Structure and function of beta2-glycoprotein I

在系统性红斑狼疮等自身免疫性疾病中，β -糖蛋白I （β - 2gpi）是抗心磷脂抗体识别磷脂抗原心磷脂的辅助因子。beta2GPI（326个氨基酸）由5个重复单元（结构域I-V）组成，每个重复单元含有60-80个氨基酸残基，与补体控制蛋白模块的短一致重复序列相对应。最近的研究表明，结构域V在与心磷脂的结合和辅助因子活性的表达中起着重要的作用。通过制备各种衍生物，研究了β - 2gpi的结构-功能关系。我们研究了不同形式的牛β - 2gpi与磷脂脂质体在不同条件下的相互作用。结果表明，n端结构域和c端结构域在beta2GPI与心磷脂的相互作用中起重要作用，而含有唾液酸的三个剩余结构域对相互作用没有显著影响。我们使用甲基营养酵母毕赤酵母构建了人类结构域V的高水平表达系统。我们发现重组蛋白具有天然二硫键和适当的折叠结构。利用该表达体系，制备了15N和13C标记域v，用于核磁共振三维结构测定。核磁共振研究正在进行中。对重组结构域V的构象、稳定性和脂质体结合活性进行了表征，并与各种β 2gpi衍生物进行了比较。结果表明，包含Trp76-Thr78的区域在与cardiolipin结合中起着关键作用。beta2GPI结构域V的缺口形式已被证明对心磷脂具有较低的能力。利用重组结构域V，我们发现纤溶蛋白可以产生缺口形式的结构域V，这说明了其生物学意义。

beta2-Glycoprotein I (beta2GPI) is a cofactor in the recognition of the phospholipid antigen cardiolipin by anti-cardiolipin antibodies in autoimmune diseases such as systemic lupus erythematosus. beta2GPI (326 amino acids) consists of five repeating units (Domains I-V), each containing 60-80 amino acid residues, corresponding to the short consensus repeat of the complement control protein module. Recent studies suggest that Domain V plays important roles in the binding to cardiolipin and expression of cofactor activity. We studied the structure-function relationship of beta2GPI by preparing various derivatives.1.We examined the interactions of various forms of bovine beta2GPI with phospholipid liposomes under different conditions. The results indicate that the N-terminal as well as C-terminal domains have an important role in the interaction of beta2GPI with cardiolipin, and that the three residual domains containing sialic acid have no significant effect on the interaction.2.We constructed a high-level expression system for human Domain V using a methylotrophic yeast, Pichia pastoris. We found that the recombinant protein as the native disulfide bonds and a proper folded structure. For the three dimensional structure determination by NMR,with this expression system, we prepared 15N and 13C labeled Domain V.The NMR studies are on going.3.Conformation, stability, and liposome-binding activity of the recombinant Domain V were characterized and compared with those of various beta2GPI derivatives. The results suggested that the region including Trp76-Thr78 has a critical role in binding to cardiolipin.4.A nicked form of domain V in beta2GPI has been shown to have a lower ability to cardiolipin. Using the recombinant Domain V,we found that plasmin can produce the nicked form of domain V,suggesting the biological significance.