Surface-exposed Glycoproteins of Hyperthermophilic Sulfolobus solfataricus P2 Show a Common N-Glycosylation Profile

Cell surface proteins of hyperthermophilic
Archaea actively participate
in intercellular communication, cellular uptake, and energy conversion
to sustain survival strategies in extreme habitats. Surface (S)-layer
glycoproteins, the major component of the S-layers in many archaeal
species and the best-characterized prokaryotic glycoproteins, were
shown to have a large structural diversity in their glycan compositions.
In spite of this, knowledge on glycosylation of proteins other than
S-layer proteins in Archaea is quite limited. Here, the N-glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin
affinity purification, HPAEC-PAD, and multiple mass spectrometry-based
techniques. Detailed analysis of SSO1273, one of the most abundant
ABC transporters present in the cell surface fraction of S.
solfataricus, revealed a novel glycan structure composed
of a branched sulfated heptasaccharide, Hex4(GlcNAc)2 plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan
of the S-layer glycoprotein of S. acidocaldarius,
this is the most complex archaeal glycan structure known today. SSO1273
protein is heavily glycosylated and all 20 theoretical N-X-S/T (where
X is any amino acid except proline) consensus sequence sites were
confirmed. Remarkably, we show that several other proteins in the
surface fraction of S. solfataricus are N-glycosylated by the same sulfated oligosaccharide and we identified
56 N-glycosylation sites in this subproteome.

嗜热古菌的细胞表面蛋白质积极参与细胞间通讯、细胞摄取和能量转换，以维持其在极端环境中的生存策略。表面（S）层糖蛋白，作为许多嗜热古菌物种S层的主体成分，同时也是已知最完善的原核生物糖蛋白，其糖基组成表现出丰富的结构多样性。尽管如此，关于古菌中除S层蛋白之外蛋白质糖基化的知识相当有限。在本研究中，通过凝集素亲和纯化、高效液相色谱-脉冲安培检测法（HPAEC-PAD）以及多种基于质谱的技术，对嗜热菌 Sulfolobus solfataricus P2 的细胞表面暴露蛋白的N-糖基化模式进行了分析。对存在于S. solfataricus 细胞表面部分中最丰富的ABC转运蛋白SSO1273的详细分析揭示了由分支硫酸化的七糖（Hex4(GlcNAc)2）加上磺基奎诺酸（sulfoquinovose）组成的糖结构，其中Hex是D-甘露糖和D-葡萄糖。由于比S. acidocaldarius的S层糖蛋白糖基多出一个单糖单元，这被认为是目前已知的最复杂的古菌糖基结构。SSO1273蛋白高度糖基化，且所有20个理论上的N-X-S/T（其中X是除脯氨酸之外的所有氨基酸）的共识序列位点均得到证实。值得注意的是，我们展示了S. solfataricus表面部分中的其他几种蛋白质也通过相同的硫酸化寡糖进行N-糖基化，并在该亚蛋白组中鉴定出56个N-糖基化位点。