Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin

The bioavailability of insulin-like growth factor (IGF)-I and -II is contro lled by six IGF-binding proteins (IGFBPs 1-6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnan cy-associated plasma protein-A (PA-PP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesi s that PAPP-A belongs to the metzincin superfamily of metalloproteinases, a ll containing the elongated zinc-binding motif HEXXHXXGXXH (His-482-His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitutio n of Glu-483 with Ala causes a complete loss of activity, defining this mot if as part of the active site of PAPP-A. Interestingly, a mutant with Glu-4 83 replaced by Gln shows residual activity. Known metzincin structures cont ain a so-called Met-turn, whose strictly conserved Met residue is thought t o interact directly with residues of the active site. By further mutagenesi s we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecit y. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separat e family. We also found that PAPP-A can undergo autocleavage, and that auto cleaved PAPP-A is inactive. A lack of unifying elements in the sequences ar ound the found cleavage sites of PAPP-A and a variant suggests steric regul ation of substrate specificity.