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

PLoS One. 2016 Oct 11;11(10):e0164593. doi: 10.1371/journal.pone.0164593. eCollection 2016.


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.

MeSH terms

  • Adipokines / chemistry
  • Adipokines / metabolism*
  • Animals
  • Antibodies / immunology
  • Binding Sites
  • Cell Line
  • Chromatography, High Pressure Liquid
  • Flow Cytometry
  • HEK293 Cells
  • Humans
  • Immunoprecipitation
  • Insulin / chemistry
  • Insulin / metabolism
  • Ligands*
  • Lysosomal-Associated Membrane Protein 1 / chemistry
  • Lysosomal-Associated Membrane Protein 1 / immunology
  • Lysosomal-Associated Membrane Protein 1 / metabolism
  • Lysosomal-Associated Membrane Protein 2 / chemistry
  • Lysosomal-Associated Membrane Protein 2 / immunology
  • Lysosomal-Associated Membrane Protein 2 / metabolism
  • Mice
  • Microscopy, Fluorescence
  • Protein Binding
  • Rats
  • Tandem Mass Spectrometry


  • Adipokines
  • Antibodies
  • CORS26 protein, mouse
  • CTRP1 protein, mouse
  • Insulin
  • Ligands
  • Lysosomal-Associated Membrane Protein 1
  • Lysosomal-Associated Membrane Protein 2