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

Abstract

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...

Multiple Domains of GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal Enzymes

“…This autoinhibition is achieved by the Notch repeat modules forming numerous interdomain contacts that allow these modules to wrap around the juxtamembrane heterodimerization domain of the receptor and mask the protease cleavage site. Aside from the Notch receptors, the only other proteins reported to contain Notch repeat modules are the metalloproteinase pregnancyassociated plasma protein-A (PAPP-A) and its homologue PAPP-A2 (27,28). These proteases act on a limited number of substrates, with PAPP-A cleaving only IGF-binding proteins 4 and 5, whereas PAPP-A2 only cleaves IGFBP-5.…”

Inhibition of the Proteolytic Activity of Pregnancy-associated Plasma Protein-A by Targeting Substrate Exosite Binding

“…The PCR product resulting from the combination of the two fragments was cloned into the HindIII/XbaI sites of pcDNA3.1(ϩ) (Invitrogen) to obtain pmPA(CCP1-C-His), encoding murine PAPP-A residues 1129 -1545 followed by the amino acid sequence GSHHH-HHH. A similar construct, encoding residues 1133-1547 of human PAPP-A, had previously been made (38). This construct did not include a His tag.…”

“…Expression constructs encoding human PAPP-A (39), murine PAPP-A (37), human PAPP-A2 (23), human PAPP-A/ PAPP-A2 chimeric proteins (PAPP-A(P2-CCP5-C), PAPP-A(P2-CCP4-C), PAPP-A(P2-CCP3-C), PAPP-A(P2-CCP2-C), PAPP-A(P2-CCP1-C), PAPP-A(P2-CCP3-4), and PAPP-A(P2-LNR3) (22,38)), a truncated variant of PAPP-A (PAPP-A(dLNR3-C) (38)), and variants of PAPP-A with single amino acids substituted into alanine, PAPP-A(D1484A), PAPP-A(D1499A), and PAPP-A(D1502A) (40)), were also used for transfection. Finally, we used a construct encoding PAPP-A in which Glu-483 of the active site was replaced with glutamine, PAPP-A(E483Q) (8).…”

Pregnancy-associated plasma protein-A2 modulates development of cranial cartilage and angiogenesis in zebrafish embryos

“…Protein modules that have known function in pappalysins, including the proteolytic domain and the C-terminus, showed higher percentages of identity (Fig. 1B,C), indicating functional conservation (Boldt et al, 2004;Boldt et al, 2001;Weyer et al, 2007).…”