A cardiac troponin T epitope conserved across phyla.
Troponin T is a thin filament protein that is important in regulating striated muscle contraction. We have raised a monoclonal antibody against rabbit cardiac troponin T, monoclonal (mAb) 13-11, that recognizes its epitope in cardiac troponin T isoforms from fish, bird, and mammal but not from frog. The number of these isoforms expressed in cardiac muscle varies among species and during development. Cardiac troponin T isoforms were not found in adult skeletal muscle, while they were expressed transiently in immature skeletal muscle. We have mapped the epitope recognized by mAb 13-11 using rabbit cardiac troponin T isoforms. Analysis of stepwise cyanogen bromide digestion, which allowed association of the epitope to regions spanning methionine residues, coupled with immunoactivity of synthetic peptides, corresponding to sequences containing methionine residues, indicated that mAb 13-11 recognized its epitope in a 17-residue sequence containing the methionine at position 68, SKPKPRPFMPNLVPPKI. Comparison of skeletal and cardiac troponin T sequences suggested that the epitope was contained within the sequence FMPNLVPPKI. Synthetic peptides PFMPNLVPPKI and FMPNLVPPKI were recognized by mAb 13-11 on slot-blots. Enzyme-linked immunosorbent assay demonstrated mAb 13-11 recognized, in order of descending affinity, the 17-, 11-, and 10-residue sequence. Preabsorption of mAb 13-11 with each of these sequences blocked the recognition of the 17-residue peptide by mAb 13-11. The domain, PFMPNLVPPKI is encoded by the 5' region of the cardiac gene exon 10 and is present in hearts across a broad range of phyla. These findings suggest that this cardiac troponin T-specific sequence confers onto myofilaments structural and functional properties unique to the heart.
Malouf
N N
NN
Department of Pathology, University of North Carolina, Chapel Hill 27514.
McMahon
D
D
Oakeley
A E
AE
Anderson
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D006868:Hydrolysis; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure; D013045:Species Specificity; D014336:Troponin / Q000235:genetics / Q000276:immunology*; D020107:Troponin T
D000911:Antibodies, Monoclonal; D000939:Epitopes; D014336:Troponin; D020107:Troponin T; D003488:Cyanogen Bromide