Abstract
Purpose :
Periaxin (Prx) is a scaffold protein and is known to play essential roles in fiber cell shape and organization; it also acts as a genetic modifier in cataractogenesis of Cx46 knockout (Cx46KO) mice. However, the mechanisms underlying periaxin functions in the lens fibers and in Cx46KO cataractogenesis remain unclear. We hypothesize that the periaxin associated proteins play key roles in fiber cell morphogenesis and cataract formation. This study aims to determine specific lens proteins that interact with periaxin in wild-type (WT) and Cx46KO lenses of both 129SvJae (129) and C56BL/6J (B6) mouse strains.
Methods :
Mouse lenses were isolated and homogenized for immunoprecipitation (IP) with anti-Prx antibody. IP protein samples were separated using SDS-PAGE gels. Protein bands from 5 kDa to over 500 kDa were excised from the gel and were subjugated to in-gel tryptic digestion and protein identification with mass spectrometry. Biochemical methods and immunolabeling were used to characterize Prx-associated protein candidates.
Results :
Based on the criteria that at least two peptides for each protein with confidence interval percentage over 99.90% were used for protein ID, a total of 239 proteins were identified in Prx IP samples across all genetic backgrounds, including 129WT, B6WT, 129 Cx46KO, and B6 Cx46KO. According to the heat map from hierarchical clustering of significantly changed proteins between 129WT versus 129 PRX KO, and 129WT versus B6WT, a group of cytoskeletal associated proteins, including Ankyrin B, Arvcf, Catenin beta-1, were identified as the potential candidates for Prx associated proteins in 129 background but not B6. While Periplakin and Phakinin were identified as the potential candidates for Prx associated proteins in B6 background but not 129.
Conclusions :
Many potential candidates of periaxin associated proteins were identified in the lens. Different proteins between 129 or B6 lens samples were identified to be associated with Prx. Some of these candidates are involved in the cataract formation in Cx46KO in 129 strain background but not in B6 strain background. Further biochemical analysis and immunostaining of Phakinin, Periplakin, Ankyrin B and ARVCF, Catenin beta-1 would lead to new insight about the mechanism for the involvement of these periaxin associated proteins in cataract formation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.