Department of Physiology, School of Medicine, UCLA, and Veterans Administration Greater Los Angeles Health Care System, VAGLAHS/West LA, Building 113, Room 324, 11301 Wilshire Boulevard, Los Angeles, California 90073, USA.
Biochemistry. 2010 Apr 13;49(14):3116-28. doi: 10.1021/bi100115a.
Bulky hydrophilic N-glycans stabilize the proper tertiary structure of glycoproteins. In addition, N-glycans comprise the binding sites for the endoplasmic reticulum (ER)-resident lectins that assist correct folding of newly synthesized glycoproteins. To reveal the role of N-glycans in maturation of the Na,K-ATPase beta(2) subunit in the ER, the effects of preventing or modifying the beta(2) subunit N-glycosylation on trafficking of the subunit and its binding to the ER lectin chaperone, calnexin, were studied in MDCK cells. Preventing N-glycosylation abolishes binding of the beta(2) subunit to calnexin and results in the ER retention of the subunit. Furthermore, the fully N-glycosylated beta(2) subunit is retained in the ER when glycan-calnexin interactions are prevented by castanospermine, showing that N-glycan-mediated calnexin binding is required for correct subunit folding. Calnexin binding persists for several hours after translation is stopped with cycloheximide, suggesting that the beta(2) subunit undergoes repeated post-translational calnexin-assisted folding attempts. Homology modeling of the beta(2) subunit using the crystal structure of the alpha(1)-beta(1) Na,K-ATPase shows the presence of a relatively hydrophobic amino acid cluster proximal to N-glycosylation sites 2 and 7. Combined, but not separate, removal of sites 2 and 7 dramatically impairs calnexin binding and prevents the export of the beta(2) subunit from the ER. Similarly, hydrophilic substitution of two hydrophobic amino acids in this cluster disrupts both beta(2)-calnexin binding and trafficking of the subunit to the Golgi. Therefore, the hydrophobic residues in the proximity of N-glycans 2 and 7 are required for post-translational calnexin binding to these N-glycans in incompletely folded conformers, which, in turn, is necessary for maturation of the Na,K-ATPase beta(2) subunit.
亲水性大的 N-聚糖稳定糖蛋白的正确三级结构。此外，N-聚糖还包含内质网（ER）驻留凝集素的结合位点，这些凝集素有助于新合成的糖蛋白的正确折叠。为了揭示 N-聚糖在 ER 中 Na,K-ATPaseβ2 亚基成熟过程中的作用，研究了在 MDCK 细胞中，阻止或修饰β2 亚基 N-糖基化对亚基运输及其与 ER 凝集素伴侣 calnexin 结合的影响。阻止 N-糖基化会使β2 亚基与 calnexin 结合，并导致亚基在 ER 中滞留。此外，当用 castanospermine 阻止聚糖-calnexin 相互作用时，完全 N-糖基化的β2 亚基仍保留在 ER 中，表明 N-聚糖介导的 calnexin 结合对于正确的亚基折叠是必需的。用环己酰亚胺停止翻译后，calnexin 结合可维持数小时，这表明β2 亚基在翻译后经历了反复的 calnexin 辅助折叠尝试。使用α1-β1 Na,K-ATPase 的晶体结构对β2 亚基进行同源建模表明，在靠近 N-糖基化位点 2 和 7 的位置存在一个相对疏水的氨基酸簇。位点 2 和 7 的联合而非单独去除会严重损害 calnexin 结合并阻止β2 亚基从 ER 中输出。同样，该簇中两个疏水氨基酸的亲水取代会破坏β2-calnexin 结合以及亚基向高尔基体的运输。因此，N-聚糖 2 和 7 附近的疏水性残基对于未完全折叠的构象中翻译后 calnexin 与这些 N-聚糖的结合是必需的，而这对于 Na,K-ATPaseβ2 亚基的成熟是必需的。
