Biosynthesis of N-acetyllactosamine glycans in human cell nucleus

PARODI P; IRAZOQUI FJ

Mendoza, Argentina

Congreso; SAIB; 2022

SAIB

O-glycosylation is a protein post-translational modification in human cells that is critical for cell physiology and involved in several pathologies. In particular, O-GlcNAc glycosylation is found in nuclear proteins with regulatory functions in the cell nucleus and showing altered expression in different cancer types. Moreover, there is no evidence about the elongation of this glycan in nuclear proteins. In the present work we study the catalytic activity of β galactosyltransferase in the cell nucleus, necessary for the elongation of O-GlcNAc glycan in N-acetyllactosamine. We detected the presence of β-4 Galactosyltransferase 1 in the nucleus of human cells such as CaCo2, HeLa and A549 by confocal microscopy including z-axis cutting. This enzyme catalyze the covalent union between galactose (using UDP-galactose as donor) and O-GlcNAc terminals (acceptor) via β 14 bond to form nuclear O-acetyllactosamine. It is important to mention that UDP-Gal is permeable to the nuclear membrane, so it is biologically available in the cell nucleus. We studied the nuclear β-galactosyltransferase activity through in vitro assays for purified nucleoplasm of different line cells (HeLa and CaCo2). This activity was measured on different glycoprotein acceptors such as GlcN-BSA and ovalbumin, and detected by using biotinylated Erythrina cristagalli lectin (ECL) that is able to bond to terminal lactosamine residues. In these studies we found an important β -galactosyltransferase activity in cell nucleoplasm. We also find constitutive presence of lactosamine residues in nuclear proteins by immunofluorescence and western blot, using ECL as probe. Finally, we measured lactosamine residues in nuclear proteins such as Lamin B1 and RNA pol2 by ELISA sandwich assays. All together show that the machinery needed for O-GlcNAc elongation is located in the human cell nucleus. We are working in additional evidences of this important biochemical discovery.