Identification and Regulation of the IGFBP-4 Protease and Its Physiological Inhibitor in Human Trophoblasts and Endometrial Stroma: Evidence for Paracrine Regulation of IGF-II Bioavailability in the Placental Bed during Human Implantation

The Lin12-Notch repeat (LNR) module of about 35 residues is a hallmark of the Notch receptor family. Three copies, arranged in tandem, are invariably present in the extracellular portion of the Notch receptors. Although their function is unknown, genetic and biochemical data indicate that the LNR modules participate in the regulation of ligand-induced proteolytic cleavage of the Notch receptor, a prerequisite to intramembrane cleavage and Notch signaling. Outside the Notch receptor family, the LNR module is present only in the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) and its homologue PAPP-A2, which also contain three copies. Curiously, LNR modules 1 and 2 are present within the proteolytic domain of PAPP-A/A2, but LNR3 is separated from LNR2 by more than 1000 amino acids. The growth factor antagonists insulin-like growth factor-binding protein (IGFBP)-4 and -5 are both substrates of PAPP-A. We provide here evidence that the PAPP-A LNR modules function together to determine the proteolytic specificity of PAPP-A. Analysis of C-terminally truncated PAPP-A mutants followed by the analysis of LNR deletion mutants demonstrated that each of the three PAPP-A LNR modules is strictly required for proteolytic activity against IGFBP-4 but not for proteolytic activity against IGFBP-5. Individual substitution of conserved LNR residues predicted to participate in calcium coordination caused elimination (D341A, D356A, D389A, D1484A, D1499A, and D1502A) or a significant reduction (D359A and E392A) of IGFBP-4 proteolysis, whereas IGFBP-5 proteolysis was unaffected. The activity of the latter mutants against IGFBP-4 could be partially rescued by calcium, and the addition of the calcium-binding protein calbindin D9k to wild-type PAPP-A eliminated activity against IGFBP-4 but not against IGFBP-5, demonstrating that the PAPP-A LNR modules bind calcium ions. We propose a model in which LNR3 is spatially localized in proximity to LNR1 and -2, forming a single functional unit.

mentioning

confidence: 71%

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

Boldt

Kjær-Sørensen

Overgaard

et al. 2004

“…Through this proteolytic activity, PAPP-A causes the release of active IGF and thereby regulates its bioavailability in several biological systems. In particular, the role of PAPP-A has been studied in ovarian follicular development (4), implantation (5), fetal development (6 -8), wound healing (9), and atherosclerosis (10,11).…”

mentioning

confidence: 99%

A Substrate Specificity-determining Unit of Three Lin12-Notch Repeat Modules Is Formed in Trans within the Pappalysin-1 Dimer and Requires a Sequence Stretch C-terminal to the Third Module

Weyer

Boldt

Poulsen

et al. 2007

Members of the pappalysin family of metzincin metalloproteinases, pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) and PAPP-A2 (pappalysin-2), regulate the bioavailability of insulin-like growth factors (IGFs) by specific proteolytic inactivation of IGF-binding proteins (IGFBPs). PAPP-A cleaves IGFBP-4 and IGFBP-5, whereas PAPP-A2 cleaves only IGFBP-5. The pappalysins contain three Lin12-Notch repeat (LNR1-3) modules, previously considered unique to the Notch receptor family in which they function to regulate receptor cleavage. In contrast to the Notch receptor where three LNR modules are tandemly arranged, LNR3 is separated by more than 1000 residues from LNR1-2 in the pappalysin sequence. Each of the three LNR modules of PAPP-A is required for proteolysis of IGFBP-4, but not IGFBP-5. However, we here find that a C-terminal truncated variant of PAPP-A, which lacks LNR3 and therefore activity against IGFBP-4, cleaves IGFBP-4 when co-expressed with a PAPP-A variant, which is mutated in the active site. This suggests that LNR3 from the inactive subunit interacts in trans with LNR1-2 of the truncated PAPP-A subunit to form a functional trimeric LNR unit. We also show that formation of such a functional LNR unit depends on dimerization, as dissociation of a mutated non-covalent PAPP-A dimer results in reduced activity against IGFBP-4, but not IGFBP-5. Using PAPP-A/PAPP-A2 chimeras, we demonstrate that PAPP-A2 LNR1-2, but not LNR3, are functionally conserved with respect to IGFBP proteolysis. Additionally, we find that a sequence stretch C-terminal to LNR3 and single residues (Asp 1521 , Arg 1529 , and Asp 1530 ) within this are required for LNR functionality.The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79) 2 is secreted as a disulfide-linked 400-kDa homodimer (1) that cleaves insulinlike growth factor binding proteins (IGFBP)-4 (2) and IGFBP-5 (3). Through this proteolytic activity, PAPP-A causes the release of active IGF and thereby regulates its bioavailability in several biological systems. In particular, the role of PAPP-A has been studied in ovarian follicular development (4), implantation (5), fetal development (6 -8), wound healing (9), and atherosclerosis (10, 11). PAPP-A is the founding member of the pappalysin family (12, 13) within the metzincin superfamily of metalloproteinases (14, 15), also including its only known homologue, PAPP-A2, which cleaves IGFBP-5, but not IGFBP-4 (16). The 1558 3 -residue PAPP-A2 shares 46% of its residues with the 1547-residue PAPP-A (17), and they display the same domain organization with a laminin G-like domain and a proteolytic domain in the N-terminal part (18), a central region of ϳ500 residues with unknown domain composition, and a C-terminal part with five complement control protein (CCP) modules that mediate cell surface adhesion of 20). The pappalysins also contain three Lin12-Notch repeat (LNR) modules, previously considered unique to the Notch receptor family. But in contrast to the Notch receptors, which...

“…Accordingly, the proteolytic activity of PAPP-A is believed to be involved in a number of physiological and pathological processes known to involve IGF signaling, e.g. ovarian follicular development (4), human implantation (5), fetal development (6 -8), wound healing (9), and atherosclerosis (10).…”

mentioning

confidence: 99%

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

Glerup

Kløverpris

Laursen

et al. 2007

The metzincin metalloproteinase pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) promotes cell growth by proteolytic cleavage of insulin-like growth factorbinding 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 disulfidebonded 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 PAPPA⅐proMBP 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 PAPPA⅐proMBP 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-A⅐proMBP complex is formed at the cell surface in vivo rather than in the circulation. Further supporting this, we show that formation of the PAPP-A⅐proMBP 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.