Purification and physical properties of nematode mini-titins and their relation to twitchin

The giant protein titin is the major component of the elastic filaments of sarcomeric muscles from vertebrates (for reviews see Wang, 1985; Maruyama, 1986). Immunoelectron microscopy using a bank of different monoclonal antibodies has shown that the titin molecule extends from the Z-band into the M-band (Fürst et al. 1988,1989). While the native titin molecule is still difficult to obtain, a defined proteolytic fragment TH, which spans the distance from the N\ line into the M line, is readily purified (Maruyama et al. 1984; Trinick et al. 1984; Wang et al. 1984; Nave et al. 1989). TII seems to be a single polypeptide of apparent molecular weight 2.1×10⁶ to 2.4×10⁶; (Kurzban and Wang, 1988; Nave et al. 1989). When oriented on mica by centrifugal force, subsequent metal shadowing demonstrates uniformly thin rods with a diameter of 3–4 nm and a length around 900 nm (Nave et al. 1989). Thus the parent titin TI probably has the same length as a half-sarcomere.

When flight and leg muscles of Locusta migratoria and other insects are subjected to a titin purification scheme, a protein of apparent molecular weight 0.6 × 10⁶ is obtained. It resembles vertebrate titin in many physical-chemical properties, but has a length of only 260 nm. In line with its reduced length, immunoelectron microscopy with rabbit antibodies locates the mini-titin at the I-band and the adjacent part of the A-band. Western blots and immunofluorescence microscopy show that mini-titin is present in the muscles of various invertebrates, including the nematodes Ascaris lumbricoides and Caenorhabditis elegans (Nave and Weber, 1990).

Recent cloning of the unc-22 gene of Caenorhabditis elegans predicts a body muscle protein of molecular weight 668520 built from repetitive domains related to the immunoglobulin superfamily. Mutants lacking the protein, which has been called twitchin, do not develop normal body muscle contraction and small regions of the myofilament organization in individual cells contract transiently in the absence of contraction in adjacent regions (Benian et al. 1989). In addition, a sizeable fraction of rabbit titin has been established by cDNA cloning (Labeit et al. 1990). The two cloning studies establish that twitchin and titin belong to the expanding family of proteins that covers the immunoglobulins, cell adhesion molecules and other muscle proteins such as C-protein, the 86K protein (Einheber and Fischman, 1990), and myosin light-chain kinase (Olson et al. 1990). Such molecules are built from repetitive 100-residue domains of two distinct types, designated motifs I and II. Among the collection of proteins in this superfamily, titin and twitchin show enhanced sequence homology.

To relate the insect mini-titins characterized by their ultrastructure and their counterparts defined immunologically in nematodes (Nave and Weber, 1990) with the twitchin molecule of C. elegans predicted by cDNA cloning (Benian et al. 1989), we have used purification schemes for titin and mini-titin (Nave et al. 1989; Nave and Weber, 1990) on the nematodes C. elegans and Ascaris. We obtain flexible and thin rods with a length of around 245 nm, which react with antibodies to insect mini-titin as well as with antibodies raised against two peptides from the predicted twitchin sequence. The combined results suggest that the mini-titins of invertebrates defined ultrastructurally correspond to the twitchins defined functionally.

Ascaris was obtained from pigs at the local slaughterhouse. Wildtype C. elegans (strain N2) and the unc-22-deficient mutants (strain E66) were kindly provided by R. Schnabel (Max Planck Institute for Developmental Biology, Tübingen, FRG) and grown as described (Brenner, 1974).

Body musculature of Ascaris was dissected. Tissue homogenization, washing of myofibrils and subsequent extraction steps were the same as those used for the isolation of minititin from Locusta migratoria flight muscle (Nave and Weber, 1990). The final extract was dialyzed extensively at 4 °C against buffer T (50 mM Tris-HCl, pH7.9, 2mM EGTA, ImM 2-mercaptoethanol, ImM NaN₃) containing 70 mM KOI and clarified by centrifugation (100 000g, Ih). The supernatant was applied to a column (1.6cm×5cm for 5g tissue) of Q-Sepharose (fast flow, Pharmacia LKB, Uppsala, Sweden) equilibrated in buffer T plus 70 mM KC1. The column was washed with several volumes of the same buffer, and then with buffer T containing 150 mM KC1. Fractions containing mini-titin were pooled, dialyzed against buffer T containing 500 mM KC1 and subjected to high-resolution gel permeation chromatography (GPC) using a TSK 6000 PW column (7.5mm×600mm, LKB) equilibrated with the same buffer. The column was developed at room temperature at a flow rate of 12mlh⁻¹. Fractions containing pure mini-titin were pooled. The same procedure was also used to isolate mini-titin from the small nematode C. elegans. Animals (lg kindly provided by Dr Schnabel) were homogenized in toto in liquid nitrogen using a pestle and mortar. Subsequent steps were as above.

Rabbit antibodies to mini-titin from Locusta migratoria have been described (Nave and Weber, 1990). Rabbit antibodies to two different peptides in the published sequence of twitchin (Benian et al. 1989) were also raised. The peptides were synthesized and purified by HPLC. They were conjugated to ovalbumin (chicken egg, Sigma A-5503) via an N-terminal cysteine with sulfo-znmaleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS, Pierce no. 22312) as crosslinker (Liu et al. 1979). The modified procedure of Green et al. (1982) was used. Briefly, 22 mg of ovalbumin, dissolved in 1 ml phosphate-buffered saline (PBS) was activated by addition of 8.2 mg MBS dissolved in 300 pl water (i.e. a 40-fold molar excess of MBS over ovalbumin). After stirring for 30 min at room temperature, activated ovalbumin was separated from free MBS by gel filtration on a PD10 column (Pharmacia LKB, Uppsala, Sweden) equilibrated in 50 mM potassium phosphate, pH 6.0. Peptides dissolved in PBS were added in a 40-fold molar excess over ovalbumin and the pH of the solution was adjusted to 7.5 with IM K₂HPO₄. The samples were stirred at room temperature for 3 h and then dialyzed against PBS. A 200 μg sample of conjugated peptide was injected on days 0,14, 28,42 and 56. Antipeptide antisera were affinity purified on peptide material conjugated to CNBr-activated Sepharose.

Gel electrophoresis on SDS-polyacrylamide gradient gels (2 % to 12 % acrylamide, 0.5 % bisacrylamide) and immunoblotting were as described (Fürst et al. 1988). Samples solubilized in SDS were incubated at 50°C for 15 min prior to electrophoresis. Immunofluorescence microscopy on frozen sections was as described (Fürst et al. 1988). In the case of C. elegans, crushed samples of wild-type and mutant animals were used.

Specimens of isolated molecules were oriented on the mica by centrifugation as described in detail (Nave et al. 1989). Rotary shadowing with tantalum/tungsten at an elevation angle of 5° and carbon at 90° was as described. Replicas were viewed with a Philips CM12 electron microscope at 80 kV.

When the myofibrillar residue of insect muscles is extracted with high salt buffer, mini-titin and myosin are solubilized. Dialysis against 80 mM KC1 leads to the precipitation of most of the myosin, and a subsequent chromatography step on DE52 provides mini-titin free of myosin in the flow-through fraction. Further gel-permeation chromatography on TSK 6000 PW yields the pure protein (Nave and Weber, 1990). When the same protocol is used on Ascaris body muscle, the protein obtained from the DE52 column is heavily contaminated by myosin. This problem is overcome by substituting Q-Sepharose for DE52. Mini-titin eluted from the column around 150 mM KC1 in the buffer specified in Materials and methods, while myosin eluted around 180 mM (Fig. 1). A number of small polypeptides present in the fractions containing mini-titin are removed by the subsequent gel-permeation step on the TSK column. Here mini-titin elutes as a narrow peak at about 17 ml (Fig. 1, lane g), which corresponds to a molecular weight of about 600 000 (for column calibration and viscosity radius calculation see Nave et al. 1989; Nave and Weber, 1990). Gel electrophoresis (Fig. 1) indicates a single polypeptide of apparent molecular weight around 600000 (see also the Western blots in Fig. 1 of Nave and Weber, 1990). Up to 0.6 mg of pure protein can be obtained from 1 g of body muscle. The same procedure can be used on C. elegans. Here the animals were homogenized in tofo in liquid nitrogen using a pestle and mortar. Although the final yield of C. elegans mini-titin varied, about 30 μg can be obtained from 1g of pelleted nematodes.

The nematode proteins were applied to mica, oriented by centrifugation (Nave et al. 1989) and subjected to metal shadowing. Fig. 2 shows that the individual molecules are visualized as long flexible rods of a uniform diameter. Using the known diameter of the myosin rod domain of 2 run, we calculated a molecular diameter of 3–4 nm. The length distribution of the Ascaris protein obtained from a histogram of 460 molecules was rather narrow, yielding an average value of 245 ±25nm (Fig. 3). Corresponding molecules from C. elegans showed a slightly more heterogeneous appearance (Fig. 2) but, owing to the low amount of protein available, no detailed analysis was made. The contour length of the molecules gave a value around 240 nm.

As the Ascaris protein was obtained at sufficient concentration, we also analyzed its circular dichroism spectrum. Fig. 4 shows that this spectrum is dominated by βstructure. Since the precise protein concentration in this experiment was not determined, we cannot give the amount of β-structure in absolute terms.

Fig. 5 shows that the two nematode proteins react in Western blotting experiments with the rabbit antibodies against insect mini-titin that were previously characterized (Nave and Weber, 1990). In immunofluorescence microscopy these antibodies decorated both the body musculature and the pharynx of wild-type C. elegans (Fig. 6). In contrast, the unc-22-deficient mutant (strain E66) lacked reactivity of the body muscle cells, while the pharynx retained decoration.

Since the combined results are in line with the possibility that mini-titin and twitchin are the same or very similar molecules, we have used the predicted amino acid sequence of C. elegans twitchin (Benian et al. 1989) to raise rabbit antibodies against defined peptides. Peptide P₁ with the sequence DLKWKPPADDGGAPIE is a consensus sequence present in various type I motifs. Peptide P₂ with the sequence DIWKQYYPQPVEIKHD covers residues 5130 to 5145 and lies in the putative myosin-kinase domain (for sequences and nomenclature, see Benian et al. 1989). Fig. 7 shows that both peptide antibodies react in Western blotting experiments with the purified proteins from Ascaris body muscle and Locusta flight muscle. Interestingly, antibody P₁ also detects the vertebrate titin polypeptides T₁ and T₂ in myofibrils from chicken breast muscle. Antibody P2 on the other hand, did not react with chicken titin.

Our results suggest that the long and flexible rod-like molecules isolated from various invertebrate muscles, which we have described in ultrastructural terms as minititins to emphasize their reduced length versus the giant vertebrate titins (see Results; and Nave and Weber, 1990), are probably twitchins as defined by the cloning of the C. elegans unc-22 gene (Benian et al. 1989). The mini-titins have the circular dichroism spectrum predicted for a protein rich in β-structure (for the β-spectrum of titin, see also Maruyama et al. 1986). More importantly, body muscle cells of the unc-22-deficient mutant of C. elegans are not decorated by antibodies raised to the insect muscle protein. Finally, antibodies P₁ and P₂ raised against two short sequences taken from the predicted protein sequence of C. elegans twitchin are general reagents that detect mini-titins of various invertebrates (e.g. Ascaris and Locusta migratoria). Antibody P₁ reacts also with vertebrate titin. The latter result conforms nicely with the recently established moderate sequence homology of C. elegans twitchin and rabbit titin (Labeit et al. 1990) and the report that a monoclonal antibody to chicken breast muscle titin decorates the myofibrils of Ascaris in immunoelectron microscopy (Matsuno et al. 1989).

The sequence relation between twitchin and titin and the IgG superfamily has introduced an important structural aspect, which we can now use for the isolated nematode molecules. A single Ig domain of approximately 100 residues resembles, as shown by X-ray crystallographic analysis, an ellipsoid with axes of 4nm×2.5nm×2.8nm. The polypeptide chain folds into two sheets formed by eight strands. About 50% of all residues are involved in antiparallel β-pleated sheet formation (see, for instance, Epp et al. 1974). Since N and C termini of each domain are located at opposite ends of the long axis, repetitive segments can easily be envisaged as forming a long rod. With 31 copies of motif I and 26 copies of motif II in the C. elegans twitchin sequence (Benian et al. 1989), one expects a length of at least 230 nm, since this value does not include the interspersed region resembling the catalytic domain of myosin light-chain kinase. Since this region of some 600 residues is again dominated by β-structure, our measured molecular length values of 240–245 nm for nematode twitchins are in good agreement with possible predictions from the sequence. The measured thickness of the rods with a value of 3–4 nm is based on corrections necessary for metal decoration. These have been done using the myosin rod as standard (see also Nave et al. 1990). Given the dimension of the repeating 100-residue domains (see above), the molecular diameter supports the argument for single monomers rather than a dimeric character for the isolated molecule. This is in line with previous hydrodynamic measurements made on titin as well as insect mini-titin. The occasional electron micrographs indicating local unravelling of titin Tn_A molecules (Nave et al. 1989; see there for nomenclature) could simply indicate breaks within the segment where the single polypeptide is displayed in eight strands (see above).

The immunofluorescence microscopical results on wildtype C. elegans and the wzic-22-deficient mutant indicate that the reaction of antibodies to mini-titin, although abolished in the body muscle cells of the mutant, is retained in the pharynx muscle cells. In line with this result we find, from immunoblotting studies of full mutant organisms, a strong reduction in the antibody-reactive polypeptide but not a total loss of reactivity. Preliminary experiments indicate that the reactivity in the 700000 molecular weight range is about 10- to 20-fold reduced (data not shown). This result is in line with the earlier observation that a rabbit antibody raised against an unc-22-β-galactosidase fusion polypeptide showed ‘drastically reduced staining of mutant animals in the body wall while the pharyngeal signal is identical to wild-type’ (Moerman et al. 1988). Differential gene expression of structural proteins in body wall muscle and pharynx seems a general feature of C. elegans. Thus, for instance, of the four known myosin heavy chains, two are specific for the body muscle and two restricted to the pharynx (Dibb et al. 1989).

After this study was completed, Lakey et al. (1990) reported a short stretch of amino acid sequence for a protein with an apparent molecular weight of 800 000 from Lethocerus muscle. The sequence clearly shows the fl-structural features established for twitchin and titin.