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.
Biochim Biophys Acta Gen Subj. 2024 Sep;1868(9):130663. doi: 10.1016/j.bbagen.2024.130663. Epub 2024 Jun 25.
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.
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.
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.
The slight difference in K346T localization may be responsible for the higher biosynthetic activity despite the reduced activity.
Our findings underscore the importance of fine intra-Golgi localization and reaction orders of glycosyltransferases for the biosynthesis of complex glycan structures in cells.
N-乙酰氨基葡萄糖基转移酶-III（GnT-III，也称为 MGAT3）在高尔基体中催化形成特定的 N-聚糖分支，双分支 GlcNAc。双分支 GlcNAc 是抑制 N-聚糖成熟的关键残基，与癌症和阿尔茨海默病的发病机制有关。然而，GnT-III 如何识别其底物以及 GnT-III 活性如何在细胞中调节仍不清楚。
使用 AlphaFold2 和结构比较，我们预测了 GnT-III 中与催化口袋中底物相互作用的关键氨基酸残基。我们还使用点突变进行了体外活性测定、凝集素印迹分析和 N-糖基化分析，以评估它们的活性。
我们的数据表明，人 GnT-III 的 E320 是催化中心。更有趣的是，我们发现了一种独特的突变体 K346T，其体外活性较低，而细胞内活性高于野生型 GnT-III。使用各种底物的酶测定表明，K346T 的底物特异性没有改变，而环己酰亚胺追踪实验表明，K346T 突变体的半衰期略短，表明突变体不稳定，可能是由于部分错误折叠。此外，基于 TurboID 的邻近标记显示，K346T 突变体的定位略微向高尔基体顺面移动，可能允许与竞争的半乳糖基转移酶预先作用。
尽管活性降低，但 K346T 定位的微小差异可能是导致更高生物合成活性的原因。
我们的发现强调了精细的高尔基体内部定位和糖基转移酶反应顺序对于细胞中复杂糖结构生物合成的重要性。
