Stanniocalcin-1 Potently Inhibits the Proteolytic Activity of the Metalloproteinase Pregnancy-associated Plasma Protein-A

Abstract

Stanniocalcin-1 (STC1) is a disulfide-bound homodimeric glycoprotein, first identified as a hypocalcemic hormone important for maintaining calcium homeostasis in teleost fish. STC1 was later found to be widely expressed in mammals, although it is not believed to function in systemic calcium regulation in these species. Several physiological functions of STC1 have been reported, although many molecular details are still lacking. We here demonstrate that STC1 is an inhibitor of the metzincin metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), which modulates insulin-like growth factor (IGF) signaling through proteolytic cleavage of IGF-binding proteins (IGFBPs). STC1 potently (Ki = 68 pm) inhibits PAPP-A cleavage of IGFBP-4, and we show in a cell-based assay that STC1 effectively antagonizes PAPP-A-mediated type 1 IGF receptor (IGF1R) phosphorylation. It has recently been found that the homologous STC2 inhibits PAPP-A proteolytic activity, and that this depends on the formation of a covalent complex between the inhibitor and the proteinase, mediated by Cys-120 of STC2. We find that STC1 is unable to bind covalently to PAPP-A, in agreement with the absence of a corresponding cysteine residue. It rather binds to PAPP-A with high affinity (KD = 75 pm). We further demonstrate that both STC1 and STC2 show inhibitory activity toward PAPP-A2, but not selected serine proteinases and metalloproteinases. We therefore conclude that the STCs are proteinase inhibitors, probably restricted in specificity to the pappalysin family of metzincin metalloproteinases. Our data are the first to identify STC1 as a proteinase inhibitor, suggesting a previously unrecognized function of STC1 in the IGF system.

Keywords: IGF-binding protein; disulfide; insulin-like growth factor (IGF); metalloprotease; metzincin; pappalysin; protease inhibitor; proteolysis; stanniocalcin.

FIGURE 1.

Co-transfection with cDNA encoding stanniocalcin-1 abrogates the proteolytic activity of PAPP-A. A, PAPP-A proteolytic activity toward radiolabeled IGFBP-4 in culture media from HEK293T cells transfected with the indicated combinations of cDNAs. Cleavage of IGFBP-4 by PAPP-A followed by separation in SDS-PAGE (12%) results in the generation of two co-migrating cleavage products of ∼16 kDa. B, PAPP-A Western blot (WB) following reducing 3–8% SDS-PAGE of culture supernatants from A.

FIGURE 2.

N-terminal sequence analysis and analytical gel filtration of purified STC1. A, Coomassie-stained 12% SDS-PAGE gel of purified STC1 under nonreducing (−) or reducing (+) conditions. N-terminal sequences (N1 and N2) determined by Edman degradation of reduced STC1 are aligned with the amino acid sequence of STC1 including the signal peptide. B, analytical gel filtration of purified STC1. Positions of the salt peak, the void volume (V₀), the total volume (V_t), and the elution position of bovine serum albumin (BSA, 66 kDa) are indicated. STC1 Western blotting of selected fractions following nonreducing 12% SDS-PAGE is shown.

FIGURE 3.

Analysis of peptides derived from purified STC1. A, MALDI mass spectra of STC1 peptides containing Cys-202. The upper-left panel shows the monomeric Ile-186–Arg-208 peptide, in which Cys-202 is cysteinylated. Reduction (lower-left panel) causes a loss of 118.8 Da, corresponding to the loss of a cysteine residue. The upper-right panel shows the dimeric Ile-186–Arg-208 peptide (of 5172.23 Da), in which Cys-202 is engaged in forming the interchain disulfide bond, responsible for covalent dimerization of the intact STC1. Following reduction (lower-right panel), all of the peptide is present in the monomeric form. B, MALDI mass spectra of the STC1 peptide containing Asn-62 before (upper panel) and after (lower panel) treatment with PNGase F. Masses of the peptides are shown and the loss of a fucose monosaccharide moiety is indicated (ΔFuc). A fraction of the peptide contains an oxidized methionine residue (MetOX). C, STC1 Western blot following 12% reducing SDS-PAGE of culture supernatants from cells transfected with cDNA encoding STC1 variants, in which the asparagine residues (Asn-24 and Asn-62) of the two potential N-glycosylation sites are substituted individually with a glutamine residue. D, the N-terminal amino acid sequence of STC1 with the two observed signal peptidase cleavage sites indicated by arrows. Disulfide bonds are shown as black lines, and the two potential N-glycosylation sites are underlined, one of which was demonstrated to be substituted with carbohydrate (Glyc).

FIGURE 4.

Unlike STC2, STC1 forms a noncovalent inhibitory complex with PAPP-A. A, schematic representations of the previously reported disulfide structure of STC1 (38) and the predicted disulfide structure of STC2. Disulfide bonds are shown as black lines. The positions of cysteine residues engaged in interchain disulfide bond formation are indicated (Dim) and asterisks indicate positions of three cysteines of STC2, which have no counterpart in STC1. The position of Cys-120 of STC2, which is required for formation of the PAPP-A·STC2 complex (19), is indicated (PAPP-A). B, PAPP-A Western blot following 3–8% nonreducing SDS-PAGE of culture supernatants from cells transfected with cDNA encoding PAPP-A incubated 0–16 h with medium from cells transfected with empty vector, STC1 cDNA, or STC2 cDNA. Note that the migration of PAPP-A is not affected by prolonged incubation with STC1, whereas all of PAPP-A has formed a covalent complex of higher molecular mass following incubation for 16 h with STC2. C, proteolytic activity toward radiolabeled IGFBP-4 in culture media from cells transfected with PAPP-A cDNA in the presence or absence of a 10-fold molar excess of purified STC1. PAPP-A and STC1 was not preincubated prior to the cleavage reaction.

FIGURE 5.

STC1 is a high affinity inhibitor of PAPP-A. A, surface plasmon resonance analysis of STC1 binding to PAPP-A. 2-Fold serial dilutions of STC1 were injected over a surface with or without antibody-captured PAPP-A. Recorded binding curves are shown in black, and a global 1:1 Langmuir fit is shown in red. The fitted constants are: k_a = 2.78 ± 0.001 × 10⁶ m⁻¹ s⁻¹ and k_d = 2.08 ± 0.004 × 10⁻⁴ s⁻¹, resulting in K_D = 7.5 × 10⁻¹¹ m. The sensorgrams are truncated, but all collected data points were used for model fitting. B, kinetic analysis of STC1 inhibition of PAPP-A-mediated IGFBP-4 cleavage. Relative initial velocities (v/v₀) of PAPP-A-mediated IGFBP-4 cleavage in the presence of increasing concentrations of STC1 are shown. The inhibition constant (K_i) was determined to be 6.8 ± 0.7 × 10⁻¹¹ m by fitting the Morrison K_i model (competitive inhibition) to the data.

FIGURE 6.

Covalent dimerization of STC2 is not required for inhibition of PAPP-A. A, STC2 Western blot (WB) following nonreducing (−) or reducing (+) 12% SDS-PAGE of culture supernatants from cells transfected with empty vector, cDNA encoding STC2, or cDNA encoding STC2(C211A). B, PAPP-A Western blot following nonreducing 3–8% SDS-PAGE of culture supernatants from cells co-transfected with PAPP-A cDNA and empty vector, cDNA encoding STC2, or cDNA encoding STC2(C211A). C, PAPP-A proteolytic activity toward IGFBP-4 in medium from cells co-transfected with PAPP-A cDNA and empty vector, STC2 cDNA, or STC2(C211A) cDNA. Results are mean ± S.D. from four independent experiments.

FIGURE 7.

STC1 inhibits IGF signaling in vitro. A, cells stably expressing the IGF1R was stimulated with combinations of IGF-I, IGFBP-4 (BP4), PAPP-A, and STC1. Phosphorylated and total IGF1R were detected in cell lysates by Western blotting (WB). β-Actin was used as loading control. B, the IGF1R phosphorylation was quantified from the Western blots and normalized to the signal with IGF alone. Results are mean ± S.D. from four independent experiments (ns, not statistically significant; *, p < 0.0001).

FIGURE 8.

STC1 and STC2 inhibit the pappalysins (PAPP-A and PAPP-A2). Proteolytic activity toward radiolabeled IGFBP-5 was assessed. The experiments were carried out in the absence or presence of STC1, STC2, or a general proteinase inhibitor (EDTA or PMSF). The following proteinases were analyzed: A, A disintegrin and metalloproteinase (ADAM)-10; B, matrix metalloproteinase-2 (MMP2); C, trypsin; D, matriptase; E, PAPP-A2.