Site-directed antibodies for probing the structure and biogenesis of phosphatidylinositol glycan-linked membrane proteins: application to placental alkaline phosphatase

An immunological approach to the study of the structure and biogenesis of the phosphatidylinositol glycan (PI-G) membrane anchor at the carboxyl terminus of human placental alkaline phosphatase (PLAP) is described. Based on the protein sequence predicted from full length PLAP cDNA, two epitopes were chosen in the region of the carboxyl terminus for the production of site-directed antibodies. The exo site represents the last nine residues of preproPLAP, (res. 505-513), which is part of the sequence that is expected to be cleaved from the nascent protein during processing and addition of the PI-G tail. A second site, the endo sequence, was selected close to the expected carboxyl terminus in mature PI-G-tailed PLAP (res. 474-484 of proPLAP). The two peptides were synthesized, polyclonal antibodies to the conjugated peptides were prepared, and the antisera were characterized. Analytical methods for both synthetic peptides and proteins are presented. Preliminary applications to the isolation and characterization of the PI-G-linked carboxyl terminus of mature PLAP and to the characterization of nascent PLAP are described. The application of both carboxyl terminal-directed antibodies, and a third antibody directed to the amino terminus of mature PLAP, in studies employing mutant forms of PLAP and to the PI-G tailing process itself are discussed. The immunological approach used here for PLAP should be applicable generally to the study of other PI-G-tailed proteins.