Characterization of amphioxus nebulin and its similarity to human nebulin.
Identification of a large molecule in muscle is important but difficult to approach by protein chemistry. In this study we isolated nebulin cDNA from the striated muscle of amphioxus, and characterized the C-terminal regions of nebulins from other chordates. Although the sequence homology with that of human is only 26%, the C-terminal region of amphioxus nebulin has similar structural motifs of 35 amino acid nebulin repeats and an SH3 domain. Using in situ indirect immunofluorescence analysis with a specific antibody raised to the bacterially produced recombinant peptide, we identified that this nebulin fragment is located in the Z-line of the sarcomere, similar to human nebulin. Pull-down and co-sedimentation assays in vitro showed that the C-terminal region binds to actin, alpha-actinin and connectin (titin). These results suggest that the C-terminal region of amphioxus nebulin plays a similar role in maintaining striated muscle structure to that of human nebulin. This is the first report of the exact location of nebulin in amphioxus muscle.
Hanashima
Akira
A
Department of Biology, Graduate School of Science, Chiba University, Chiba 263-8522, Japan.
Kubokawa
Kaoru
K
Kimura
Sumiko
S
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002816:Chordata, Nonvertebrate / Q000235:genetics*; D003001:Cloning, Molecular; D064211:Connectin; D006801:Humans; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D012518:Sarcomeres / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D000199:Actins; D064211:Connectin; D009124:Muscle Proteins; C577285:TTN protein, human; C035620:nebulin; D011494:Protein Kinases