used dimedone-based probes, allowing for a rapid trapping of sulfenylated proteins. Mass spectrometry analysis of BCN-Bio1 labeled proteins revealed the first insight into the Plasmodium falciparum trophozoite sulfenylome, identifying 102 proteins containing 152 sulfenylation sites. Comparison with Plasmodium proteins modified by S-glutathionylation and S-nitrosation showed a high overlap, suggesting a common core of proteins undergoing redox regulation by multiple mechanisms. Furthermore, parasite proteins which were identified as targets for sulfenylation were also identified as being sulfenylated in other organisms, especially proteins of the glycolytic cycle. This study suggests that a number of Plasmodium proteins are subject to redox regulation and it provides a basis for further investigations into the exact structural and biochemical basis of regulation, and a deeper understanding of cross-talk between post-translational modifications.

Keywords: 9-hydroxymethylbicyclo[6.1.0]nonyne (BCN-Bio1); Cysteine modification; Malaria; Post-translational modification; Redox proteomics; Sulfenylation.

Figure 1 –

Schematic workflow of pull-down protocol Sulfenylated proteins of P. falciparum trophozoites were covalently labeled by BCN-Bio1, Free thiols were blocked by N-ethylmaleimide (NEM). Enrichment using avidin beads was performed. Throughout stringend washing steps unspecifically bound proteins were removed. Eluted proteins were analysed using multidimensional protein identification technique (MudPIT). Sulfenylated peptides labeld by BCN-Bio1 can be detected in MudPIT analysis by a mass shift of +392.1759 Da.

Figure 2 –

A: Gel electrophoresis of pull-down steps. Samples collected during pull-down were subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie brilliant blue. B: Immunodetection of pull-down eluates from 3 biological replicates using α-biotin.

Figure 3 -

Functional classification and enrichment of sulfenylated proteins in the trophozoite stage of P. falciparum. Sulfenylated proteins identified were clustered according to their gene ontology (GO) annotations in biological process (A) and molecular function (B). The number in brackets indicates the enrichment of the respective GO slim term compared to all P. falciparum proteins. GO slim enrichment analysis was performed using the online enrichment tool of PlasmoDB (www.plasmodb.org)

Figure 4 -

Venn diagram depicting the overlap between P. falciparum proteins susceptible to different redox modifications Pull-down experiments identified 101 targets for sulfenylation, 491 target proteins for S-glutathionylation, and 317 target proteins for S-nitrosation [15,16]. 55 proteins were identified containing all 3 PTM. The Venn diagram was generated using the online tool http://bioinformatics.psb.ugent.be/webtools/Venn/.