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-TnC(2b ') (2094 nt, 141 aa and 15.9 kDa), -C-4 ' (1667 nt, 155 aa and 17.3 kDa), -C-5 (2884 nt, 149 aa and 17.3 kDa), -C-6 (2439 nt, 155 nt and 17.4 kDa) and -C-6x (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-TnC(1) (814 nt, 150 aa and 17.1 kDa), -C-2a(639 nt, 152 aa and 17.2 kDa), -C-2b'. (2136 nt, 155 aa and 17.5 kDa), -C-3 (1046 nt, 150 aa and 16.9 kDa), -C-4'. (842 nt, 108 aa and 12.1 kDa) and one partial (3 ') cDNA, designated Ha-TnC4 ', (563 nt and 57 aa). Ha-TnC(1), -C-2a, and -C-2b ' corresponded to lobster TnC sequences in the GenBank protein database (Ha-TnC(1), -C-2a, and -C-2b). Alternative splicing appeared responsible for TnC(2b ') and -C-2b ''; TnC(4 '), -C-4 ''; and-C-4 ''; and TnC(6) and -C-6x. The deduced amino acid sequences differed primarily in the terminal regions and EF-hands land III. Ha-TnC(6x) 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-TnC(1), -C-2a, -C-2b ', -C-2b '' and - C-3 were expressed primarily in skeletal muscles; Ha-TnC(5) was expressed in heart: and Ha-TnC(4) and -C-6 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. (C) 2010 Elsevier Inc. All rights reserved.