Allelic Amino Acid Substitutions Affect the Conformation and Immunoreactivity of Germ-Cell Alkaline Phosphatase Phenotypes

Abstract

The gene encoding placental alkaline phosphatase (PLAP) displays a well-documented allelic polymorphism. Likewise, different phenotypes exist for the PLAP-related germ-cell alkaline phosphatase (GCAP). We investigated the extent to which various allelic GCAP positions are critical in determining the enzymatic, structural, and immunological properties of GCAP phenotypes. Three homozygous GCAP phenotypes [JEG3, BeWo, and wild-type (wt) GCAP] were analyzed and compared with a "core" GCAP mutant that contains the seven amino acid substitutions that are consistently different between PLAP and GCAP but are common to the three known allelic GCAP genotypes. Although some substitutions could influence the electrophoretic behavior of the phenotypes, the allelic differences did not affect the kinetic properties of GCAP. However, they did affect the immunoreactivity and conformation of the variants as detected with a panel of 18 epitope-mapped monoclonal antibodies (MAbs) to PLAP. The selective immunoreactivity of the PLAP/GCAP-discriminating MAb C2 was critically dependent on the nature of the allelic residues 133 and 361 in GCAP. Residue 133 was also important for the general stability of the molecule because BeWo and wt GCAP, which have Asn133 and Val133, respectively, instead of Met133, showed a consistently reduced heat stability compared to core GCAP and JEG3. Because the core GCAP mutant consistently shows the characteristics of wt GCAP, its use as an antigen should allow the generation of monoclonal antibodies to GCAP that will not cross-react with PLAP and whose immunoreactivity will only marginally be influenced by allelic GCAP variation.