Characterization of the in vitro expressed autoimmune rippling muscle disease immunogenic domain of human titin encoded by TTN exons 248-249

Biochem Biophys Res Commun. 2011 Aug 5;411(3):501-5. doi: 10.1016/j.bbrc.2011.06.139. Epub 2011 Jun 29.

Abstract

Autoimmune rippling muscle disease (ARMD) is an autoimmune neuromuscular disease associated with myasthenia gravis (MG). Past studies in our laboratory recognized a very high molecular weight skeletal muscle protein antigen identified by ARMD patient antisera as the titin isoform. These past studies used antisera from ARMD and MG patients as probes to screen a human skeletal muscle cDNA library and several pBluescript clones revealed supporting expression of immunoreactive peptides. This study characterizes the products of subcloning the titin immunoreactive domain into pGEX-3X and the subsequent fusion protein. Sequence analysis of the fusion gene indicates the cloned titin domain (GenBank ID: EU428784) is in frame and is derived from a sequence of N2-A spanning the exons 248-250 an area that encodes the fibronectin III domain. PCR and EcoR1 restriction mapping studies have demonstrated that the inserted cDNA is of a size that is predicted by bioinformatics analysis of the subclone. Expression of the fusion protein result in the isolation of a polypeptide of 52 kDa consistent with the predicted inferred amino acid sequence. Immunoblot experiments of the fusion protein, using rippling muscle/myasthenia gravis antisera, demonstrate that only the titin domain is immunoreactive.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Autoimmune Diseases / genetics
  • Autoimmune Diseases / immunology*
  • Connectin
  • DNA, Complementary / genetics
  • Exons
  • Humans
  • Immunodominant Epitopes / genetics
  • Immunodominant Epitopes / immunology
  • Molecular Sequence Data
  • Muscle Proteins / genetics
  • Muscle Proteins / immunology*
  • Muscular Diseases / genetics
  • Muscular Diseases / immunology*
  • Protein Kinases / genetics
  • Protein Kinases / immunology*
  • Protein Structure, Tertiary

Substances

  • Connectin
  • DNA, Complementary
  • Immunodominant Epitopes
  • Muscle Proteins
  • TTN protein, human
  • Protein Kinases

Supplementary concepts

  • Rippling muscle disease, 1