Characterization of GPI anchor biosynthesis genes.

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Our goal is to clone all genes involved in GPI anchor biosynthesis in mammalian cells and to characterize each reaction steps. In the present study, we analyzed first and second reaction steps. We identified human homologue of yeast GPI2 and showed that it is the gene defective in class C mutant termed PIG-C.PIG-C cDNA encoded a 297 amino-acid protein that is expressed in the endoplasmic reticulum. PIG-C protein is hydrophobic, probably spanning the membrane six or seven times. It has no significant homology to other proteins with known functions. So, it is not possible to predict its function from the primary structure. We found that two other gene products involved in the first step, PIG-A and PIG-H,form a protein complex in the endoplasmic reticulum and that they both have cytoplasmic orientation. Since PIG-A may be a catalytic component of N-acetylglucosaminyl transferase that mediates the first step, this supports the notion that the first step occurs on the cytoplasmic side of the endoplasmic reticulum.We generated a CHO cell mutant that is defective in the second step and using this mutant expression cloned PIG-L cDNA that complements the defective second step. PIG-L cDNA encoded a 252 amino-acid protein that is expressed in the endoplasmic reticulum. PIG-L protein had cytoplasmic orientation, supporting the notion that the second step also occurs on the cytoplasmic side of the endoplasmic reticulum.