Fig 1: Interaction mechanism between PAPP-A and IGFBP5.a Structural depiction of the IGFBP5 anchor peptide binding to PAPP-A with the highlight of the K128 residue anchor site (left) and the active site (right). The structure shows that the IGFBP5 anchor peptide resides next to the zinc-coordination site of PAPP-A. b Binding affinity between the IGFBP5 anchor peptide and WT PAPP-A measured by fluorescence polarization (FP) assay. The N-terminal Alexa Fluor 568 conjugated IGFBP5121-143 (5 nM) was incubated with serial dilutions of WT PAPP-A (5.65 μM to 33 nM) in PBS buffer at room temperature. c Binding affinity between the FL IGFBP5 and PAPP-A (E483A) measured by fluorescence polarization (FP) assay. FAM-labeled IGFBP5 (5 nM) was incubated with serial dilutions of PAPP-A (E483A) (10 μM–50 nM) in PBS buffer at room temperature. In b, c the FP values were recorded at 10 min. Data shown is the representative of three replicates. The data were analyzed in GraphPad Prism 9.1.2. d Quantification of the WT PAPP-A proteolytic activity efficiency towards WT IGFBP5, IGFBP5 K128A and IGFBP5 K128D in the gel-based assay. Quantitative comparison was performed across samples from the same experiment with gels run in parallel. Bars are the mean ± standard deviation of n = 3 independent replicates (analyzed in GraphPad Prism 9.1.2). The single data representative of IGFBP5 K128A, IGFBP5 K128D were shown in Supplementary Fig. 13c. e SDS-PAGE gel-based WT PAPP-A proteolytic activity assay demonstrating the pivotal role of the IGFBP5 anchor peptide. WT PAPP-A has efficient cleavage towards WT IGFBP5, while the chimeric IGFBP5 with residues 121-143 replaced by the corresponding IGFBP4 residues 114-134 shows almost no response to WT PAPP-A. Data shown is the representative of 3 independent replicates. In d, e, 500 nM WT IGFBP5 or chimeric protein were incubated with WT PAPP-A in dose-response. The reactions were performed at 37 °C for 4 h. Source data are provided as a Source Data file.
Fig 2: PAPP-A substrate selectivity towards IGFBP4 and IGFBP5.a, b Proteolytic activity data suggest that the cleavage for IGFBP4, but not for IGFBP5, depends on the proper interaction between the LG and CCP2 domains. Quantifications of different PAPP-A truncations and the triple mutant (L1254A/F1257A/H1211A) proteolytic activity in gel-based assay towards IGFBP4 and IGFBP5 are shown in a and b, respectively. The single data representatives of the above gel-based assay are shown in Supplementary Fig. 16. c, d Proteolytic activity assay showed that PAPP-A dimer formation is crucial for IGFBP4 cleavage, but not for IGFBP5. Quantifications of WT PAPP-A, PAPP-A1100-1111*, PAPP-A1100-1135*, PAPP-A4C4S proteolytic activity towards substrates IGFBP4 and IGFBP5 in the gel-based assay are shown in c and d, respectively. The single data representatives of the assay in c, d are shown in Supplementary Fig. 17a, b. e, f Co-expression of PAPP-A (1132) and FL PAPP-A (E483A) significantly rescued IGFBP4 cleavage. Quantifications of FL PAPP-A, catalytic dead mutant PAPP-A (E483A), PAPP-A (1132), and the co-expressed PAPP-A (E483A)/PAPP-A (1132) heterodimer proteolytic activity towards IGFBP4 and IGFBP5 in the gel-based assay are shown in e and f, respectively. The single data representatives of the assay in e, f are shown in Supplementary Fig. 18b, c. For all proteolytic activity assay, 400 nM IGFBP4/700 nM IGF1, or 500 nM WT IGFBP5 were incubated with different proteases in dose-response. The reactions were performed at 37 °C for 4 h. In all quantifications, bars are the mean ± standard deviation of n = 3 independent replicates. Quantitative comparison was performed across samples from the same experiment with gels run in parallel. Source data are provided as a source data file.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human IGFBP-4 Protein, CF