Abstract
The metzincin metalloproteinase pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) promotes cell growth by proteolytic cleavage of insulin-like growth factor-binding proteins 4 and 5, causing the release of bound insulin-like growth factors. PAPP-A binds an unknown cell-surface heparan sulfate proteoglycan, suggesting that it controls insulin-like growth factor signaling spatially. In human pregnancy, the majority of PAPP-A circulates as a disulfide-bonded complex with its inhibitor, the proform of eosinophil major basic protein (proMBP). Interestingly, Ser-62 of proMBP is substituted with a glycosaminoglycan (GAG) chain, possibly a heparan sulfate type, and the PAPP-A.proMBP complex is unable to bind to the cell surface. We show here that proMBP detaches surface-bound PAPP-A in a process that depends on the proMBP GAG and also on the formation of intermolecular disulfide bonds between PAPP-A and proMBP. Unlike what was expected, we demonstrate that the GAG of proMBP is not required for PAPP-A.proMBP complex formation and that proMBP residues His-137, Ser-178, Arg-179, and Asn-181 are important for the recognition of PAPP-A. Using a mouse model, we find that the half-life of circulating PAPP-A and proMBP in complex is severalfold higher than both of the uncomplexed proteins, further suggesting that the PAPP-A.proMBP complex is formed at the cell surface in vivo rather than in the circulation. Further supporting this, we show that formation of the PAPP-A.proMBP complex at the cell surface proceeds rapidly compared with the slow rate of complex formation in solution. Because both PAPP-A and proMBP are expressed ubiquitously, this model may be applicable to many tissues in which insulin-like growth factor bioavailability is locally regulated.
Highlights
The metzincin metalloproteinase pregnancy-associated plasma protein-A (PAPP-A,2 pappalysin-1) cleaves insulin-like growth factor-binding proteins 4 and 5 [1, 2], which generally function to antagonize the activity of insulin-like growth factors (IGF)-I and -II
We find that the half-life of circulating PAPP-A and proform of eosinophil major basic protein (proMBP) in complex is severalfold higher than both of the uncomplexed proteins, further suggesting that the PAPP-A1⁄7proMBP complex is formed at the cell surface in vivo rather than in the circulation
We show that formation of the PAPP-A1⁄7proMBP complex at the cell surface proceeds rapidly compared with the slow rate of complex formation in solution
Summary
Mutagenesis—Site-directed mutagenesis of proMBP cDNA was carried out by overlap extension PCR [26] using proMBP cDNA contained in pcDNA3.1ϩ (pcDNA3.1-proMBP [15]) as the template. Complex was detected with a biotinylated, proMBP-specific Flow Cytometry—Transfected cells detached with PBS conmonoclonal antibody (PM-5A) followed by incubation with taining 5 mM EDTA were washed with cold Dulbecco’s modiperoxidase-conjugated avidin. In this assay, sample dilution fied Eagle’s medium (Invitrogen) containing 2% fetal bovine and washing after sample incubation were carried out using serum When these three residues were substituted individually by alanine, the ability to form the PAPP-A1⁄7proMBP complex was reduced by 50 – 60% (Fig. 2B) These mutants and wild-type proMBP migrated in nonreducing SDS-PAGE (Fig. 3A), and all showed similar CD spectra (Fig. 3B). All Abrogation of PAPP-A Surface Binding Requires the ProMBP GAG—
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