Verification of two potential glycosylation sites in human ncu-g1
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- Master's theses (KBM) 
Glycosylation is an important postranslational modification that greatly affects protein function. N-glycosylation is one class of this modification that has been characterized widely for some proteins. This glycosylation type involves the addition of glycans to asparagine amino acids. The most common protein acceptor motif for N-glycosylation is N-X-S/T. NCUG1 is a highly glycosylated novel protein. According to the mentioned consensus motif, hNCU-G1 contains seven predicted glycosylation sites. The aim of this study was to investigate some of the potential glycosylation sites on the hNCU-G1 amino acid sequence, and to study the effect of these sites on the protein molecular weight, expression, and subcellular localization. Although prediction programs gave variable results concerning potential glycosylation sites on the hNCU-G1 protein sequence, they agreed on four of these sites. We changed asparagine amino acid of two of these potential sites to alanine using a site directed mutagenesis kit. These sites were located at positions 65 and 230 of the hNCU-G1 amino acid sequence. We transiently transfected Hela cells with the wild type hNCU-G1 and the two Nglycosylation mutants (65mut and 230mut), then analyzed the expressed proteins by western immunoblotting and confocal microscopy. Both methods showed lower expression of the 65mut, as opposed to a very high expression of the 230mut. Two glycoforms of NCU-G1 proteins with the molecular weights of 62 and 75 kDa were detected. In addition, the 230mut was found to lower the apparent molecular weight of the 75 kDa glycoform by around 3 kDa. hNCU-G1 was found to localize in the cytoplasm, and it was also detected in the nuclei of the 230mut. In conclusion, we hypothesize that glycosylation at position 65 of the hNCU-G1 amino acid sequence possibly affects its stability, folding, or antigenicity, and that glycosylation at position 230 possibly affects its antigenicity, turnover or conformation. In addition, the 230mut glycosylation site influenced the nuclear import, although this effect is not well understood. Finally, we expect that hNCU-G1 has more than one glycoform, with molecular weights that are cell and organelle specific. To this end, we recommend further characterization of more single and multiple glycosylation mutants. It would be of great value to study the exact mechanisms by which the glycosylation mutants exert their effects.