Filippo Cristina Amat di San, Ardon Orly, Longo Nicola
Department of Pediatrics and Pathology, University of Utah, Salt Lake City, UT 84132, USA.
Biochim Biophys Acta. 2011 Mar;1812(3):312-20. doi: 10.1016/j.bbadis.2010.11.007. Epub 2010 Nov 29.
Primary carnitine deficiency is caused by impaired activity of the Na(+)-dependent OCTN2 carnitine/organic cation transporter. Carnitine is essential for entry of long-chain fatty acids into mitochondria and its deficiency impairs fatty acid oxidation. Most missense mutations identified in patients with primary carnitine deficiency affect putative transmembrane or intracellular domains of the transporter. Exceptions are the substitutions P46S and R83L located in an extracellular loop close to putative glycosylation sites (N57, N64, and N91) of OCTN2. P46S and R83L impaired glycosylation and maturation of OCTN2 transporters to the plasma membrane. We tested whether glycosylation was essential for the maturation of OCTN2 transporters to the plasma membrane. Substitution of each of the three asparagine (N) glycosylation sites with glutamine (Q) decreased carnitine transport. Substitution of two sites at a time caused a further decline in carnitine transport that was fully abolished when all three glycosylation sites were substituted by glutamine (N57Q/N64Q/N91Q). Kinetic analysis of carnitine and sodium-stimulated carnitine transport indicated that all substitutions decreased the Vmax for carnitine transport, but N64Q/N91Q also significantly increased the Km toward carnitine, indicating that these two substitutions affected regions of the transporter important for substrate recognition. Western blot analysis confirmed increased mobility of OCTN2 transporters with progressive substitutions of asparagines 57, 64 and/or 91 with glutamine. Confocal microscopy indicated that glutamine substitutions caused progressive retention of OCTN2 transporters in the cytoplasm, up to full retention (such as that observed with R83L) when all three glycosylation sites were substituted. Tunicamycin prevented OCTN2 glycosylation, but it did not impair maturation to the plasma membrane. These results indicate that OCTN2 is physiologically glycosylated and that the P46S and R83L substitutions impair this process. Glycosylation does not affect maturation of OCTN2 transporters to the plasma membrane, but the 3 asparagines that are normally glycosylated are located in a region important for substrate recognition and turnover rate.
原发性肉碱缺乏症是由钠依赖性OCTN2肉碱/有机阳离子转运体活性受损引起的。肉碱对于长链脂肪酸进入线粒体至关重要，其缺乏会损害脂肪酸氧化。在原发性肉碱缺乏症患者中鉴定出的大多数错义突变影响转运体的推定跨膜或细胞内结构域。例外情况是位于靠近OCTN2推定糖基化位点（N57、N64和N91）的细胞外环中的P46S和R83L替代。P46S和R83L损害了OCTN2转运体向质膜的糖基化和成熟。我们测试了糖基化对于OCTN2转运体向质膜成熟是否必不可少。用谷氨酰胺（Q）替代三个天冬酰胺（N）糖基化位点中的每一个都会降低肉碱转运。一次替代两个位点会导致肉碱转运进一步下降，当所有三个糖基化位点都被谷氨酰胺替代（N57Q/N64Q/N91Q）时，这种下降完全消除。对肉碱和钠刺激的肉碱转运的动力学分析表明，所有替代都降低了肉碱转运的Vmax，但N64Q/N91Q也显著增加了对肉碱的Km，表明这两个替代影响了转运体中对底物识别重要的区域。蛋白质印迹分析证实，随着天冬酰胺57、64和/或91逐渐被谷氨酰胺替代，OCTN2转运体的迁移率增加。共聚焦显微镜检查表明，谷氨酰胺替代导致OCTN2转运体在细胞质中逐渐滞留，当所有三个糖基化位点都被替代时，会完全滞留（如R83L所观察到的）。衣霉素可阻止OCTN2糖基化，但不会损害其向质膜的成熟。这些结果表明OCTN2在生理上是糖基化的，并且P46S和R83L替代会损害这一过程。糖基化不影响OCTN2转运体向质膜的成熟，但通常被糖基化的三个天冬酰胺位于对底物识别和周转率重要的区域。
