Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology
October 11, 2016
PLoS One. 2016 Oct 11;11(10):e0164593. doi: 10.1371/journal.pone.0164593. eCollection 2016. Li Y1, Ozment T2, Wright GL1, Peterson JM1,3. 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.
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.
- 1Quillen College of Medicine, Department of Biomedical Sciences, East Tennessee State University, Johnson City, Tennessee, United States of America.
- 2Quillen College of Medicine, Department of Internal Medicine, East Tennessee State University, Johnson City, Tennessee, United States of America.
- 3College of Public Health, Department of Health Sciences, East Tennessee State University, Johnson City, Tennessee, United States of America.