The K346T mutant of GnT-III bearing weak in vitro and potent intracellular activity.

Autor: Hashimoto Y; Graduate School of Natural Science and Technology, Gifu University, Gifu 501-1193, Japan., Kawade H; Graduate School of Natural Science and Technology, Gifu University, Gifu 501-1193, Japan., Bao W; Glyco-Biochemistry Laboratory, Institute for Glyco-core Research (iGCORE), Gifu University, Gifu 501-1193, Japan., Morii S; Graduate School of Integrated Sciences for Life, Hiroshima University, Higashihiroshima 739-8530, Japan., Nakano M; Graduate School of Integrated Sciences for Life, Hiroshima University, Higashihiroshima 739-8530, Japan., Nagae M; Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Japan; Laboratory of Molecular Immunology, Immunology Frontier Research Center (IFReC), Osaka University, Suita 565-0871, Japan., Murakami R; Glycoanalytical Chemistry Laboratory, Institute for Glyco-core Research (iGCORE), Gifu University, Gifu 501-1193, Japan., Tokoro Y; Glyco-Biochemistry Laboratory, Institute for Glyco-core Research (iGCORE), Gifu University, Gifu 501-1193, Japan., Nakashima M; Glyco-Biochemistry Laboratory, Institute for Glyco-core Research (iGCORE), Gifu University, Gifu 501-1193, Japan., Cai Z; Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai 981-8558, Japan., Isaji T; Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai 981-8558, Japan., Gu J; Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai 981-8558, Japan., Nakajima K; Glycoanalytical Chemistry Laboratory, Institute for Glyco-core Research (iGCORE), Gifu University, Gifu 501-1193, Japan., Kizuka Y; Graduate School of Natural Science and Technology, Gifu University, Gifu 501-1193, Japan; Glyco-Biochemistry Laboratory, Institute for Glyco-core Research (iGCORE), Gifu University, Gifu 501-1193, Japan. Electronic address: kizuka.yasuhiko.k8@f.gifu-u.ac.jp.
Jazyk: angličtina
Zdroj: Biochimica et biophysica acta. General subjects [Biochim Biophys Acta Gen Subj] 2024 Sep; Vol. 1868 (9), pp. 130663. Date of Electronic Publication: 2024 Jun 25.
DOI: 10.1016/j.bbagen.2024.130663
Abstrakt: Background: N-Acetylglucosaminyltransferase-III (GnT-III, also designated MGAT3) catalyzes the formation of a specific N-glycan branch, bisecting GlcNAc, in the Golgi apparatus. Bisecting GlcNAc is a key residue that suppresses N-glycan maturation and is associated with the pathogenesis of cancer and Alzheimer's disease. However, it remains unclear how GnT-III recognizes its substrates and how GnT-III activity is regulated in cells.
Methods: Using AlphaFold2 and structural comparisons, we predicted the key amino acid residues in GnT-III that interact with substrates in the catalytic pocket. We also performed in vitro activity assay, lectin blotting analysis and N-glycomic analysis using point mutants to assess their activity.
Results: Our data suggested that E320 of human GnT-III is the catalytic center. More interestingly, we found a unique mutant, K346T, that exhibited lower in vitro activity and higher intracellular activity than wild-type GnT-III. The enzyme assays using various substrates showed that the substrate specificity of K346T was unchanged, whereas cycloheximide chase experiments revealed that the K346T mutant has a slightly shorter half-life, suggesting that the mutant is unstable possibly due to a partial misfolding. Furthermore, TurboID-based proximity labeling showed that the localization of the K346T mutant is shifted slightly to the cis side of the Golgi, probably allowing for prior action to competing galactosyltransferases.
Conclusions: The slight difference in K346T localization may be responsible for the higher biosynthetic activity despite the reduced activity.
General Significance: Our findings underscore the importance of fine intra-Golgi localization and reaction orders of glycosyltransferases for the biosynthesis of complex glycan structures in cells.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023. Published by Elsevier B.V.)
Databáze: MEDLINE
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