Identification and confirmation of SUMOylation-modified proteins in Giardia trophozoites

Abstract

Posttranslational modification by the small ubiquitin-related modifier (SUMO) is one of the crucial cellular processes in <i>Giardia lamblia</i>, a protozoan pathogen. In this study, 5 candidate SUMO substrate proteins of <i>G. lamblia</i> trophozoites were chosen based on their enrichment through affinity chromatography using a SUMO-interaction motif: never in mitosis A-related kinase (NEK), aminoacyl-histidine dipeptidase (AHD), protein disulfide isomerase 2 (PDI2), alcohol dehydrogenase 3, and ornithine carbamoyltransferase. Transgenic <i>Giardia</i> trophozoites expressing 1 of the 5 candidate SUMO substrate proteins were constructed, and their expression was confirmed by western blot using hemagglutinin-tag. Arginine deiminase (ADI) protein was expressed in <i>Giardia</i> trophozoites as a positive control. Cell extracts were processed for affinity chromatography using SUMO-interaction motif resin. As expected, the SUMOylated form of ADI was detected in the affinity chromatography extracts of ADI-expressing cells. Among the 5 candidate proteins, SUMOylated forms of NEK, AHD, and PDI2 were identified in the affinity chromatography extracts. These results suggest that NEK, AHD, and PDI2 activity is modulated via SUMOylation in <i>Giardia</i> trophozoites.