Z-band Alternatively Spliced PDZ Motif Protein (ZASP) Is the Major O-Linked β-N-Acetylglucosamine-substituted Protein in Human Heart Myofibrils

We studied O -linked β- N -acetylglucosamine ( O -GlcNAc) modification of contractile proteins in human heart using SDS-PAGE and three detection methods: specific enzymatic conjugation of O -GlcNAc with UDP- N -azidoacetylgalactosamine (UDP-GalNAz) that is then linked to a tetramethylrhodamine fluorescent tag and CTD110.6 and RL2 monoclonal antibodies to O -GlcNAc. All three methods showed that O -GlcNAc modification was predominantly in a group of bands ∼90 kDa that did not correspond to any of the major myofibrillar proteins. MALDI-MS/MS identified the 90-kDa band as the protein ZASP (Z-band alternatively spliced PDZ motif protein), a minor component of the Z-disc (about 1 per 400 α-actinin) important for myofibrillar development and mechanotransduction. This was confirmed by the co-localization of O -GlcNAc and ZASP in Western blotting and by immunofluorescence microscopy. O -GlcNAcylation of ZASP increased in diseased heart, being 49 ± 5% of all O -GlcNAc in donor, 68 ± 9% in end-stage failing heart, and 76 ± 6% in myectomy muscle samples (donor versus myectomy p < 0.05). ZASP is only 22% of all O -GlcNAcylated proteins in mouse heart myofibrils. Background: We studied O -GlcNAc-modified protein in sarcomeric proteins of human heart. Results: ZASP (Z-band alternatively spliced PDZ motif protein) accounts for 50–80% of O -GlcNAcylated protein. Conclusion: ZASP is the major O -GlcNAc-substituted protein in human heart muscle, and its levels increase in pathological muscle. Significance: Modulation of O -GlcNAcylation in ZASP may have a role in mechanotransduction in the heart.