Study of O-Glcnacylation of Contractile Proteins in Cardiac Myofibrils by Enzymatic Labelling

Abstract

Phosphorylation occurs on serine and threonine residues and plays important roles in the regulation of contractile proteins. In heart failure changes in levels of phosphorylation are reported in a number of cardiac sarcomeric proteins. O-linked-N-acetylglucosamine (O-GlcNAc) modification is another possible posttranslational modification on serine and threonine residues and recent publications reported mapping of O-GlcNAc modification sites in some rat contractile proteins, including myosin heavy chain (MHC), actin, cardiac troponin I and myosin light chains (MLC1 and MLC2). O-GlcNAc modification on normal donor hearts (49yr F and unknown) and hypertrophic obstructive cardiomyopathy myectomy samples (33yr M and 42yr M) were studied. Cardiac myofibrils were isolated and the O-GlcNAc groups labelled using an enzymatic labelling system in the presence of PUGNAc (inhibits O-GlcNAc removal enzyme O-GlcNAcase) and protease inhibitors. This method allows coupling of an azido modified N-acetylgalactosamine (UDP-GalNAz) to O-GlcNAc using the mutant enzyme Y289L β1,4-galactosyltransferase (Y289L GalT). The labelled groups were detected by reacting the azide group with an alkyne bearing the tetramethylrhodamine (TAMRA) fluorescent tag for direct imaging following SDS-PAGE. The gel was post-stained with a total protein stain for analysis with densitometry. The labelling process showed no impact on myofibril protein profiles when native and labelled myofibrils were compared. Preliminary results showed that O-GlcNAcylation profiles vary between samples with a total of 7 proteins identified. Strong TAMRA signals from α-actinin and MLC1 were observed in all the four myofibrils samples. In three of the samples the proteins actin, tropomyosin (Tm) and myosin binding protein-C (MyBP-C) were positively labelled. MHC and desmin O-GlcNAcylation were observed in one of the four subjects. This enzymatic labelling method will be investigated further for possibility of quantification and methods for mapping sites of these modifications with mass spectrometry will be explored.