Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology

Abstract

Methods: We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and insulin as a control ligand was performed on the H4IIE rat hepatoma cell line.

Results: Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and Insulin-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the insulin receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells.

Conclusion: The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3.

Relevance: The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.

Fig 1. CTRP3 binds to hepatocytes in vitro.

H4IIE hepatocytes were plated on Millicell EZ SLIDE (Millipore) and allowed to adhere for 48 H in standard growth medium. The cells were treated with recombinant FLAG tagged CTRP3, CTRP1, or vehicle the cells were then incubated with rabbit anti-FLAG primary antibody followed by fluorochrome-conjugated secondary antibody. Cells were then mounted with an anti-fade mounting medium with DAPI, and immunofluorescence was visualized.

Fig 2. CTRP3 binds to hepatocytes in vitro.

H4IIE hepatocytes were grown in standard media and then treated for 1 H ± CTRP3-FLAG (5 μg/ml). The cells were then washed and collected in PBS, fixed in 4% formaldehyde, and then incubated with rabbit anti-FLAG antibody followed by fluorochrome-conjugated secondary antibody and analyzed for mean fluorescent intensity (MFI) by flow cytometry. Representative image (20% of points shown) of raw flow data from vehicle treated (A) or CTRP3-FLAG treated (B). C, the MFI ± CTRP3 normalized to vehicle for each independent replicate. D, Percent of the cells that were positive from each experiment. Data for C & D are from 3 independent replicates performed on different days with separate lots of recombinant CTRP3-FLAG protein with each replicate performed in triplicate and reported as mean ± SD. Raw flow cytometry files are attached as supplementary data (S1 File). * p < vs. 0.0001 vehicle.

Fig 3. CTRP3 increases oxygen consumption.

Cells were pre-incubated with 5 μg/ml recombinant CTRP3 or vehicle for 1 H before being placed into the XFe24 Extracellular Flux Analyzer (Seahorse Bioscience) and oxygen consumption rate (OCR) was determined. OCR was measured in the absence (A) or after the addition of 200 μM palmitate (B) added at 15 minutes (vertical line). C, Area under the curve was calculated (mean OCR value at each interval*time) for each treatment. Data represents the mean ± SD. Data represents pooled data from 3 independent experiments each performed in triplicate, *p < 0.05 vs. vehicle + palmitate.

Fig 4. H4IIE rat hepatoma cells were treated with TriCEPs conjugated to Insulin or CTRP3.

A, The binding of ligands to cell surface receptors was detected by Steptavidin FITC (The TriCEPS™ reagent contains biotin) and analyzed for mean fluorescent intensity (MFI). Representative image (20% of points shown) of raw flow data from vehicle treated. B, The samples were analyzed by Mass spectrometry (Dualsystems Biotech, AG) and the adjusted p-value (ANOVA, adjusted for multiple comparisons) for the differential abundance of each protein was plotted against the magnitude of the fold enrichment between insulin and CTRP3 samples. Proteins are considered significant if fold change >2 and p<0.05. Two proteins (LAMP1 and LIMPII) were statistically significant. Raw data for Fig 4, S2 File.

Fig 5. Blocking LAMP1 suppresses CTRP3 binding.

H4IIE hepatocytes were grown to confluence in standard media and then treated for overnight ± CTRP3-FLAG (2.5 μg/well) and ± polyclonal LAMP1 antibody (10 μg/well, to block potential CTRP3 binding sites). The cells were then washed and collected in PBS, fixed in 4% formaldehyde, and then incubated with rabbit anti-FLAG antibody followed by fluorochrome-conjugated secondary antibody and analyzed for mean fluorescent intensity (MFI) by flow cytometry. A-D, Representative flow data from H4IIE hepatocytes. A MFI >100 was taken as positive and is indicated in the flow plots with a gray bar. A, pooled cells with no primary antibody (isotype or negative control). B, cell incubated with vehicle, C, recombinant CTRP3-FLAG protein, or D, co-incubated with recombinant CTRP3-FLAG protein plus poly-clonal anti-LAMP1. E, Percent of the cells that were positive from each experiment. F, the MFI ± CTRP3 normalized to vehicle for each independent replicate. Data for E & F are from 3 independent replicates performed on different days with separate lots of recombinant CTRP3-FLAG protein with each independent replicate performed in triplicate and reported as mean ± SD. Raw flow cytometry files are attached as supplementary data (S3 File). * p < 0.001 vs. vehicle; ** p < 0.001 vs. CTRP3+LAMP1.

Fig 6. Co-Immunoprecipitation (Co-IP) and Immunoblot Analysis.

H4IIE cells were treated overnight with vehicle, FLAG peptide, or recombinant FLAG tagged CTRP3 (rCTRP3). Total protein homogenate (A & C) or immunoprecipitate (B & D) were separated by SDS-page gel and transferred to nitrocellulose membrane. A & B, after immunoblot total protein was visualize by brief incubation with Ponceau S staining solution (5% acetic acid & 0.1% Ponceau S). C, LAMP1 in total protein homogenate was similar between treatments. D, Co-IP results showing LAMP1 binds to CTRP3. Samples were immunoprecipated with anti-FLAG Affinity Gel followed by immunoblotting with antibodies against LAMP1.