P140 Peptide in Lupus Figure 1. Peptide P140 binds the constitutively-expressed chaperone HSC70 protein (A) Biotin-labeled P140 and 131-151 peptides form a stable and specific, dose-dependent complex with HSC70 expressed on the surface of CBA/J and MRL/lpr splenocytes. The same results were obtained with LNCs from normal and lupus mice. There is no binding when a biotin-labeled scrambled form of P140 peptide (ScP140 Btn) is used instead of P140 peptide, and the binding is inhibited by an excess of free, non-biotinylated peptides (see the quantification by densitometry). (B) Direct binding to recombinant HSC70 protein of P140 and non-phosphorylated 131–151 peptides used at increasing concentrations (1.56 to 12.5 mM), as measured by surface plasmon resonance experiments. Analysis was performed using the simple 1:1 Langmuir binding model, the fitting to each model was judged by the x2 value and randomness of residue distribution was compared to the theoretical model. (C) Subcellular localization of HSC70 shown by immunoelectron microscopy. Fixed and permeabilized MRL/lpr PBLs were incubated overnight with the anti-HSC70 rat monoclonal antibody 1B5, followed by incubation with goat anti-rat IgG conjugated to ultra-small gold particles. Gold particles were enhanced using a silver kit. Cells were then treated for transmission electron microscopy with conventional methods (bar = 500 nm). (D) Co- localization, at the surface of non-permeabilized MRL/lpr splenocytes, of P140 peptide and HSC70 staining as shown by fluorescent microscopy: P140 followed using biotinylated peptide and FITC-labeled streptavidin, and HSC70 followed using the R-phycoerythrin-labeled anti-HSC70 rat monoclonal antibody 1B5. DAPI (49,69-diamidino-2-phenylindole) was used to label the nucleus (bar = 3 mm). (E) Binding in a dose-dependent manner of biotin- labeled P140 peptide (but not of ScP140 Btn) to the surface of MRL/lpr splenocytes as measured at 4uC by FACS analysis (the results obtained at 20 and 37uC are shown in Figure S4). (F) Cell-surface HSC70 expression among different CBA/J (black line) and MRL/lpr (red) cell subsets. (1) B220+TCRb2 = B cells; (2) B220+TCRb+ = activated T cells frequent in lupus mice, including CD42CD82 (DN) T cells, the number of which is raised in MRL/lpr mice; (3) B2202TCRb2 = naive cells; (4) B2202TCRb+ = T cells. Binding visualized with an isotypic control antibody is shown (right panel, in green). doi:10.1371/journal.pone.0005273.g001 stained with colloidal blue. This procedure led us to identify a single specific protein band (Figure 1A), which was identified by nano LC-MS/MS [15] as the heat-shock cognate 71-kDa protein, also termed HSC70 or Hsp/HSC73 protein (Figure S1). Twenty- six unique peptides matched between this newly characterized P140-receptor and HSC70 covered 58% of the theoretical HSC70 sequence. Identification of several discriminating peptides allowed us to clearly discard other Hsps, such as the inducible Hsp70/ Hsp72. HSC70 recovered from the cell surface in these conditions was the only protein specifically bound by P140 in a dose- dependent manner (Figure 1A). HSC70 also formed a stable complex with the non-phosphorylated peptide 131–151 but not with the scrambled peptide P140 (ScP140; Figure 1A). Formation of the complex was hampered by competing P140 (Figure 1A). To assess P140 binding to HSC70, we investigated the binding parameters and kinetics by surface plasmon resonance. HSC70 was immobilized onto the sensor chip via one of its four thiol groups, and different concentrations of P140 and 131–151 peptides were injected in the flux followed by a dissociation phase (see the overlay surface plasmon resonance plots of the raw data in Figure 1B). The association rate constants (kon) and dissociation rate constants (koff) were 530 M21s21 and 3.8861023s21 for the PLoS ONE | www.plosone.org 2 April 2009 | Volume 4 | Issue 4 | e5273