Huuskonen J, Jauhiainen M, Ehnholm C, Olkkonen V M
Department of Biochemistry, National Public Health Institute, Helsinki, Finland.
J Lipid Res. 1998 Oct;39(10):2021-30.
Plasma phospholipid transfer protein (PLTP) plays a critical role in lipoprotein metabolism and reverse cholesterol transport. We have studied the biosynthesis and secretion of PLTP using a stably transfected inducible HeLa cell line. Pulse-chase analysis revealed that: i) the major secreted forms of PLTP carry complex N-glycans; ii) N-glycosylation is crucial for PLTP secretion; iii) Endo H-resistant forms of PLTP could not be enriched using a 20 degrees C temperature block, indicating that the transport of PLTP from the endoplasmic reticulum to the Golgi apparatus is exceptionally sensitive to low temperatures; and iv) treatment of the PLTP-producing cells with the reducing agent dithiothreitol caused a reversible secretion arrest, suggesting a role of disulfide bonds in the correct folding of PLTP. Transient expression of C-terminally truncated PLTP variants in COS cells demonstrated that: i) the 30 C-terminal amino acids are dispensable for PLTP secretion, whereas deletion of 35-50 residues results in a complete absence of secretion; and ii) the deletion of 30 C-terminal amino acid residues almost completely abolished the phospholipid transfer activity of PLTP. The present study describes for the first time the biosynthesis of phospholipid transfer protein and provides tools for detailed elucidation of the structure-function relationships in the protein.
血浆磷脂转运蛋白（PLTP）在脂蛋白代谢和胆固醇逆向转运中起关键作用。我们利用稳定转染的可诱导HeLa细胞系研究了PLTP的生物合成和分泌。脉冲追踪分析显示：i）PLTP的主要分泌形式带有复杂的N-聚糖；ii）N-糖基化对PLTP的分泌至关重要；iii）使用20℃温度阻滞无法富集Endo H抗性形式的PLTP，这表明PLTP从内质网到高尔基体的转运对低温异常敏感；iv）用还原剂二硫苏糖醇处理产生PLTP的细胞会导致可逆的分泌停滞，这表明二硫键在PLTP的正确折叠中起作用。在COS细胞中C末端截短的PLTP变体的瞬时表达表明：i）30个C末端氨基酸对于PLTP的分泌是可有可无的，而缺失35-50个残基会导致完全没有分泌；ii）缺失30个C末端氨基酸残基几乎完全消除了PLTP的磷脂转移活性。本研究首次描述了磷脂转运蛋白的生物合成，并为详细阐明该蛋白的结构-功能关系提供了工具。
