STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE PIG-A PROTEIN THAT IS MUTATED IN PAROXYSMAL-NOCTURNAL HEMOGLOBINURIA

There is now convincing evidence that the Pig-a gene is mutated in pat ients with paroxysmal nocturnal hemoglobinuria (PNH), a disease in whi ch one or more clones of hematopoietic cells have incomplete assembly of glycosylphosphatidylinositol (GPI) anchors and absence of GPI-linke d protein expression on the cell surface. Little is known, however, ab out the Pig-a protein product that is necessary for GPI anchor bioasse mbly. Relatively few substitution (missense) Pig-a gene mutations have been identified, but we noted two apparent clusters at codons 128-129 and 151-156 and hypothesized that these might represent critical regi ons of the Pig-a protein. We therefore used site-directed mutagenesis to create conservative mutations in the Pig-a protein, then performed structural and functional analysis. Each Pig-a mutation generated a Pi g-a protein of normal size and stability, but certain mutations had su bstantial deleterious effects on protein function. Conservative mutati on of codons histidine 128 (H128), serine 129 (S129), and serine 155 ( S155) had greatly diminished function, while mutations of flanking res idues had no effect on function. Our results represent the first struc ture/function analysis of the Pig-a protein, and suggest that codons H 128, S129, and S155 represent critical regions of the Pig-a protein. O ur results also suggest a means by which transgenic mice with a ''part ial knock-out'' of Pig-a function could be generated, which would allo w investigation of PNH in an animal model.