Fig 1.
PA interacts with C1q in a salt-sensitive manner and specifically binds the C1q CLR.
(A) The AcPA peptide was adsorbed to a microtiter plate and incubated with a constant amount of C1q in the presence of varying amounts of NaCl. Bound C1q was detected with a goat polyclonal anti-C1q sera followed by HRP-conjugated anti-goat sera. Data represent the means of three independent experiments. Error bars denote SEM. (B) C1q and biotinylated PA in the absence or presence of the crosslinker BS3 were loaded onto a 4–12% SDS-PAGE gel, (left panel) transferred to nitrocellulose and probed with antibody to detect C1q (green) and streptavidin conjugate to detect PA (red). An identical gel was stained with bio-safe Coomassie blue, and visualized by Odyssey imaging (right panel). Molecular size markers (in kDa) are indicated to the left and chains of C1q (A, B, C, A-B, C-C) are indicated to the right. (C) Interactions between AcPA and the CLR of C1q as measured by surface plasmon resonance (SPR). The CLR of C1q was immobilized onto the surface of a dextran sensor chip and AcPA was injected at the indicated concentrations. Data analysis consisted of globally fitting the SPR sensorgrams for different peptide concentrations with a 1:1 model taking into account mass transport. KD values were calculated from koff/kon for each experiment and were averaged from at least two separate experiments.
Fig 2.
PA binds to MBL and ficolins and does not displace C1s from C1q.
(A) The AcPA and CP2 peptides were adsorbed to a microtiter plate and incubated with a constant amount of MBL or K55Q. Bound MBL was detected with a polyclonal goat anti-MBL sera followed by HRP-conjugated anti-goat sera. (B) The various ficolins indicated in the figure were adsorbed to the microtiter plate and incubated with biotinylated PA followed by a neutravidin conjugate. BSA was used as a negative control for binding. (C) Partially purified C1 was mixed with increasing amounts of CP or PA and added to a microtiter plate coated with monoclonal antibody to C1q. After washing, C1s signal present in intact C1 complexes was measured by ELISA using polyclonal antibody to C1s (CP, red line; PA, blue line) or polyclonal antibody to C1q (green line) to confirm C1q was bound to the plate. Data represent the means of three independent experiments. Error bars denote SEM.
Table 1.
Hemolytic assay in factor B-depleted serum of arginine and glutamic acid substitution peptides.
Fig 3.
Pegylated versions of PA inhibit complement activity in a hemolytic assay.
(A) Hemolytic assays using factor B-depleted serum were performed with PA dissolved in DMSO and its pegylated derivatives dissolved in water. Factor B-depleted serum was incubated with 0.77mM of each peptide and then added to sensitized sheep erythrocytes. (B) Titration of increasing amounts of PA and PA-dPEG24 in the hemolytic assay. Water and DMSO were used as vehicle controls in the presence of factor B-depleted serum. GVBS++ is a buffer- only control. Values are the means of three independent experiments. Error bars represent the SEM.
Fig 4.
Linear mode MALDI-TOF mass spectrometry analysis of PA-dPEG24 oligomeric state.
PA-dPEG24 in acidified water was analyzed by linear mode mass spectrometry. The spectrum shows two peaks for PA-dPEG24 with a monomer peak at 2778 and a dimer form at 5553.
Fig 5.
CD and 1D 1H NMR of PA-dPEG24.
(A) The PA-dPEG24 peptide at 0.2mg/mL in PBS was analyzed on a Jasco J-815 CD spectrometer. (B) The PA-dPEG24 peptide at 1.8mg/mL in PBS with 10% D2O was analyzed on a Bruker Avance 400 MHz spectrometer at a temperature of 294 K.
Fig 6.
PA-dPEG24 inhibits complement activation in rat serum, mouse plasma and non-human primate serum.
Hemolytic assays using (A) Wistar rat serum, (B) mouse plasma and (C) cynomolgus monkey serum were performed with increasing amounts PA-dPEG24 dissolved in 100mM Na2HPO4 with 0.9% NaCl buffer for rat serum, 10mM Na2HPO4 with 0.9% NaCl for cynomolgus monkey serum and 0.9% NaCl for mouse plasma. Serum or plasma were incubated with peptide and then added to human AB erythrocytes. Values are the means of three independent experiments. Error bars represent the SEM.
Fig 7.
PA-dPEG24 inhibits complement activation in vivo.
(A) Rats were IV administered PA-dPEG24 at 10 (n = 2) or 20mg/mL (n = 2) in 0.9% NaCl or received a vehicle control (n = 2) or were sham treated (n = 2). B, Rats were IV administered PA-dPEG24 at 10mg/mL (n = 4), 20mg/mL (n = 4) or 30mg/mL (n = 4) in 10mM Na2HPO4, 0.9% NaCl or received a vehicle control (n = 3). At the indicated time points post-administration, blood was collected and plasma isolated. Plasma was tested in hemolytic assays with human AB erythrocytes. Error bars represent the SEM.
Fig 8.
Model of PA inhibition of C1/MBL function.
Our data favors a model in which PIC1 derivatives functionally disrupt the C1s-C1r-C1r-C1s/MASP interaction with the CLR of C1q/MBL, respectively. By PIC1 binding to the serine protease interaction site on CLR, the serine proteases are hypothesized to be in a suboptimal conformation for autoactivation and initiation of the classical and/or lectin pathways of complement.