Deciphering roles of ppGalNAc-transferases in cellular nucleus

CEJAS, RB; LORENZ, VIRGINIA; IRAZOQUI FJ

Congreso; SAIB 2015; 2015

Sociedad Argentina de Investigaciones Bioquímicas (SAIB)

Polypeptide GalNAc-transferases (ppGalNAc-Ts) catalyze the covalent linkage of α-GalNAc to Ser/Thr in initiation of O-GalNAc glycosylation in Golgi apparatus. The aim of present work was to study subcellular ppGalNAc-T localization as well as nuclear O-GalNAc glycosylation. The presence of ppGalNAc-T3 in nucleus of several human cell lines was analyzed by confocal microscopy and subcellular fractionation. Purified ppGalNAc-T3 was immobilized in a chromatographic column and interaction with nuclear proteins was observed by SDS-PAGE, and identified by mass spectrometry. In order to elucidate nuclear O-GalNAc glycosylation by endogenous ppGalNAc-Ts, we purified cellular nucleus that conserves their integrity and are functionally active. Nuclei were incubated with UDP-GalNAc and nuclear protein glycosylation was analyzed by Western Blotting employing HPA lectin to detect GalNAcαSer/Thr terminals. We observed high levels of O-GalNAc glycosylation which was reduced when this sample was treated with α-GalNAc glycosidase. UDP-GlcNAc was assayed as control. In conclusion, we observed nuclear localization of ppGalNAc-T3 that can interacts with nucleoproteins and nuclear protein O-GalNAc glycosylation. This posttranslational modification as well as the interaction of ppGalNAc-Ts with histones and ARN related proteins could have relevant roles in transcriptional regulation.