Abstract
Nebulin, a family of giant myofibrillar proteins of 600-900 kDa, contains a large number of highly conserved sequence repeats of 31-38 amino acids. To investigate the significance of this repeat, human skeletal muscle nebulin cDNA fragments encoding two, six, seven, eight, or fifteen repeat modules were expressed in high yield as nonfusion proteins in Escherichia coli with the pET3d plasmid vector. F-actin cosedimentation and solid phase binding assays demonstrated that all nebulin fragments, except the smallest two-module 67-mer, bound to muscle actin with high affinity under physiological ionic conditions. Solid phase binding assays also revealed that a six-module fragment, NB5, binds to myosin and C-terminal protein but fails to bind to tropomyosin, troponin, and tubulin. Furthermore, the binding of NB5 to actin was inhibited by both tropomyosin and troponin. Immunoelectron microscopic localization of NB5 indicated that this N-terminal region fragment is situated near the distal end of thin filaments in the sarcomere. These results indicate that nebulin is a giant protein with an unprecedently large number of actin-binding sites along its length and is anchored at the C terminus to the Z line in the sarcomere. Nebulin may function as a multifunctional template protein that regulates the length of thin filaments and participates in muscle activities by interacting with actin and myosin filaments in the sarcomere of skeletal muscles.
Highlights
Cloning, Expression, and Protein Interaction of Human Nebulin Fragments Composed of Varying Numbers of Sequence Modules*
To test this hypothesis further, we have cloned and excroscopic localization of NB5 indicated that this N- pressed six human nebulin fragments containing 2-15 modterminalregionfragment is situated near the distal ules in Escherichia coli using the pET3d expression vector end of thin filaments in the sarcomere
The successful cloning and expression of human nebulin fragments in E. coli has made it feasible to explore the functional significance of the sequence repeats of nebulin (Wang et al, 1990; Jin and Wang, 1991)
Summary
Expression, and Protein Interaction of Human Nebulin Fragments Composed of Varying Numbers of Sequence Modules*. AS served sequencerepeatsof 31-38 aminoacids.TO an alternative approach, we have employed molecular cloning investigate the significanceof this repeat, human skel- techniques to prepare smaller nebulin fragments for detailed etal muscle nebulin cDNA fragments encotdwinog, six, structure-function studies This approach was made possible seven, eight, or fifteen repeat moduleswere expressed by the recent cloning and sequencing studies of human skelin high yieldas nonfusion proteins inEscherichia coli etal muscle nebulin cDNAs (Stedman et al, 1988a, 198813; with the pET3d plasmid vector.F-actin cosedimenta- Zeviani et al, 1988; Wang et al, 1990). Further- building block for the putative actin-binding domains of nemore, the binding of NB5 to actin was inhibited by bulin (Wang et al, 1990;Jin and Wang, 1991) To test this hypothesis further, we have cloned and excroscopic localization of NB5 indicated that this N- pressed six human nebulin fragments containing 2-15 modterminalregionfragment is situated near the distal ules in Escherichia coli using the pET3d expression vector end of thin filaments in the sarcomere. The orifunctional template protein that regulates the length entation of nebulin polypeptides in the sarcomere was of thin filaments and participates in musclaectivities elucidated by immunoelectron microscopy with fragmentby interacting with actin and myosin filaments in the specific antibodies
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