Title : Complex of pregnancy-associated plasma protein-A and the proform of eosinophil major basic protein
Abstract :
Disulfide structure and carbohydrate attachment
Pregnancy-associated plasma protein-A ( PAPP-A ) is a metzincin superfamily metalloproteinase responsible for cleavage of insulin-like growth factor-binding protein-4 , thus causing release of bound insulin-like growth factor
PAPP-A is secreted as a dimer of 400 kDa but circulates in pregnancy as a disulfide-bound 500-kDa 2:2 complex with the proform of eosinophil major basic protein ( pro-MBP), recently shown to function as a proteinase inhibitor of PAPP-A
Except for PAPP-A2 , PAPP-A does not share global similarity with other proteins
Three lin-notch (LNR or LIN-12) modules and five complement control protein modules (also known as SCR modules) have been identified in PAPP-A by sequence similarity with other proteins , but no data are available that allow unambiguous prediction of disulfide bonds of these modules
To establish the connectivities of cysteine residues of the PAPP-A
pro- MBP complex, biochemical analyses of peptides derived from purified protein were performed
The PAPP-A subunit contains a total of 82 cysteine residues , of which 81 have been accounted for
The pro-MBP subunit contains 12 cysteine residues , of which 10 have been accounted for
Within the 2:2 complex, PAPP-A is dimerized by a single disulfide bond; pro- MBP is dimerized by two disulfides, and each PAPP-A subunit is connected to a pro-MBP subunit by two disulfide bonds
All other disulfides are intra chain bridges
We also show that of 13 potential sites for N-linked carbohydrate substitution of the PAPP-A subunit , 11 are occupied
The large number of disulfide bonds of the PAPP-A
pro- MBP complex imposes many restraints on polypeptide folding, and knowledge of the disulfide pattern of PAPP-A will facilitate structural studies based on recombinant expression of individual, putative PAPP-Adomains
Furthermore, it will allow rational experimental design of functional studies aimed at understanding the formation of the PAPP-A
pro- MBP complex, as well as the inhibitory mechanism of pro-MBP