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.

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Publication Details

Journal
Biology Open
Published
2026-11-02
DOI
https://doi.org/10.1242/bio.062865
Primary Topic
Glycosylation and Glycoproteins Research
Type
article
Field-Weighted Citation Impact
0.00

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article

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

Masamichi Nagae, Robiul Hasan Bhuiyan, Hiromune Ando, Akira Togayachi et al.
1 links
Biology Open
Glycosylation and Glycoproteins Research
article

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

Masamichi Nagae, Robiul Hasan Bhuiyan, Hiromune Ando, Akira Togayachi, Yuhsuke Ohmi, Mariko Kambe, Koichi Furukawa, Hirokazu Yagi, Chihiro Sato, Akira Takeda, Mohammad Abul Hasnat, Takako Ito, Keiko Furukawa, Farhana Yesmin, Orie Tajima, Hiroka Koyanagi, Ken Kitajima, Yuka Imao
article en

Abstract

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.

Biology OpenVol. 15(11)
Shahjalal University of Science and Technology (BD), Chubu University (JP), Nagoya City University (JP), NYU Langone Health (US), Soka University (JP), Gifu University (JP), Glycan and Life Systems Integration Center (JP), Institute for Glyco-core Research (JP), Nagoya University (JP), Tohoku Medical and Pharmaceutical University (JP), University of Chittagong (BD)
Japan Agency for Medical Research and Development, Chubu University
Openalex Percentile: Top 25%
Glycosylation and Glycoproteins Research
DOIOpen Access PDFOpenAlex
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