在内质网(ER)相关降解过程中,游离N-聚糖(FNG)是通过植物胞质溶胶中的胞浆肽N-聚糖酶(cPNGase)和内切-β-N-乙酰葡糖胺糖苷酶(ENGase)的组合从错误折叠的新生糖蛋白产生的。所得的高甘露糖型(HMT)-FNG,在还原端携带一个GlcNAc残基(GN1-FNG),在发育中的植物细胞中普遍存在。在之前的研究中,我们发现HMT-FNG有助于蛋白质折叠并抑制β-淀粉样蛋白原纤维的形成,提示FNG可能参与蛋白质折叠系统的生物功能。然而,这些HMT-FNG是否出现在急诊室,参与蛋白质折叠的细胞器,仍然不清楚。相反,我们还报道了植物复合型(PCT)-GN1-FNG的存在,在非还原端携带Lewisa表位,表明这些FNG已经在高尔基体中得到了充分的处理。由于植物ENGase对HMT-N-聚糖有活性,但对携带β1-2木糖基和/或α1-3岩藻糖基残基的PCT-N-聚糖没有活性,这些PCT-GN1-FNG似乎不是通过ENGase活性从含有PCT-N-聚糖的完全加工的糖蛋白产生的.有趣的是,在细胞外间隙中发现了PCT-GN1-FNG,表明在细胞质中形成的HMT-GN1-FNG可能被转运回ER并通过蛋白质分泌途径在高尔基体中加工。作为阐明PCT-GN1-FNG产生机制的第一步,我们分析了植物微粒体中游离寡糖的结构,证明了在微粒体中可以发现HMT-FNGs(Man9-7GlcNAc1和Man9-8GlcNAc2),几乎由急诊室组成。
During endoplasmic reticulum (ER)-associated degradation, free N-glycans (FNGs) are produced from misfolded nascent glycoproteins via the combination of the cytosolic peptide N-glycanase (cPNGase) and endo-β-N-acetylglucosaminidase (ENGase) in the plant cytosol. The resulting high-mannose type (HMT)-FNGs, which carry one GlcNAc residue at the reducing end (GN1-FNGs), are ubiquitously found in developing plant cells. In a previous study, we found that HMT-FNGs assisted in protein folding and inhibited β-amyloid fibril formation, suggesting a possible biofunction of FNGs involved in the protein folding system. However, whether these HMT-FNGs occur in the ER, an organelle involved in protein folding, remained unclear. On the contrary, we also reported the presence of plant complex type (PCT)-GN1-FNGs, which carry the Lewisa epitope at the non-reducing end, indicating that these FNGs had been fully processed in the Golgi apparatus. Since plant ENGase was active toward HMT-N-glycans but not PCT-N-glycans that carry β1-2xylosyl and/or α1-3 fucosyl residue(s), these PCT-GN1-FNGs did not appear to be produced from fully processed glycoproteins that harbored PCT-N-glycans via ENGase activity. Interestingly, PCT-GN1-FNGs were found in the extracellular space, suggesting that HMT-GN1-FNGs formed in the cytosol might be transported back to the ER and processed in the Golgi apparatus through the protein secretion pathway. As the first step in elucidating the production mechanism of PCT-GN1-FNGs, we analyzed the structures of free oligosaccharides in plant microsomes and proved that HMT-FNGs (Man9-7GlcNAc1 and Man9-8GlcNAc2) could be found in microsomes, which almost consist of the ER compartments.