Myelin protein zero (P0), a glycosylated single-pass transmembrane protein, is essential in the formation and maintenance of peripheral nervous system (PNS) compact myelin. P0 in Xenopus (xP0) exists primarily as a dimeric form that remains stable after various physical and chemical treatments. In exploring the nature of the interactions underlying the dimer stability, we found that xP0 dimer dissociated into monomer during continuous elution gel electrophoresis and conventional SDS-PAGE, indicating that the dimer is stabilized by non-covalent interactions. Furthermore, as some of the gel-purified monomer re-associated into dimer on SDS-PAGE gels, there is likely a dynamic equilibrium between xP0 dimer and monomer in vivo. Because the carbohydrate and fatty acyl moieties may be crucial for the adhesion role of P0, we used sensitive mass spectrometry approaches to elucidate the detailed N-glycosylation and S-acylation profiles of xP0. Asn92 was determined to be the single, fully-occupied glycosylation site of xP0, and a total of 12 glycans was detected that exhibited new structural features compared with those observed from P0 in other species: (1) the neutral glycans were composed mainly of high mannose and hybrid types; (2) five of twelve were acidic glycans, among which three were sialylated and the other two were sulfated; (3) none of the glycans had core fucosylation; and (4) no glucuronic acid, hence no HNK-1 epitope, was detected. The drastically different carbohydrate structures observed here support the concept of the species-specific variation in N-glycosylation of P0. Cys152 was found to be acylated with stearoyl (C18:0), whereas palmitoyl (C16:0) is the corresponding predominant fatty acyl group on P0 from higher vertebrates. We propose that the unique glycosylation and acylation patterns of Xenopus P0 may underlie its unusual dimerization behaviour. Our results should shed light on the understanding of the phylogenetic development of P0's adhesion role in PNS compact myelin.

中文翻译：

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爪蟾外周神经髓鞘蛋白零的分子特征：非共价结合二聚体和单体之间的平衡

髓鞘蛋白零 (P0) 是一种糖基化的单程跨膜蛋白，对于周围神经系统 (PNS) 致密髓鞘的形成和维持至关重要。非洲爪蟾 (xP0) 中的 P0 主要以二聚体形式存在，经过各种物理和化学处理后仍保持稳定。在探索二聚体稳定性背后相互作用的性质时，我们发现 xP0 二聚体在连续洗脱凝胶电泳和常规 SDS-PAGE 过程中解离成单体，表明二聚体通过非共价相互作用稳定。此外，由于一些凝胶纯化的单体在 SDS-PAGE 凝胶上重新结合成二聚体，因此体内 xP0 二聚体和单体之间可能存在动态平衡。因为碳水化合物和脂肪酰基部分可能对 P0 的粘附作用至关重要，我们使用灵敏的质谱方法来阐明 xP0 的详细 N-糖基化和 S-酰化谱。Asn92 被确定为 xP0 的单个、完全占据的糖基化位点，总共检测到 12 个聚糖，与从 P0 观察到的其他物种相比，它们表现出新的结构特征：（1）中性聚糖主要由高甘露糖和杂种类型；(2)十二个中有五个是酸性聚糖，其中三个是唾液酸化的，另外两个是硫酸化的；(3) 没有一个聚糖具有核心岩藻糖基化；(4)未检测到葡萄糖醛酸，因此未检测到HNK-1表位。这里观察到的完全不同的碳水化合物结构支持 P0 的 N-糖基化的物种特异性变异的概念。发现 Cys152 被硬脂酰 (C18:0) 酰化，而棕榈酰 (C16: 0) 是来自高等脊椎动物的 P0 上相应的主要脂肪酰基。我们认为非洲爪蟾 P0 独特的糖基化和酰化模式可能是其不寻常的二聚化行为的基础。我们的结果应该阐明对 P0 在 PNS 致密髓鞘中的粘附作用的系统发育发展的理解。