The GalNAc-T Activation (GALA) Pathway: Drivers and markers

The enzymes GALNTs add GalNAc sugar to Ser and Thr residues, forming the Tn glycan. GALNTs are activated by trafficking from Golgi to ER, a process driven by the Src kinase and negatively regulated by ERK8. This GALNTs activation (aka GALA) pathway induces high Tn levels and is a key driver of liver tumor growth. Recently, Tabak and colleagues have contested our previous data that EGF stimulation can induce GALNTs relocation. Here, we show that relocation induced by EGF is actually detectable in the very images acquired by Tabak et al. Furthermore, we show that over-expression of EGFR strongly enhances EGF-induced relocation and that EGFR appears required to drive relocation induced by ERK8 depletion. Direct co-localisation of GALNT with the ER marker Calnexin is observed after EGF stimulation. We furthermore propose that quantification of O-glycosylation of the ER resident protein PDIA4 provides a mean to quantify GALA independently of imaging. In sum, we demonstrate that the claimed non-reproducibility was due to experimental imaging conditions, that EGFR is indeed a driver of GALA and propose additional markers to facilitate the study of this pathway.GALNTs家族糖基转移酶（GALNTs）可将N-乙酰半乳糖胺（GalNAc）单糖连接至丝氨酸（Serine，Ser）与苏氨酸（Threonine，Thr）残基，进而生成Tn聚糖（Tn glycan）。GALNTs的激活依赖于其从高尔基体（Golgi）向内质网（Endoplasmic Reticulum，ER）的转运过程，该过程由Src激酶（Src kinase）驱动，并受细胞外调节蛋白激酶8（ERK8）负向调控。该GALNTs激活通路（又称GALA通路）可诱导高水平Tn聚糖生成，是肝脏肿瘤生长的关键驱动因素。近期，Tabak及其团队对我们此前提出的"表皮生长因子（Epidermal Growth Factor，EGF）刺激可诱导GALNTs发生转位"的实验数据提出了质疑。本研究证实，EGF诱导的GALNTs转位现象实际上可在Tabak等人所获取的原始成像数据中被检测到。进一步研究表明，表皮生长因子受体（Epidermal Growth Factor Receptor，EGFR）的过表达可显著增强EGF诱导的GALNTs转位，且EGFR似乎是ERK8敲低所诱导的转位过程所必需的调控因子。在EGF刺激后，可观察到GALNT与内质网标志物钙连蛋白（Calnexin）的直接共定位现象。本研究同时提出，对内质网驻留蛋白PDIA4的O-糖基化（O-glycosylation）水平进行定量分析，可在不依赖成像实验的前提下实现对GALA通路活性的量化检测。综上，本研究证实此前所谓的"实验无法重现"实则源于实验成像条件的差异，明确了EGFR确实是GALA通路的驱动因子，并提出了可用于该通路研究的新型辅助标志物。