Medical Faculty, Institute of Biochemistry II, University of Cologne, 50931 Köln, Germany.
Glycobiology. 2013 Aug;23(8):935-45. doi: 10.1093/glycob/cwt030. Epub 2013 May 1.
The apical transmembrane glycoprotein MUC1 is endocytosed to recycle through the trans-Golgi network (TGN) or Golgi complex to the plasma membrane. We followed the hypothesis that not only the known follow-up sialylation of MUC1 in the TGN is associated with this process, but also a remodeling of O-glycan core structures, which would explain the previously described differential core 2- vs core 1-based O-glycosylation of secreted, single Golgi passage and recycling membrane MUC1 isoforms (Engelmann K, Kinlough CL, Müller S, Razawi H, Baldus SE, Hughey RP, Hanisch F-G. 2005. Glycobiology. 15:1111-1124). Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles. To address this novel observation, we used recombinant epitope-tagged MUC1 (MUC1-M) and mutant forms with abrogated clathrin-mediated endocytosis (MUC1-M-Y20,60N) or blocked recycling (palmitoylation-defective MUC1-M-CQC/AQA). We show that the CQC/AQA mutant transits the TGN at significantly lower levels, concomitant with a strongly reduced shedding from the plasma membrane and its accumulation in endosomal compartments. Intriguingly, the O-glycosylation of the shed MUC1 ectodomain subunit changes from preponderant sialylated core 1 (MUC1-M) to core 2 glycans on the non-recycling CQC/AQA mutant. The O-glycoprofile of the non-recycling CQC/AQA mutant resembles the core 2 glycoprofile on a secretory MUC1 probe that transits the Golgi complex only once. In contrast, the MUC1-M-Y20,60N mutant recycles via flotillin-dependent pathways and shows the wild-type phenotype with dominant core 1 expression. Differential radiolabeling of protein with [(35)S]Met/Cys or glycans with [(3)H]GlcNH2 in pulse-chase experiments of surface biotinylated MUC1 revealed a significantly shorter half-life of [(3)H]MUC1 when compared with [(35)S]MUC1, whereas the same ratio for the CQC/AQA mutant was close to one. This finding further supports the novel possibility of a recycling-associated O-glycan processing from Gal1-4GlcNAc1-6(Gal1-3)GalNAc (core 2) to Gal1-3GalNAc (core 1).
顶端跨膜糖蛋白 MUC1 被内吞作用再循环通过反式高尔基体网络 (TGN) 或高尔基体复合物到质膜。我们遵循这样的假设，即不仅 MUC1 在 TGN 中的已知后续唾液酸化与这个过程有关，而且 O-聚糖核心结构的重塑也与这个过程有关，这可以解释先前描述的分泌的、单次高尔基体通过和再循环膜 MUC1 同工型的不同核心 2- 与核心 1- 基于的 O-糖基化 (Engelmann K, Kinlough CL, Müller S, Razawi H, Baldus SE, Hughey RP, Hanisch F-G. 2005. Glycobiology. 15:1111-1124)。跨膜和分泌的 MUC1 探针显示出与运输相关的 O-聚糖核心谱的变化。为了解决这一新的观察结果，我们使用了重组表位标记的 MUC1 (MUC1-M) 和突变形式，其阻断网格蛋白介导的内吞作用 (MUC1-M-Y20,60N) 或阻断再循环 (棕榈酰化缺陷的 MUC1-M-CQC/AQA)。我们表明，CQC/AQA 突变体在 TGN 中的转运水平显著降低，同时从质膜的脱落和其在内体区室中的积累也大大减少。有趣的是，脱落的 MUC1 胞外结构域亚基的 O-糖基化从占优势的唾液酸化核心 1 (MUC1-M) 改变为非再循环 CQC/AQA 突变体上的核心 2 聚糖。非再循环 CQC/AQA 突变体的 O-糖蛋白谱类似于单次通过高尔基体复合物的分泌性 MUC1 探针上的核心 2 糖蛋白谱。相比之下，MUC1-M-Y20,60N 突变体通过 flotillin 依赖途径再循环，并表现出显性核心 1 表达的野生型表型。表面生物素化 MUC1 的脉冲追踪实验中用 [(35)S]Met/Cys 或 [(3)H]GlcNH2 对蛋白质进行的差异放射性标记表明，与 [(35)S]MUC1 相比，[(3)H]MUC1 的半衰期明显更短，而 CQC/AQA 突变体的同一比例接近 1。这一发现进一步支持了一种新的可能性，即再循环相关的 O-聚糖加工从 Gal1-4GlcNAc1-6(Gal1-3)GalNAc (核心 2) 到 Gal1-3GalNAc (核心 1)。
