Mammalian skeletal muscle C-protein: purification from bovine muscle, binding to titin and the characterization of a full-length human cDNA

DIETER O. FURST UWE VINKEMEIER KLAUS WEBER binding to titin and the characterization of a full-length human cDNA - We report a fast method for the isolation of homogeneous C-protein from bovine skeletal muscle. In electron micrographs C-protein appears as short rods with a relatively uniform length of about 50 nm. Protein sequencing shows a single N-terminal sequence. Radiolabelled C-protein strongly decorates titin II and myosin rods but not myosin heads. Binding to titin II is retained in preparations lacking titin-associated proteins. Antibodies to bovine C-protein were used to screen a Agtll cDNA library constructed from fetal human skeletal muscle. Clone HC38 is 3833 bp long and encodes a protein of 1138 amino acid residues. The start of the predicted sequence fits the N-terminal sequence of the bovine protein. All partial sequences obtained from the bovine protein (348 residues) and the sequence deduced from a partial chicken cDNA (Einheber and Fischman, 1990) can be aligned along the human sequence. The sequences of human and chicken C-proteins share 50% identity and 70% similarity. Along the repeat patterns of the human protein the fibronectin (Fn)-like domains are better conserved than the immunoglobulin (Ig)-like domains. Regions of strong divergence between chicken fast C-protein and human slow C-protein may represent differences in C-protein isoforms. Titin is an important component of the myofibril (for reviews see Wang, 1985; Maruyama, 1986; Trinick, 1991). Immunoelectron microscopy with a bank of 14 monoclonal antibodies shows that titin molecules have half-sarcomere length and span the distance from the Z band to the M band (Flirst et al., 1988, 1989). Isolated titin II molecules have a narrow length distribution of about 900 nm but lack the Z-band anchoring domain due to the proteolysis that is necessary to extract the native molecules. Purified titin II molecules are a string carrying a single globular head. This seems to reflect two M band proteins, which have been described as titin-associated proteins (Nave et al., 1989). On the basis of these structural results one can discern functionally distinct parts of the titin molecule. The Zband binding region is currently not directly available to molecular analysis. Over the I band portion titin molecules show high elasticity (Maruyama et al., 1985; Furst et al., 1988; Itoh et al., 1988; Whiting et al., 1989) and are then arranged parallel to the A band (Trinick et al., 1984). Finally the molecules end within the M band where they seem firmly embedded via specific M-band proteins (Nave et al., 1989). cDNA cloning of a part of titin situated in the A band has shown a regular pattern of 100-residue repeats, which reflect similar domains in immunoglobulins (class II domains) and fibronectin (class I domains) (Labeit et al., 1990). These 100residue repeats probably explain certain electron micrographs indicating that titin is built from a linear array of 4.3 nm globular domains (Trinick et al., 1984; Whiting et al., 1989). Titin molecules seem to have an additional repeat pattern of 42 nm, which may be important in understanding the titin-A band disposition. Several monoclonal antibodies identify in immunoelectron microscopy a 42 to 43 nm repeat pattern in the A band (Furst et al., 1989). These repetitive epitopes seem to coincide with some of the striations of the A band known to harbor two myosin-associated proteins: C-protein and 86K protein (Sjostrom and Squire, 1977; Craig and Offer, 1976; Dennis et al., 1984; Bahler et al., 1985a,b; Furst et al., 1989). C-protein has been characterized extensively as a myosin-associated protein recognizing the rod portion of the myosin molecule (Moos et al., 1975; Starr and Offer, 1978) and its disposition along the A band has been well characterized (Craig and Offer, 1976; Dennis et al., 1984; Bennett et al., 1986). Several reports indicate that C-protein could play a role in modulating muscle contraction (Offer et al., 1973; Moos et al., 1978; Moos and Feng, 1980; Hartzell and Titus, 1982) or D. O. Furst and others in thick filament assembly (Offer et al., 1973). Our results on some repetitive titin epitopes opened the possibility that C-protein could connect as a missing link the titin strings at multiple sites to the A band (Furst et al., 1989). During our studies on C-protein and its possible interaction with titin, Einheber and Fischman (1990) reported a partial cDNA clone encoding about 80% of the fast isoform of chicken C-protein. Both Cprotein and titin belong to a superfamily of proteins built from domains that share sequence similarity with immunoglobulin (class II domains) and fibronectin (type I domains). Here we describe a fast and convenient procedure for the purification of C-protein from bovine slow muscle, which has facilitated the physical-chemical characterization of the molecule. We report a strong and specific binding of radiolabelled C-protein to titin and myosin rod and provide a complete C-protein sequence deduced from a human cDNA clone. Materials and methods Purification of bovine skeletal muscle C-protein Bovine muscle (Musculus iliacus) was removed immediately after slaughter. It was chopped into small pieces, quickly frozen in liquid nitrogen and stored at 70C. After quick thawing of 100 g of this material in ice-cold LSB (low salt buffer: 100 mM KC1, 2 mM MgCl2, 5 mM EGTA, 1 mM 2mercaptoethanol, 1 mM NaN3, 10 mM Tris-maleate, pH 6.8) containing 2 mM Na2P2O7% the tissue was homogenized for 2 x 30 s with a Polytron homogenizer. The following protease inhibitors were present in LSB and all subsequent buffers: 1 mM PMSF, 10 mg/ml trypsin inhibitor II (T9253, Sigma Chemical Co., St. Louis, MO, USA), and 5 mM E-64 (E3132, Sigma). Myofibrils were harvested (15 min at 3,000g), washed 3 times with LSB, and resuspended in extraction solution (0.6 M KC1, 2 mM MgCl2, 2 mM EGTA, 1 mM 2-mercaptoethanol, 1 mM NaN3, 10 mM imidazole-HCl, pH 7.0) for 35 min. The supernatant obtained after centrifugation (20,000 g for 50 min) was extensively dialyzed against buffer A (2 mM EGTA, 1 mM 2-mercaptoethanol, 1 mM NaN3, 50 mM Tris-HCl, pH 7.9) containing 70 mM KG and subsequently clarified by centrifugation (100,000 g for 50 min). To the supernatant a saturated ammonium sulfate solution was slowly added to 40% saturation. After stirring for a further 20 min and centrifugation (15,000 g, 15 min) the resulting pellet was discarded and solid ammonium sulfate was added to the supernatant to 60% saturation. The pellet obtained after centrifugation (15,000 g for 15 min) was dissolved in about 15 ml of buffer A containing 300 mM KG and immediately desalted on a Sephadex G-25 column (50 cm x 1.5 cm, equilibrated in buffer A containing 70 mM KG). The proteincontaining fractions were incubated for 30 min with 20 g of DEAE-cellulose (DE-52, Whatman Biosystems Ltd, Maidstone, England) equilibrated in the same buffer. The unbound protein fraction obtained after a short (...truncated)