Titin A-band-specific Monoclonal Antibody Tit1 5H1.1. Cellular Titin As a Centriolar Protein in Non-muscle Cells

Abstract

We report the development of a new mouse anti-titin monoclonal antibody, named MAb Tit1 5H1.1, using the synthetic peptide corresponding to an amino acid sequence in the A-band of the titin molecule as immunogen. In the human skeletal muscle, MAb Tit1 5H1.1 reveals a clearly striated staining pattern, reacting with the A-band of the sarcomere. Electrophoretic, immunoblotting, and amino acid sequence analyses with ESI-MS/MS of human skeletal muscle tissue proved the target antigen of MAb Tit1 5H1.1 to be titin. The antibody reacts with titin also in non-muscle cells, producing a punctate pattern in cytoplasm and the nucleus. The most striking finding was a clear reaction of MAb Tit1 5H1.1 with centrioles in all cell types investigated so far. Immunocytochemical co-localization study with ninein-specific antibodies confirmed that the target antigen of MAb Tit1 5H1.1 is a centriole-associated protein. Experiments of the inhibition of synthesis of titin using titin siRNA duplex for the destruction of titin mRNA have shown a decreased staining of centrioles by MAb Tit1 5H1.1 in non-muscle cells and support the proposal that the target antigen of MAb is indeed titin. We suggest this anti-titin monoclonal antibody could be a valuable tool in the study of titin function and its subcellular location, both in muscle and non-muscle cells.

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

“…Previously [1] we have shown that in the human skeletal muscle, MAb Tit15H1.1 reveals a clearly striated staining pattern reacting with the A-band of the sarcomere. Western blot and amino acid sequence analyses with the ESI-MS/MS of human skeletal muscle tissue samples proved the target antigen of MAb Tit15H1.1 to be titin.…”

“…This amino acid sequence is located between positions 27969–27987 of the A- region of human full-size titin molecule corresponding for the most part to the C-terminus of fibronectin type-III domain 103(SMART:Fn3 domain annotation, SwissProt: Q8WZ42). Previously [1] we have shown that the full-size peptide incubated overnight with MAb 5 H1.1 inhibits fully its reaction with antigen. To narrow down the epitope of MAb Tit1 5 H1.1, the supernatants inhibited with subpeptides AVNKYG, IGEPLE, EPLESD, PLESDSV and ESDSVV were used in immunofluorescence studies.…”

“…The full-size amino acid sequence originally used to prepare anti-titin monoclonal antibody MAb Tit1 5 H1.1 was a 19 amino-acid-long peptide with the sequence (NH2)AVNKYGIGEPLESDSVVAK(COOH) [1]. This amino acid sequence is located between positions 27969–27987 of the A- region of human full-size titin molecule corresponding for the most part to the C-terminus of fibronectin type-III domain 103(SMART:Fn3 domain annotation, SwissProt: Q8WZ42).…”

“…Previuosly, we have shown [1] that MAb 5 H1.1 reacts with titin also in human non-muscle cells, producing a punctate pattern in cytoplasm and in the nucleus. The most striking finding was a clear reaction of MAb Tit1 5 H1.1 with centrioles in all cell types investigated.…”

“…We have reported previously [1] on the development of a new mouse anti-titin monoclonal antibody, named MAb Titl 5 H1.1, using the synthetic peptide N-AVNKYGIGEPLESDSVVAK-C corresponding to an amino acid sequence in the A-band of the titin molecule as immunogen. In the human skeletal muscle, MAb Tit1 5 H1.1 reveals a clearly striated staining pattem, reacting with the A-band of the sarcomere.…”