O-GlcNac Modification of Human Cardiac α-Actinin

Abstract

We measured O-GlcNAcylation of myofibrillar proteins from human hearts by two assays: (1) Western blotting with an antibody (CTD110.6) specific to O-GlcNAc modified proteins and (2) specific enzymatic labelling of O-GlcNAc with N-acetylgalactosamine (UDP-GalNAz) by mutant enzyme Y289L beta-1,4-galactosyltransferase (Y289L GalT), which allows coupling of tetramethylrhodamine (TAMRA) fluorescent tag for direct imaging following SDS-PAGE. In every sample, the predominant modified protein was alpha-actinin, which made up 65±4.48% of the enzymatically labelled myofibrillar proteins. We found that alpha-actinin exists as two bands on 12% SDS-PAGE. EA-53 antibody to alpha-actinin revealed that the faster migrating alpha-actinin band is on average <1.4±0.31% of the slower migrating band. Interestingly, CTD110.6 showed signals from both bands with similar intensities (mean ratio=1.02±0.10), indicating that O-GlcNAc modification is more abundant in the minor band (>80x more concentrated). This was confirmed by the enzymatic labelling method; O-GlcNAc was often not detectable in the slower migrating band, with an average ratio of faster/slower migrating band of 2.45±0.49. Further tests showed that the higher mobility band was not a degradation product of the lower mobility band. O-GlcNAc can be removed from the myofibril proteins by beta-N-acetylglucosaminidase. Using CTD110.6 detection, increasing enzyme concentration decreased the faster migrating band to near zero, but the slower migrating band did not show the same trend, suggesting that the antibody may cross react slightly with unmodified alpha-actinin. We conclude that O-GlcNAcylation is highly concentrated in the low abundance faster migrating species of alpha-actinin. The possibility that the two bands represent two isoforms of alpha-actinin differentially modified by the potentially regulatory O-GlcNAc modification is being investigated.