%0 Journal Article
%T The K346T mutant of GnT-III bearing weak in vitro and potent intracellular activity.
%A Hashimoto Y
%A Kawade H
%A Bao W
%A Morii S
%A Nakano M
%A Nagae M
%A Murakami R
%A Tokoro Y
%A Nakashima M
%A Cai Z
%A Isaji T
%A Gu J
%A Nakajima K
%A Kizuka Y
%J Biochim Biophys Acta Gen Subj
%V 1868
%N 9
%D 2024 Sep 25
%M 38936637
%F 4.117
%R 10.1016/j.bbagen.2024.130663
%X 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.
CONCLUSIONS: Our findings underscore the importance of fine intra-Golgi localization and reaction orders of glycosyltransferases for the biosynthesis of complex glycan structures in cells.