Implication of N-glycosylations on B4GALNT1 enzyme in its molecular assembly and function

B4GALNT1 is a glycosyltransferase essential for the synthesis of complex gangliosides and plays important roles in the nervous system. Although N-glycosylation is thought to regulate B4GALNT1, its functional significance has remained unclear. In this study, we generated human B4GALNT1 mutants carrying single or combined substitutions at three N-glycosylation sites (N1-N3) and analyzed their enzymatic activity, intracellular localization, and molecular assembly using transfected B78 mouse melanoma cells. Loss of N-glycosylation reduced GM2 expression and enzymatic activity in a site-dependent manner, with multiple-site mutants showing greater impairment. Golgi localization was also progressively disrupted as the number of mutated N-glycosylation sites increased. Non-reducing immunoblotting demonstrated that loss of the N1 glycosylation site markedly impaired B4GALNT1 dimer formation, with additional defects caused by the combined loss of the N3 and N2 sites. Predicted dimeric B4GALNT1 structures generated by AlphaFold2 revealed the spatial arrangement of N-glycans, suggesting a dominant role for the N1 site and a supportive role for the N3 site in stabilizing intermolecular contacts. These results indicated that N-glycosylation sites in the three-dimensional structure of B4GALNT1 are critical for the molecular integrity and enzymatic activity.