Cloning and tissue expression of eleven troponin-C isoforms in the American lobster, Homarus americanus

Troponin-C is the Ca2+-binding subunit of the troponin regulatory complex in striated muscles. As TnC isoforms can influence the Ca2+-activation properties of fiber phenotypes, the diversity and tissue distribution of TnC cDNAs were assessed in the American lobster, Homarus americanus. We cloned ten full-length cDNAs and one partial cDNA coding for distinct TnC isoforms. Five were sequenced from expressed sequence tag clones and were designated Ha-TnC2b′ (2094 nt, 141 aa and 15.9 kDa), - C4′ (1667 nt, 155 aa and 17.3 kDa), - C5 (2884 nt, 149 aa and 17.3 kDa), - C6 (2439 nt, 155 nt and 17.4 kDa) and - C6x (2171 nt, 154 aa and 16.9 kDa). The remainder were cloned using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends: five full-length cDNAs, designated Ha-TnC1 (814 nt, 150 aa and 17.1 kDa), - C2a (639 nt, 152 aa and 17.2 kDa), - C2b″ (2136 nt, 155 aa and 17.5 kDa), - C3 (1046 nt, 150 aa and 16.9 kDa), - C4″ (842 nt, 108 aa and 12.1 kDa) and one partial (3′) cDNA, designated Ha-TnC4‴ (563 nt and 57 aa). Ha-TnC1, - C2a, and - C2b′ corresponded to lobster TnC sequences in the GenBank protein database (Ha-TnC1, - C2a, and - C2b). Alternative splicing appeared responsible for TnC2b′ and - C2b″; TnC4′, - C4″ and - C4‴; and TnC6 and - C6x. The deduced amino acid sequences differed primarily in the terminal regions and EF-hands I and III. Ha-TnC6x had a highly divergent 76 aa proline-rich N-terminal sequence. Tissue expression of the Ha-TnC isoforms was analyzed qualitatively by endpoint PCR. Ha-TnC1, - C2a, - C2b′, - C2b″ and - C3 were expressed primarily in skeletal muscles; Ha-TnC5 was expressed in heart; and Ha-TnC4 and - C6 variants were expressed in muscles and other tissues. The number and diversity of TnC sequences suggest the potential for varying the Ca2+-activated properties of the troponin-tropomyosin regulatory complex through differential expression of TnC isoforms.