Jerkovic Lidija, Voegele Andreas F, Chwatal Sabine, Kronenberg Florian, Radcliffe Catherine M, Wormald Mark R, Lobentanz Eva M, Ezeh Benjie, Eller Patricia, Dejori Norbert, Dieplinger Benjamin, Lottspeich Friedrich, Sattler Wolfgang, Uhr Manfred, Mechtler Karl, Dwek Raymond A, Rudd Pauline M, Baier Gottfried, Dieplinger Hans
Division of Genetic Epidemiology, Department of Medical Genetics, Clinical and Molecular Pharmacology, Innsbruck Medical University, Schoepfstrasse 41, A-6020 Innsbruck, Austria.
J Proteome Res. 2005 May-Jun;4(3):889-99. doi: 10.1021/pr0500105.
Hydrophobic vitamins are transported in human plasma and extravascular fluids by carrier proteins. No specific protein has been described so far for vitamin E, which plays a crucial role in protecting against oxidative damage and disease. We report here the purification of a 75-kDa glycoprotein with vitamin E-binding properties by stepwise chromatography of lipoprotein-depleted human plasma and monitoring of vitamin E (alpha-tocopherol)-binding activity. Partial sequencing identified this protein as afamin, a previously described member of the albumin gene family with four or five potential N-glycosylation sites. Glycosylation analysis indicated that >90% of the glycans were sialylated biantennary complex structures. The vitamin E-binding properties were confirmed using recombinantly expressed afamin. Qualitative and quantitative analysis of plasma and extravascular fluids revealed an abundant presence of this protein not only in plasma (59.8+/-13.3 microg/mL) but also in extravascular fluids such as follicular (34.4+/-12.7 microg/mL) and cerebrospinal (0.28+/-0.16 microg/mL) fluids, suggesting potential roles for afamin in fertility and neuroprotection. Afamin is partly (13%) bound to plasma lipoproteins. Afamin and vitamin E concentrations significantly correlate in follicular and cerebrospinal fluids but not in plasma. The vitamin E association of afamin in follicular fluid was directly demonstrated by gel filtration chromatography and immunoprecipitation which complements the in vitro findings for purified native and recombinant afamin.
疏水性维生素通过载体蛋白在人体血浆和血管外液中运输。迄今为止，尚未发现针对维生素E的特异性蛋白，而维生素E在预防氧化损伤和疾病方面起着关键作用。我们在此报告，通过对去除脂蛋白的人血浆进行分步色谱分离并监测维生素E（α-生育酚）结合活性，纯化出一种具有维生素E结合特性的75 kDa糖蛋白。部分测序确定该蛋白为afamin，它是白蛋白基因家族中先前描述的成员，具有四到五个潜在的N-糖基化位点。糖基化分析表明，超过90%的聚糖是唾液酸化的双天线复杂结构。使用重组表达的afamin证实了其维生素E结合特性。对血浆和血管外液的定性和定量分析表明，这种蛋白不仅大量存在于血浆中（59.8±13.3μg/mL），也存在于血管外液中，如卵泡液（34.4±12.7μg/mL）和脑脊液（0.28±0.16μg/mL），这表明afamin在生育和神经保护方面可能发挥作用。Afamin部分（13%）与血浆脂蛋白结合。在卵泡液和脑脊液中，afamin与维生素E浓度显著相关，但在血浆中不相关。通过凝胶过滤色谱和免疫沉淀直接证明了卵泡液中afamin与维生素E的结合，这补充了纯化的天然和重组afamin的体外研究结果。
