Mechanism of biosynthesis of protein GPI anchors.

点击翻译按钮获取中文摘要

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.