Titin A-band-specific monoclonal antibody Tit1 5H1.1. Cellular Titin as a centriolar protein in non-muscle cells.
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.
Institute of General and Molecular Pathology, University of Tartu, Tartu, Estonia. aavo-valdur.mikelsaar@ut.ee
Aavo-Valdur
AV
Mikelsaar
Alar
A
Sünter
Peeter
P
Toomik
Ruth
R
Mikelsaar
Ingrid
I
Kalev
Anu
A
Kõiveer
Andres
A
Piirsoo
Kalmer
K
Karpson
Erkki
E
Juronen
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D000918:Antibody Specificity; D002477:Cells / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002502:Centrioles / Q000187:drug effects / Q000378:metabolism*; D064211:Connectin; D000939:Epitopes; D005260:Female; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization
D000911:Antibodies, Monoclonal; D064211:Connectin; D000939:Epitopes; D009124:Muscle Proteins; D034741:RNA, Small Interfering; C577285:TTN protein, human; D011494:Protein Kinases