Amino acid sequence of human pregnancy-associated plasma protein A derived from cloned cDNA

Abstract

The amino acid sequence of human pregnancy-associated plasma protein-A (PAPP-A), a component of the circulating complex with the proform of eosinophil major basic protein (proMBP), has been determined from partial protein sequencing and from sequencing of cloned cDNA. The PAPP-A monomer contains 1547 amino acid residues, but is derived from a larger precursor of placental origin. PAPP-A contains 82 Cys residues, which are all bridged, 14 putative sites for N-glycosylation, and 7 putative sites for attachment of glycosaminoglycan groups. The C-terminal part of PAPP-A contains 5 approximately 60-residue motifs related to the short consensus repeats of complement proteins and selectins. The SCRs presently known can be grouped into three classes: complement-type, class I; selectin-type, class II; PAPP-A-type, class III. PAPP-A further contains three approximately 26-residue motifs, related to the lin-notch motifs of proteins regulating early tissue differentiation, and, in addition, a putative Zn2+ binding site similar to that found in many metalloproteinases has been identified. Apart from these features, the PAPP-A sequence is not related to other known protein sequences.

Localization of disulfide bridges and free sulfhydryl groups in human eosinophil granule major basic protein

“…Curiously, proMBP exists as a dimer in the complex with PAPP-A, a disulfide bond between the 2 residues corresponding to Cys2 in MBP being responsible in part [18]. In addition, at least one Cys residue in the pro-piece of proMBP further links to PAPP-A [18,28]. The complete arrangement of disulfide bonds in the complex (PAPPNproMBP) is not yet known.…”

Expression of Recombinant Human Pregnancy-associated Plasma Protein-A and Identification of the Proform of Eosinophil Major Basic Protein as Its Physiological Inhibitor

“…Plasmid Construction-A PAPP-A expression plasmid was constructed from three overlapping partial PAPP-A cDNA clones, 29-2, pPA3, and pPA1 (8,9). The BbeI-EcoRI fragment of 29-2, encoding PAPP-A residues 6 -228, 3 was excised by partial and full digestion, respectively, and ligated to a nucleotide fragment encoding an artificial signal peptide (MKDSCITVMAMALLSGFFFFAPASSYAA) plus residues 1-5 of PAPP-A.…”

“…2), and secreted abundantly into the culture medium as a dimer of the expected size (Fig. 1), the PAPP-A propeptide (8,9) is required neither for folding nor secretion. Often a propeptide functions to retain the proteolytic activity of a zymogen, which becomes active in the extracellular compartment only after propeptide cleavage (37).…”

“…In reducing SDS-PAGE, the PAPP-A subunit has an apparent molecular mass of 200 kDa (7), and its 1547-residue sequence is known from cloned cDNA (8). PAPP-A shows no global homology to any protein, but it contains sequence motifs, including an elongated zinc binding motif (HEXXHXXGXXH) at position 482-492 (8).…”

“…This motif and a structurally important methionine residue, also thought to reside in PAPP-A at position 556, are strictly conserved within the metzincins, a superfamily of zinc peptidases: astacins, adamalysins (or reprolysins), serralysins, and matrixins (matrix metalloproteinases) (8 -11). PAPP-A does not conform to other defining features of the individual established metzincin superfamily members, and interestingly, in PAPP-A the linear distance between the zinc binding motif and the conserved Met residue is 63 amino acids (8), whereas in other metzincins this distance is between 7 and 44 residues (11).…”