Identification of novel PGRP3 protein -protein interactions using yeast two hybrid system

Abstract

AbstractBackgroundPeptidoglycan recognition protein-3 (PGRP3) is a pattern recognition receptor that binds peptidoglycan to elicit an immune response. How PGRP3 mediates these immunomodulatory effects remains unknown. Identifying the proteins that interact with PGRP3 will help begin to address this important knowledge gap. Therefore, in this study our objective was to identify and validate novel PGRP3-protein interactions.MethodsPGRP3-protein interactions were identified using a yeast two-hybrid system. PGRP3 cloned into a pGBKT7 DNA-BD vector and transformed into a Y2HGold yeast strain served as the bait. A normalized universal human cDNA library cloned in a pGADT7 AD vector and pre-transformed into an Y187 yeast strain served as the prey. Y2HGold and Y187 yeast strains were mated and diploids were plated on selective media. True positive clones were sent in for DNA sequencing and corresponding proteins were identified through NCBI Blast. Two proteins were selected for validation. His-tagged PGRP3 expressing 293T cells were then co-transfected with expression vectors containing the identified proteins tagged with c-Myc. Lysates from co-transfected cells were subjected to anti-His and anti-Myc co-immunoprecipitation and analyzed with western blots.ResultsFour unique proteins— MBNL3, RBP5, ATXN2, and GPATCH8—were identified from our yeast two-hybrid screen. Co-transfection and co-immunoprecipitation assays successfully confirmed MBNL3 and RBP5 as positive interactors with PGRP3.ConclusionsBased on the known functions of MBNL3, RBP5, ATXN2, and GPATCH8, we hypothesize that PGRP3 may be involved in RNA processing, endocytic trafficking, and PPARγ pathways related to innate immunity.