Biochimica et biophysica acta. General subjects · 2024
3D structural insights into the effect of N-glycosylation in human chitotriosidase variant G102S.
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Xu Xiao, Manabe Noriyoshi, Ohno Shiho, Komatsu Sachiko, Fujimura Tsutomu, Yamaguchi Yoshiki
Аннотация
BACKGROUND: N-glycosylation is a key post-translational modification critical for protein function and stability. Chitotriosidase-1 (CHIT1), belonging to glycoside hydrolase family 18, is clinically utilized as a biomarker of Gaucher disease. A G102S variant is common in some populations, but the implications of this missense mutation on CHIT1 function and in disease pathology are unknown. We have investigated the effects of the G102S mutation on the N-glycosylation, structure, and activity of CHIT1.
METHODS: Three recombinant CHIT1 proteins, wild-type (WT), G102S, and N100Q+G102S double mutants, were expressed, purified, and analyzed for glycosylation using SDS-PAGE, MALDI-MS, PNGase F treatment, and lectin blotting. NMR and LC-MS/MS were employed to characterize glycan structures. Enzymatic assays and molecular dynamics simulations were used to assess the effects of mutations on CHIT1 function and dynamics.
RESULTS: The G102S mutation introduced a new N-glycosylation site at N100, confirmed by SDS-PAGE and MALDI-MS, and the composition of the N-glycan structures was verified by lectin blotting, NMR, and MS. Both G102S and N100Q+G102S proteins exhibited reduced catalytic efficiency compared to WT. Molecular dynamics simulations suggested that G102S mutation induces significant structural changes and reduces stability, particularly without N-glycan, likely impairing substrate binding and enzymatic activity.
CONCLUSION: Our findings indicate that the common G102S mutation affects the structure and function of CHIT1, partially by introducing a new N-glycosylation site. They provide a foundation for further research on the impact of N-glycosylation on its hydrolase activity and structural dynamics, with potential implications for understanding the role of CHIT1 in Gaucher disease.
METHODS: Three recombinant CHIT1 proteins, wild-type (WT), G102S, and N100Q+G102S double mutants, were expressed, purified, and analyzed for glycosylation using SDS-PAGE, MALDI-MS, PNGase F treatment, and lectin blotting. NMR and LC-MS/MS were employed to characterize glycan structures. Enzymatic assays and molecular dynamics simulations were used to assess the effects of mutations on CHIT1 function and dynamics.
RESULTS: The G102S mutation introduced a new N-glycosylation site at N100, confirmed by SDS-PAGE and MALDI-MS, and the composition of the N-glycan structures was verified by lectin blotting, NMR, and MS. Both G102S and N100Q+G102S proteins exhibited reduced catalytic efficiency compared to WT. Molecular dynamics simulations suggested that G102S mutation induces significant structural changes and reduces stability, particularly without N-glycan, likely impairing substrate binding and enzymatic activity.
CONCLUSION: Our findings indicate that the common G102S mutation affects the structure and function of CHIT1, partially by introducing a new N-glycosylation site. They provide a foundation for further research on the impact of N-glycosylation on its hydrolase activity and structural dynamics, with potential implications for understanding the role of CHIT1 in Gaucher disease.