Z-band alternatively spliced PDZ motif protein (ZASP) is the major O-linked β-N-acetylglucosamine-substituted protein in human heart myofibrils

J Biol Chem. 2013 Feb 15;288(7):4891-8. doi: 10.1074/jbc.M112.410316. Epub 2012 Dec 27.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / chemistry*
  • Adaptor Proteins, Signal Transducing / metabolism
  • Adaptor Proteins, Signal Transducing / physiology*
  • Alternative Splicing
  • Amino Acid Sequence
  • Antibodies, Monoclonal / chemistry
  • Fluorescent Antibody Technique, Indirect / methods
  • Gene Expression Regulation*
  • Heart / physiology*
  • Humans
  • LIM Domain Proteins / metabolism
  • LIM Domain Proteins / physiology*
  • Microscopy, Fluorescence / methods
  • Molecular Sequence Data
  • Myofibrils / metabolism*
  • Peptides / chemistry
  • Signal Transduction
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Adaptor Proteins, Signal Transducing
  • Antibodies, Monoclonal
  • LDB3 protein, human
  • LIM Domain Proteins
  • Peptides
  • Acetylglucosamine