Sda碳水化合物表位及其生物合成B4GALNT2酶在健康结肠中表达，并在结肠癌中下调至不同程度。人B4GALNT2基因驱动共享相同跨膜和腔结构域的长和短蛋白同种型(LF-B4GALNT2和SF-B4GALNT2)的表达。两种同种型都是反式高尔基体蛋白，由于其延伸的细胞质尾部，LF-B4GALNT2也定位在高尔基体后囊泡中。支持Sda和B4GALNT2在胃肠道中表达的控制机制是复杂的，尚未完全了解。这项研究揭示了B4GALNT2腔结构域中存在两个不寻常的N-糖基化位点。第一个非典型N-X-C位点在进化上是保守的，并被复合型N-聚糖占据。我们使用定点诱变探索了这种N-聚糖的影响，并表明每个突变体的表达水平略有降低，稳定性受损，酶活性降低。此外，我们观察到突变体SF-B4GALNT2在内质网中部分错位,而突变体LF-B4GALNT2仍位于高尔基体和后高尔基体囊泡中。最后,我们表明，在两种突变的同种型中，同二聚体的形成受到了极大的损害。在每个单体上具有N-聚糖的LF-B4GALNT2二聚体的AlphaFold2模型证实了这些发现，并表明每个B4GALNT2同工型的N-糖基化控制了它们的生物活性。
The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.
