{Reference Type}: Journal Article
{Title}: The K346T mutant of GnT-III bearing weak in vitro and potent intracellular activity.
{Author}: Hashimoto Y;Kawade H;Bao W;Morii S;Nakano M;Nagae M;Murakami R;Tokoro Y;Nakashima M;Cai Z;Isaji T;Gu J;Nakajima K;Kizuka Y;
{Journal}: Biochim Biophys Acta Gen Subj
{Volume}: 1868
{Issue}: 9
{Year}: 2024 Sep 25
{Factor}: 4.117
{DOI}: 10.1016/j.bbagen.2024.130663
{Abstract}: <B>BACKGROUND: </B>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.<BR><B>METHODS: </B>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.<BR><B>RESULTS: </B>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.<BR><B>CONCLUSIONS: </B>The slight difference in K346T localization may be responsible for the higher biosynthetic activity despite the reduced activity.<BR><B>CONCLUSIONS: </B>Our findings underscore the importance of fine intra-Golgi localization and reaction orders of glycosyltransferases for the biosynthesis of complex glycan structures in cells.