Identification of Novel PGRP3 Interactomes

Objectives: Peptidoglycan recognition protein 3 (PGRP3), is a crucial mediators of tissue homeostasis. It surveils the environment and discriminates normobionts from pathobionts. PGRP3 is expressed by antigen presenting cells in the gingiva. We have previously shown elevated levels of PGRP3 in saliva and tissue of periodontitis patients. Functionally, PGRP3 is considered anti-inflammatory and antimicrobial. PGRP3 interferes with IFN-Υ activity in the gut; and in the skin it limits TH17 over activation by promoting T-reg accumulation. Both these mechanisms have functional relevance in periodontitis. Critically, how PGRP3 mediates these immune-modulatory effects remains unknown. Therefore, our objective was to identify and validate novel PGRP3-protein interactions
Methods: PGRP3-protein interactions were identified using a yeast two-hybrid system. With PGRP3 as bait, a commercially available normalized universal human cDNA library was used as prey. Positive colonies (blue) were isolated and subjected to endonuclease restriction digestion analysis. DNA sequencing was done on true positive clones and corresponding proteins were identified through NCBI Blast. His-tagged PGRP3 overexpressing 293T cells were there co-transfected with expression vectors containing the identified protein(s) tagged with c-MYC. Lysates from co transfected cells were subjected to a HIS pull down and analyzed with western blots.

Results: Four unique and novel PGRP3 interacting proteins, namely ATXN2, RBP5, GPATCH8, and MBNL3, were identified. Co-transfection and co-immunoprecipitation assays in a mammalian cell system successfully confirmed the protein interactions identified through the yeast two hybrid assay.
Conclusions: Here we have identified and validated hitherto unknown novel interacting partners of PGRP3. ATXN2, MBNL3 &, GPATCH8 are known to be critical for RNA processing. RBP5 has known transporter activity, while ATXN2 is also known for endocytic trafficking. Therefore it’s reasonable to hypothesize that PGRP3 may exert its effects by altering RNA processing and endocytic trafficking. However the exact effect of these interactions needs further elucidation.