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.
方法:使用AlphaFold2和结构比较,我们预测了GnT-III中与催化袋中底物相互作用的关键氨基酸残基。我们还进行了体外活性测定,使用点突变体进行凝集素印迹分析和N-糖组学分析以评估其活性。
结果:我们的数据表明,人GnT-III的E320是催化中心。更有趣的是,我们发现了一个独特的突变体,K346T,与野生型GnT-III相比,其表现出更低的体外活性和更高的细胞内活性。使用各种底物的酶分析表明,K346T的底物特异性没有变化,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的邻近标记表明,K346T突变体的定位稍微向高尔基体的顺侧移动,可能允许对竞争半乳糖基转移酶的事先作用。
结论:尽管活性降低,但K346T定位的微小差异可能是生物合成活性较高的原因。
结论:我们的发现强调了高尔基体内精细定位和糖基转移酶反应顺序对于细胞内复杂聚糖结构生物合成的重要性。