Initially, PRP-1 was isolated from the neurosecretory granules of bovine neurohypophysis by the method described (3,41) followed by its chemical synthesis (42).

Antiserum for PRP1 was generated (43), then affinity chromatography purified, AminoLink Plus Immobilization kit instructions (44894; Thermo Fisher Scientific, Waltham, MA, USA) were followed for protein sample desalting with Zeba Spin columns (89891; Thermo Fisher Scientific).

The human JJ012 chondrosarcoma cell line was received from Dr Joel Block's Laboratory (Rush University, Chicago IL, USA). JJ012 chondrosarcoma cells were cultured as previously described (8). The medium composition: Dulbecco's modified Eagle's medium (DMEM), supplemented with F12, 10% fetal bovine serum (FBS), 25 μg/ml ascorbic acid, 100 ng/ml insulin, 100 nM hydrocortisone and 1% penicillin/streptomycin.

Adherent cells were grown directly on coverslips with 5×10⁵ cells/coverslip in 6-well clusters, where they were cultured overnight at 37°C in an incubator. Twenty-four hours later the medium was removed and samples were fixed in 1 ml of 4% formaldehyde solution, (F8775; Sigma-Aldrich St. Louis, MO, USA) in phosphate-buffered saline (PBS), pH 7.4 1X Gibco, (10010-023) PBS for 15 min in the incubator. Samples were washed with PBS twice, then were permeabilized with PBS/Triton X-100 (T9284; Sigma-Aldrich), 1% for 5 min at room temperature. Detergent was removed and non-specific sites were blocked in PBS containing 2% bovine serum albumin (BSA, A2153; Sigma-Aldrich) at room temperature for 30 min. Samples were further incubated overnight in cold room along with all primary antibodies for the experiment, followed by two consecutive washes the next morning and incubation in BSA solution with secondary antibodies at room temperature for 2 h along with Zenon complex and two washes with PBS, for 10 min each. Second fixation step with formaldehyde for 15 min at room temperature in the dark was performed, followed by two washing steps.

PRP-1 serum antibody and Zenon rabbit IgG, Alexa Fluor 488 (Z-25302; Molecular Probes, Eugene, OR, USA) were mixed according to the manual and the procedures. The mixture was incubated for 10 min at room temperature with labeling reagent A, then another 10 min incubation with the blocking reagent B and 1 ml of the resulting mixture was applied to each well. Cells were stained with 3 μM of 4′,6-diamino-2-phenylindole dihydrochloride (DAPI, D1306; Thermo Fisher Scientific) for nuclear staining or 10 min at room temperature, the washed with PBS twice. The samples on coverslips were mounted in Antifade mounting medium, followed by microscopy. ProLong Gold Antifade reagent (P10144; Life Technologies) was applied as a liquid mountant directly to fluorescently labeled cells on microscope slides. The reagent contains chemicals to protect fluorescent dyes from fading during fluorescence microscopy.

For plasma membrane staining wheat germ agglutinin Alexa Fluor 594 conjugate was used (W11262; Thermo Fisher Scientific); TLR1 rabbit antibody (ab180798; Abcam); goat anti-rabbit H&L (DyLight 550) (ab96884; Abcam); mouse anti-MUC5B, Abcam (ab77995); goat anti-mouse IgG secondary antibody Alexa Fluor 647 (A2124; Life Technologies).

Image acquisition was performed by the Analytical Imaging Core Facility at DRI/SCCC, University of Miami (FL, USA).

Zeiss 200M, ApoTome fluorescent microscope, DAPI 49, GFP 38HE, Cy3 43, Cy5 50 filter cubes, heated stage, Orca II ERG Hamamatsu b/w 14 bit camera and AxioVision acquisition software were used. The coverslips were placed in regular 35-mm Petri dishes and the cells grown on them, covered with medium. Once the cells were grown, the coverslips were taken out, the cells were fixed, stained and mounted on the glass slides. For imaging controls secondary antibodies were used without the primaries.

MUC5B protein was measured with human mucin -5 subtype (MUC5B) ELISA kit (MyBioSource, San Diego, CA, USA) (MBS 704534-48T).

The cells were trypsinized once they reached confluency and then seeded in 6-well clusters at a concentration of 1×10⁶ cells/ml. PRP-1 was added only to the experimental samples but not to controls. The overnight incubation in 5% CO₂ incubator at 37°C was followed by cell wash with ice-cold PBS with added protease inhibitor. The cell lysis buffer (C2978; Sigma-Aldrich) was supplemented with the protease inhibitor in a 1:100 ratio. The cells were collected with a scraper and centrifuged at 15,000 × g at 4°C. The supernatant was collected and the protein concentration was measured. The pellets were frozen at −80°C until loading on the gel (20 μg/lane). Polyacrylamide gel electrophoresis and western blotting reagents were supplied by Lonza, Inc. (Allendale, NJ, USA), and all the related procedures followed the company's protocol. The catalog numbers for the reagents and the suppliers are listed below for convenience, although they were reported in our previous communication (8). PAGEr™ Gold Precast Gels (59502; 10% Tris-Glycine; Lonza); ECL reagent (RPN2109; GE Healthcare, Little Chalfont, UK); Western Blocker solution (W0138; Sigma-Aldrich); ProSieve QuadColor Protein marker (4.6–300 kDa, 00193837; Lonza); 20X Reducing Agent for ProSieve ProTrack Dual Color Loading buffer (00193861; Lonza); ProTrack Loading buffer (00193861; Lonza); ProSieve ProTrack Dual Color Loading buffer EX running buffer (00200307; Lonza); ProSieve EX Western Blot Transfer buffer (00200309; Lonza); Immobilon^®-P PVDF Membranes (P4188; Sigma-Aldrich).

Rabbit polyclonal anti-TLR6 (ab37072), MW 92 kDa (Abcam); rabbit anti-TLR1 cell (2209), MW 86 kDa (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-TLR1 (ab68158), MW 90 kDa (Abcam); mouse anti-TLR2 [TL2.1], (ab9100), MW 90 kDa (Abcam); Mouse anti-TLR3 (TLR3.7) (sc-32232), MW 104 kDa (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-TLR4 (25) (sc-293072), MW 95-120 kDa (Santa Cruz Biotechnology); mouse anti-TLR5 (19D759.2), (sc-57461), MW 110-120 kDa (Santa Cruz Biotechnology); rabbit anti-TLR7, (5632), MW 140 kDa (Cell Signaling Technology); mouse TLR 8 (9A6), (sc-135584), MW 119.8 kDa (Santa Cruz Biotechnology); rabbit anti-TLR9, (5845), MW 130 kDa (Cell Signaling Technology); mouse TLR10 (2A11), sc-293300, MW 90 kDa (Santa Cruz Biotechnology); mouse anti-tubulin, (T5168; Sigma-Aldrich); rabbit anti-TRIF/TICAM1, NBP2-31189, MW 75 kDa (Novus Biologicals, Littleton, CO, USA); mouse anti-TICAM2, MW 21 kDa (Santa Cruz Biotechnology); rabbit anti-TRAF6 (3566R-100), MW 54 kDa (BioVision, Inc., Milpitas, CA, USA); goat anti-rabbit IgG, HRP-linked (7074; Cell Signaling Technology); anti-mouse IgG, HRP-linked (7076; Cell Signaling Technology).

Nuclear Hormone Receptor Assays: PathHunter^® NHR Protein Interaction (Pro) and Nuclear Translocation (NT) assays monitor the activation of a nuclear hormone receptor in a homogeneous, non-imaging assay format using a technology developed by DiscoveRx called enzyme fragment complementation (EFC). The company described NHR Pro assay detects of protein-protein interactions between an actvated NHR protein and a nuclear fusion protein containing steroid receptor co-activator peptide (SRCP). When bound by ligand, the NHR will migrate to the nucleus and recruit the SRCP domain, whereby complementation occurs, generating a unit of active β-galactosidase (β-gal) and production of chemiluminescent signal.

The PathHunter^® β-arrestin assay based on activation of a GPCR using a method developed by DiscoveRx called enzyme fragment complementation (EFC) with β-galactosidase (β-gal) as the functional reporter (44). In brief, according to the manufacturer's protocol: the enzyme is split into two inactive complementary portions (EA for enzyme acceptor and ED for enzyme donor) expressed as fusion proteins in the cell. EA is fused to β-arrestin and ED is fused to the GPCR of interest. When the GPCR is activated and β-arrestin is recruited to the receptor, ED and EA complementation occurs, restoring β-gal activity which is measured using chemiluminescent PathHunter^® detection reagents.

The GPCR max panel % agonist was calculated as 100% (mean of test samples - mean of vehicle control)/mean Max control ligand - mean of vehicle control). For antagonist mode assays, percentage inhibition was calculated using the following formula: % Inhibition =100% × (1 – (mean RLU of test sample − mean RLU of vehicle control)/(mean RLU of EC80 control − mean RLU of vehicle control). For the orphan max panel, % agonist activity was calculated as 100% × (mean of test sample − mean of vehicle control)/mean of vehicle control.

To determine if a compound is potentially acting as an agonist to activate the receptor and induce arrestin recruitment the following factors should be considered: Is the % activity >30%? If so, is the compound mean RLU >Baseline RLU + 3 × Baseline SD.

Inhibition of GPCR activation by a compound acting as an antagonist of ligand binding results in a decrease in β-arrestin recruitment to the target GPCR. The NHR panel measures agonist interactions during a 6-h period and antagonist are preincubated for 1 h prior to agonist challenge. To determine if a compound is potentially acting as an antagonist to inhibit receptor activation the following factors should be considered: Is the % inhibition >35%, if so, is the compound mean RLU <EC80 RLU − 3 × EC80 SD.

Activation of Orphan GPCR by a compound acting as an agonist will result in an increase in β-arrestin recruitment to the target orphan GPCR. To determine if a compound is potentially acting as an agonist to activate an orphan receptor and induce arrestin recruitment the following factor should be considered: Is the % activity >50%. If so, is the compound mean RLU >Baseline RLU + 3 × Baseline SD.

This technology from Dualsystems Biotech AG (Zurich, Switzerland) was implemented to detect PRP-1 receptor or interacting partners. Specific cell surface protein receptors are involved in the drug, peptide ligand mediated physiological responses. The TriCEPS method is based on the Ligand-based receptor capture (LRC) technology where special reagent can be coupled to a ligand of interest, which allows to capture the ligand when bound to corresponding receptors. One can picture TriCEPS with three arms: one that binds to amino group containing ligands, a second for the ligand-based capture of glycosylated and a third one with biotin tag for purifying receptor peptides to be analyzed by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., insulin) receptor.

This procedure implemented processing of ligand and the identification of receptor candidates (3 ligand and 3 control samples, 300 μg control ligand or 300 μg ligand of interest to 120 μl of 25 mM HEPES pH 8.2, 1.5 μl (150 μg) TriCEPS v.3 was added to both reactions and mixed immediately by pipetting up and down using a 200 μl pipette, then incubated at 20°C under gentle agitation (350 rpm) in a ThermoMixer for 90 min. Cell preparation and oxidation 1.2×10⁸ cells were utilized for the experiment in triplicates. Cells were centrifuged at 300 × g for 5 min at 4°C, then were resuspended in 49 ml LRC buffer. The oxidation agent 1 ml (75 mM sodium metaperiodate) were added to a final concentration of 1.5 mM metaperiodate, followed by incubation for 15 min at 4°C. The mild oxidant sodium metaperiodate generates aldehydes from carbohydrates that link to the proteins of cell surface. When the ligand binds to the receptor, the hydrazine group formed a bond with the aldehyde for protein labeling.

The LRC-TriCEPS samples were analyzed on a Thermo LTQ Orbitrap XL spectrometer. The samples were processed in data dependent acquisition mode in a 90-min gradient with 10 cm C18 packed column. The statistical ANOVA model was applied to the six remaining samples in the CaptiRec dataset. With models of Gaussian distribution the system tests each protein for differential abundance in all pairwise comparisons of ligand and control samples and calculates P-values. P-values are undergoing multiple comparisons to control the experiment-wide false discovery rate (FDR). Then, this adjusted P-value from each individual protein is plotted against the magnitude of the fold enrichment between the two experimental conditions. The receptor candidate space is defined based on the criteria where the area in the volcano plot that is limited by an enrichment factor of ≥4-fold and an FDR-adjusted P≤0.01.

These custom designed assays by Qiagen (Valencia, CA, USA) served as sensitive gene expression profiling tool for real-time PCR analyses. Assay utilized RT2 SYBR-Green qPCR Master Mixes. Mature RNA isolated using RNA extraction according to the manufacturer's instructions. RNA quality was determined using a spectrophotometer and was reverse transcribed using a cDNA conversion. The cDNA in combination with RT2 SYBR-Green qPCR Master Mix (cat. no. 330529) was used with RT2 qPCR assays. Ct values were uploaded on web portal at http://www.qiagen.com/geneglobe. Samples were assigned to control and test groups. Ct values were normalized based on a manual selection of reference genes. The data analysis web portal calculates fold change/regulation using ∆∆Ct method, in which ∆Ct is calculated between gene of interest (GOI) and an average of housekeeping genes (HKG) followed by ∆∆Ct calculations [∆Ct(experiment) − ∆Ct(control)]. Fold change is then calculated using the 2^−∆∆Ct formula.

The search for PRP-1 binding partners started with DiscoverX platform of G protein coupled receptors, (GPCR) in agonist, (Table I) and antagonist modes (Table II). There was no indication that PRP-1 was G protein coupled, neither that this peptide was agonist for orphan receptors (Table III) or agonist/antagonist for nuclear receptor in nhrMax panels (Table IV). MUC5B was identified as PRP-1 receptor binding partner in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. We proceeded further in the attempt to identify binding partners for PRP-1 using TriCEPS Ligand-receptor capture (LRC) technology from Dualsystems Biotech AG (45). LRC was used to identify novel ligand-receptor interactions. After the TriCEPS coupled ligand (PRP-1) bound to its targets in or at the cell membrane the second arm of TriCEPS coupled to the glycans of that target receptor. The third arm of TriCEPS was used to isolate the proteins that are bound to TriCEPS (Fig. 1A). In the next step the isolated proteins were subjected to a trypsin digest. The resulting peptides of the digest were identified and quantified using liquid chromatography, tandem mass spectrometry (LC-MS/MS) (45,46). Then, the quantified peptides from the control reaction (transferrin as ligand) were compared to the ligand of interest (PRP-1) reaction (labelled in the volcano plot as peptide). The proteins that were 4-fold enriched in one of the treatments compared to the other treatments were considered as the binding partners of the ligand used. When the cells were treated with TriCEPS coupled transferrin, the transferrin receptor protein (TFR1) was enriched (left side of the volcano plot), whereas in the ligand of interest treated samples the MUC5B was enriched. Thus, MUC5B was identified as receptor for PRP-1 (Fig. 1B). The data of the experiment was presented in biological triplicates. The P-value obtained for every protein was plotted against the log2 of the magnitude of the fold enrichment. The space for positive control receptors and high-confidence receptor candidates was designated and visualized based on (fold-change >4) significant enrichment (adjusted P<0.01). True positive receptor candidates that contain only few tryptic peptides can be enriched substantially but will rarely get adjusted P<0.01. For the final selection of receptor candidates for follow-up investigations, all proteins in the receptor space should be viewed based on the following parameters: proteins that were increased >4 times (log2=2 on the x-axis) with adjusted P<0.01 [−log (0.01)=2] were considered to be in the receptor space (white). The protter figure (Fig. 1C) usually displays peptides belonging to the ligand's receptor as being enriched (shown in highlighted stripes) compared with the control sample (no ligand). Protter is an interactive tool for protein data analysis (47). The experimental results with MUC5B ELISA confirmed the binding, PRP-1 at 1 μg/ml detected MUC5B presence at 440 ng/ml in the cell lysates of human JJ012 chondrosarcoma cells (Fig. 1D). Toll like receptors TLR1/2 and TLR6 were identified by western blot as binding interaction partners with PRP-1 in human chondrosarcoma JJ012 cell line lysates. Polyacrylamide gel electrophoresis, immunoblot results indicated that PRP-1 caused strong upregulation of TLR1 and TLR2 in comparison to untreated control. TLR3 expression was present but weak and data is not shown. TLR4 and TLR5 were expressed, but PRP-1 did not have any effect (Fig. 2). TLR6 protein expression was also increased in dose-dependent manner in PRP-1 treated samples. TLR7 was not expressed at all in the cell line, whereas TLR8, 9 and 10 were expressed but there was no indication of PRP-1 effect (Fig. 2). Thus, TLR1/2 and TLR6 were identified as interacting binding partners for PRP-1. Fig. 2 depicts the PRP-1 action on the protein expression of the adaptors TICAM1 (TRIF) and TICAM2 (TRAM). PRP-1 upregulated in dose-dependent manner the adaptor TICAM2 but not TICAM1. Toll like receptors TLR1/2 and TLR6 and MUC5B were detected with PRP-1 in the nucleus of human chondrosarcoma cells by immunocytochemistry. Since the experiments with immunoblot and TriCEPS indicated that cell surface receptors TLR1/2, TLR6 and gel forming mucin MUC5B were the binding partners for PRP-1, the immunocytochemistry experiments followed next; not only to prove PRP-1 endogenous presence in chondrosarcoma, but also its cellular colocalization with the binding partners. The images are displayed in Fig. 3. PRP-1 antibody, isolated from rabbit serum and affinity chromatography purified was labeled with Zenon Alexa Fluor 488 IgG complex (green) and manifested its presence in the nucleus (labeled with DAPI in blue) of the chondrosarcoma cells (Fig. 3A). The green speckles and dots can be seen both inside and outside the nucleus. In the separate experiment without PRP-1 we have demonstrated that MUC5B is present in the nucleus of these cells as well (Fig. 3B). The plasma membrane is seen in red and MUC5B, which was labeled with DyLight 488 is green. The composite image (Fig. 3C) demonstrates nuclear localization of both MUC5B (left panel) and PRP-1 (right panel) in aqua green color on the background of blue nucleus. Fig. 3D illustrates the presence of TLR1 receptor, (labeled in yellow with H&L DyLight 550) in the nucleus and around it. Fig. 3E depicts colocalization experiment of TLR6 and PRP-1 and whereas it was problematic to show TLR1 and PRP-1 colocalization in the previous figure due to spectral overlaps, here TLR6 receptor nuclear and cytoplasmic localization is demonstrated with PRP-1, which was stained with Zenon Alexa Fluor 488 IgG (green). H&L DyLight 550 was used as a secondary antibody (yellow) for TLR6. The immunoblot experiment with the nucleolin antibody, marker for nucleoli indicated that PRP-1 was not located in the nucleoli, as no changes in nucleolin protein expression was observed on PRP-1 treatment (Fig. 3F).

RT2 qPCR custom designed primer assays were performed by Qiagen to understand the effect of PRP-1 on gene expression of TLR receptors and MUC5B. Mature RNA was isolated using RNA extraction according to the manufacturer's instructions. RNA was subjected to spectrophotometrical quality control and then reverse transcribed to cDNA. RT2 SYBR-Green qPCR Master Mix was used with RT2 qPCR assays. In this study, 7 genes (TLR1, TLR2, TLR6, MUC5B, c-Myc and 2 housekeeping genes GAPDH and ACTB) were profiled on three samples with technical triplicates. c-Myc was included, as we wanted to confirm its drastic downregulation after PRP-1 treatment in luciferase assay (4) and western blot experiments, reported earlier (10). The heat map, clustergram of average Ct values across the gene of each sample, with the magnitude of gene expression scale below, is presented in Fig. 4. As evident from the figure, there is dose-dependent effect of PRP-1 on the expression of the above mentioned genes, except the control housekeeping genes. The TLR1 receptor was well expressed in the cells treated with 10 μg/ml, whereas no expression was detected at 1 μg/ml peptide treatment. TLR2, TLR6, MUC5B demonstrated high expression with 1 μg/ml treatment when compared to nontreated control. c-Myc expression went down drastically when treated with 10 μg/ml PRP-1. The data analysis web portal calculated fold change/regulation using ∆∆Ct method, in which ∆Ct is calculated between gene of interest (GOI) and an average of housekeeping genes (HKG) followed by ∆∆Ct calculations (∆Ct (experiment) − ∆Ct (control). Fold change is then calculated using the 2^−∆∆Ct formula. The system detected only statistically significant upregulation (P<0.0001) of TLR2 for the samples treated with 1 μg/ml PRP-1, with 5.26-fold upregulation when calculated with the ∆∆Ct.