Molecular characterization of avian muscle titin

• Published in: The Journal of biological chemistry 1993-10, Vol.268 (30), p.22900-22907
• Main Authors: Tan, K.O., Sater, G.R., Myers, A.M., Robson, R.M., Huiatt, T.W.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding and carrier proteins; Biological and medical sciences; Blotting, Southern; Chick Embryo; Chickens; CHIMIE; Cloning, Molecular; Connectin; Conserved Sequence; DNA, Complementary - genetics; DNA, Complementary - metabolism; Escherichia coli; Fundamental and applied biological sciences. Psychology; IMMUNOLOGIE; INMUNOLOGIA; Membrane Proteins - biosynthesis; Molecular Sequence Data; MUSCLE; Muscle Proteins - biosynthesis; Muscle Proteins - genetics; Muscle Proteins - isolation & purification; MUSCULOS; Myocardium - metabolism; PEPTIDE; PEPTIDOS; POLLO; POULET; Protein Kinases; Proteins; QUIMICA; Rabbits; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Repetitive Sequences, Nucleic Acid; Restriction Mapping; SECUENCIA NUCLEICA; Sequence Homology, Amino Acid; SEQUENCE NUCLEIQUE
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)41611-0

Titin is an approximately 3000-kDa polypeptide that constitutes a set of elastic filaments that connect thick filaments to the Z-line in vertebrate striated muscle myofibrils. To characterize the primary structure of titin, three overlapping cDNA clones comprising 2.4 kilobases of avian muscle titin coding sequence were obtained from a cDNA library constructed from embryonic chick cardiac muscle RNA size-selected for large transcripts. Expression of one cDNA clone in Escherichia coli produced a fusion protein that reacted specifically with titin antibodies, and titin antiserum affinity-purified against this fusion protein reacted specifically with titin on immunoblots of chicken cardiac and skeletal muscle myofibrils. Indirect immunofluorescence localization with the fusion protein-specific antibodies demonstrated that the cDNA sequence was from the region of titin located in the myofibrillar A-band adjacent to the A/I junction. Derived amino acid sequences demonstrated a repeating pattern of fibronectin type III and immunoglobulin C2 motifs, as shown previously for a portion of rabbit skeletal muscle titin located in the central region of the A-band and for other myofibrillar proteins that bind to myosin. Differences between the rabbit and chicken titin sequences included a unique, serine-rich region in one motif, which represents a potential phosphorylation site. This is the first report of sequence information for avian titin from a previously uncharacterized portion of the titin molecule.