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 (Asp1521, Arg1529, and Asp1530) within this are required for LNR functionality.
Highlights
Disulfide-linked 400-kDa homodimer [1] that cleaves insulinlike growth factor binding proteins (IGFBP)-4 [2] and insulin-like growth factors (IGFs)-binding proteins (IGFBPs)-5 [3]
As expected, when either PAPP-A variant was expressed alone, no activity against IGFBP-4 could be detected in the culture medium (Fig. 1A, lanes 3 and 4), and activity against IGFBP-5 was detected only with culture medium containing the truncated PAPP-A variant, PA(1–1477) (Fig. 1B, lane 3)
Upon co-expression, proteolytic activity against IGFBP-4 was rescued, corresponding to the formation within the dimer of one proteolytic domain with activity toward IGFBP-4 (Fig. 1A, lane 5). This is most likely explained by an interaction of LNR3 from the inactivated subunit (E483A) with LNR1–2 from the truncated PAPP-A subunit, PA(1–1477), suggesting that the three Lin12-Notch repeat (LNR) modules interact in trans within the PAPP-A dimer (Fig. 1C)
Summary
Plasmid Construction and Mutagenesis—Single (PA(K1509A), PA(K1511A), PA(K1512A), PA(D1521A), PA(D1525A), PA(R1529A), PA(D1530A), PA(E1535A), PA(R1538A), PA(K1539A), PA(D1540A), PA(R1542A)) and triple (PA(C381A/ C652A/C1130A)) point mutants of the 1547-residue human PAPP-A polypeptide were obtained using three pBluescript II SKϩ vectors each containing PAPP-A cDNA corresponding to residues 1– (pB1– 407), –988 (pB408 –988), or 989 – 1547 (pB989 –1547) [12]. To assay the proteolytic activity of dissociated wild-type PAPP-A or the PA(C381A/C652A/ C1130A) mutant dimer, a 96-well plate coated with mAb 234-5 was incubated (1 h at 37 °C) with 100 l of culture medium containing 25 nM proteinase, washed in PBST, and incubated (1 h at 37 °C) with PBS with or without an additional 0.5 M NaCl. Following washing and equilibration in 50 mM Tris-HCl, 100 mM NaCl, 1 mM CaCl2, pH 7.5, reaction mixtures containing radiolabeled substrate (as described above) were added directly to the wells (total sample volume of 50 l). When assaying the IGFBP-4 proteolytic activity of PAPP-A/PAPP-A2 chimeric proteins, proteinase levels were adjusted to have the same activity against IGFBP-5 (40% cleavage after 2 h incubation), and were tested for IGFBP-4 proteolytic activity by time course experiments (reaction mixtures as described above using a total sample volume of 70 l). Recorded signals were subtracted from the background signal, determined by the injection of PAPP-A variants over a surface containing mAb 9E10 only
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