Mechanism of biosynthesis of protein GPI anchors.

本研究旨在阐明GPI锚生物合成的分子机制，并取得了以下结果.我们纯化了GPI-GlcNAc转移酶，它介导GPI生物合成途径的第一步。该酶由六种蛋白质组成：四种以前报道过的蛋白质和两种新的蛋白质。新的组分之一是PIG-P，它是酶所必需的，因为PIG-P缺陷的突变小鼠细胞系在GPI生物合成中完全缺陷。另一个新的成分是DPM 2，它被认为是多萜醇-磷酸-甘露糖合酶的调节成分。DPM 2缺陷细胞的GPI-GlcNAc转移酶活性是野生型细胞的三分之一，表明GPI-GlcNAc转移酶也受DPM2.2调节。我们发现，人的甘露醇-磷酸-甘露糖合酶由DPM 1，DPM 2和DPM 3组成，DPM 2稳定DPM 3，而DPM 3反过来又稳定催化剂DPM1.3。我们克隆了PIG-M的cDNA，并证明PIG-M编码一个甘露糖转移酶，该酶将第一个甘露糖转移到GPI上，PIG-M在内质网的内腔结构域中有一个功能重要的DXD基序，表明第一个甘露糖转移到内质网的内腔侧.我们证明PIG-O参与磷酸乙醇胺向第三甘露糖的转移。PIG-O最有可能是一种催化成分，与PIG-F相关，PIG-F也参与同一步骤。PIG-O的稳定表达依赖于PIG-F。我们的特点是哺乳动物基因GAA 1，GPI转酰胺酶的一个组成部分。人和小鼠GAA 1基因有12个外显子，跨度为4kb。人和小鼠GAA 1基因分别存在于8q24.3和15 E。这些GAA 1基因的内含子8为小类。GPI转酰胺酶的另一组分GPI 8具有在来自其他生物体和半胱氨酸蛋白酶家族成员的GPI 8中保守的功能必需的半胱氨酸，这表明该半胱氨酸是GPI转酰胺酶的活性位点。

We aimed to clarify molecular mechanisms of GPI anchor biosynthesis and obtained following results.1. We purified GPI-GlcNAc transferase that mediates the first step in GPI biosynthesis pathway. The enzyme consisted of six proteins : four previously reported and two new components. One of the new components was PIG-P that is essential for the enzyme because mutant mouse cell line defective in PIG-P is completely deficient in GPI biosynthesis. The other new component was DPM2 that is known as a regulatory component of dolichol-phosphate-mannose synthase. GPI-GlcNAc transferase activity of DPM2-deficient cells was one-third that of wild-type cells, indicating that GPI-GlcNAc transferase is also regulated by DPM2.2. We found that human dolichol-phosphate-mannose synthase consists of DPM1, DPM2 and DPM3, that DPM2 stabilizes DPM3 and that DPM3, in turn, stabilizes catalytic DPM1.3. We cloned PIG-M cDNA and demonstrated that PIG-M encodes a mannosyltransferase that transfers the first mannose to GPI.PIG-M had a functionally important DXD motif within a lumenal domain, suggesting that the first mannose is transferred on the lumenal side of the endoplasmic reticulum.4. We demonstrated that PIG-O is involved in transfer of phosphoethanolamine to the third mannose. PIG-O that is most likely a catalytic component is associated with PIG-F that is also involved in the same step. Stable exprrssion of PIG-O was dependent upon PIG-F.5. We characterized mammalian genes for GAA1, a component of GPI transamidase. Human and mouse GAA1 genes had 12 exons and spanned 4 kb. Human and mouse GAA1 genes are present in 8q24.3 and 15E, respectively. Intron 8's of those GAA1 genes were of minor class. GPI8, the other component of GPI transamidase, had a functionally essential cysteine that is conserved among GPI8 from other organisms and members of a cystein protease family, suggesting that this cystein is active site of GPI transamidase.