Epitope of titin a-band-specific monoclonal antibody Tit1 5 H1.1 is highly conserved in several Fn3 domains of the titin molecule. Centriole staining in human, mouse and zebrafish cells

Background

Previously we have reported on the development of a new mouse anti-titin monoclonal antibody, named MAb Titl 5 H1.1, using the synthetic peptide N-AVNKYGIGEPLESDSVVAK-C which corresponds to an amino acid sequence in the A-region of the titin molecule as immunogen. In the human skeletal muscles, MAb Titl 5 H1.1 reacts specifically with titin in the A-band of the sarcomere and in different non-muscle cell types with nucleus and cytoplasm, including centrioles. In this report we have studied the evolutionary aspects of the binding of MAb Tit1 5 H1.1 with its target antigen (titin).

Results

We have specified the epitope area of MAb Tit1 5 H1.1 by subpeptide mapping to the hexapeptide N-AVNKYG-C. According to protein databases this amino acid sequence is located in the COOH-terminus of several different Fn3 domains of the A-region of titin molecule in many organisms, such as human being, mouse, rabbit, zebrafish (Danio rerio), and even in sea squirt (Ciona intestinalis). Our immunohisto- and cytochemical studies with MAb Tit1 5 H1.1 in human, mouse and zebrafish tissues and cell cultures showed a striated staining pattern in muscle cells and also staining of centrioles, cytoplasm and nuclei in non-muscle cells.

Conclusions

The data confirm that titin can play, in addition to the known roles in striated muscle cells also an important role in non-muscle cells as a centriole associated protein. This phenomenon is highly conserved in the evolution and is related to Fn3 domains of the titin molecule. Using titin A-band-specific monoclonal antibody MAb Tit1 5 H1.1 it was possible to locate titin in the sarcomeres of skeletal muscle cells and in the centrioles, cytoplasm and nuclei of non-muscle cells in phylogenetically so distant organisms as Homo sapiens, Mus musculus and zebrafish (Danio rerio).