Definition, expression, and characterization of a protein domain in the N-terminus of pregnancy-associated plasma protein-A distantly related to the family of laminin G-like modules.
Although pregnancy-associated plasma protein-A (PAPP-A), a modulator of insulin-like growth factor (IGF) activity through its cleavage of IGF-binding protein (IGFBP)-4 and -5, has been known for more than two decades, knowledge about its domain architecture is still incomplete. Using position-specific iterative BLAST, we have identified distant relatives of the PAPP-A N-terminal sequence stretch of 250 residues. We present evidence that a protein domain with weak similarity to known laminin G-like (LG) modules is contained within this region, and we propose that PAPP-A and PAPP-A2 are new and unique members in the group of LG proteins as the pappalysins represent the first examples where LG modules are associated with proteinases. Fourteen beta-strands characteristic for the LG structure were tentatively located within the PAPP-A LG (PA-LG) module using secondary structure prediction and sequence alignment. Upon mammalian expression of PAPP-A truncation mutants, we defined domain boundaries showing that PA-LG is an autonomously folding unit, which spans the first 243 residues. We were unable to express PAPP-A variants which lack the PA-LG module, suggesting a possible role in stabilization of the proteolytic domain. To obtain larger amounts of protein for functional and structural analysis, the defined PA-LG domain was expressed in bacteria and folded in vitro. In addition, the availability of recombinant PA-LG module may potentially improve diagnostic assays based on the measurement of PAPP-A antigen, and also facilitate the study of PAPP-A in animal model systems.
Boldt
Henning B
HB
Department of Molecular Biology, Science Park, University of Aarhus, Denmark.
Glerup
Simon
S
Overgaard
Michael T
MT
Sottrup-Jensen
Lars
L
Oxvig
Claus
C
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000378:metabolism; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism; D007797:Laminin / Q000737:chemistry* / Q000145:classification*; D008969:Molecular Sequence Data; D019151:Peptide Library; D011247:Pregnancy; D011266:Pregnancy-Associated Plasma Protein-A / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D018974:Insulin-Like Growth Factor Binding Protein 4; D018975:Insulin-Like Growth Factor Binding Protein 5; D007797:Laminin; D019151:Peptide Library; C089141:laminin gamma 1; D011266:Pregnancy-Associated Plasma Protein-A