Cell surface adhesion of pregnancy‐associated plasma protein‐A is mediated by four clusters of basic residues located in its third and fourth CCP module

Abstract

The metalloproteinase pregnancy‐associated plasma protein‐A (PAPP‐A) cleaves a subset of insulin‐like growth factor binding proteins (IGFBP), which inhibit the activities of insulin‐like growth factor (IGF). Through this proteolytic activity, PAPP‐A is believed to regulate IGF bioavailability in several biological systems, including the human reproductive system and the cardiovascular system. PAPP‐A adheres to mammalian cells by interactions with glycosaminoglycan (GAG), thus targeting the proteolytic activity of PAPP‐A to the cell surface. Based on site‐directed mutagenesis, we here delineate the PAPP‐A GAG‐binding site in the C‐terminal modules CCP3 and CCP4. Using heparin affinity chromatography, commonly employed in such studies, we define three clusters of arginines and lysines of CCP3, which are important for the interaction of PAPP‐A with heparin. In a model of PAPP‐A CCP3‐CCP4, basic residues of these sequence clusters form a contiguous patch located on one side of the structure. Binding to the unknown, natural cell surface receptor of PAPP‐A, assessed by flow cytometry, also depends on residues of these three basic clusters. However, single or double residue substitutions generally have a modest effect on PAPP‐A heparin binding assessed by chromatography, but cell surface adhesion was critically reduced by several of these substitutions, emphasizing the relevance of analysis by flow cytometry. The contributions of positively charged residues located in CCP4 were all minor when analyzed by heparin affinity chromatography. However, the mutation of CCP4 residues Arg1459 and Lys1460 to Ala almost abrogated cell surface adhesion. Furthermore, when acidic residues of the homologous proteinase PAPP‐A2 (Asp1547, Glu1555 and Glu1567) were introduced into the corresponding positions in the sequence of PAPP‐A, located in each of the three basic clusters of CCP3, binding to heparin was strongly impaired and cell surface binding was abrogated. This explains, at least in part, why PAPP‐A2 lacks the ability of cell surface adhesion, and further emphasizes the role of the basic clusters defined in PAPP‐A.

Immunofluorometric Point-of-Care Assays for the Detection of Acute Coronary Syndrome-Related Noncomplexed Pregnancy-Associated Plasma Protein A

“…Anticoagulation treatment with either unfractionated heparin or low-molecular-weight heparin is commonly used in the clinical management of ACS. PAPP-A contains glycosaminoglycan binding site that actively binds heparin and thus heparin may interfere some PAPP-A assays (12,20 ). The new assays described here with heparin plasma samples gave results that were very similar to results obtained with serum samples, which indicates that these assays are not affected by heparin.…”

The Lin12-Notch Repeats of Pregnancy-associated Plasma Protein-A Bind Calcium and Determine Its Proteolytic Specificity

“…The secreted protein is a disulfide-linked 400-kDa homodimer of 1547 residue subunits. Its proteolytic domain of ϳ300 residues is located in the N-terminal half of the subunit, and five complement control protein modules (CCP1-5), which mediate cell surface adhesion (16,17), are located near the C terminus (Fig. 1A).…”

Cell Surface Detachment of Pregnancy-associated Plasma Protein-A Requires the Formation of Intermolecular Proteinase-Inhibitor Disulfide Bonds and Glycosaminoglycan Covalently Bound to the Inhibitor

“…Mapping experiments localized the site responsible for specific surface binding to complement control protein (CCP) modules 3 and 4 in the C-terminal region of PAPP-A, which contains five consecutive CCP modules (11,12). These modules, originally known from proteins associated with the complement system, are responsible for interactions with glycosaminoglycans (GAGs) in several other proteins (13,14).…”