Purification and Physical Properties of Nematode Mini-Titins and Their Relation to Twitchin

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

RUDIGER NAVE*, DIETER FURST, UWE VINKEMEIER and KLAUS WEBER

Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, P.O. Box 2841, D-3400 Goettingen, FRG •Present address: Byk Gulden, Postfach 6500, D-7750 Konstanz, FRG

Summary

We have isolated mini-titin from the nematodes well as the pharynx of wild-type C. elegans in Ascaris lumbricoides and Caenorhabditis elegans immunofluorescence microscopy. In the twitchin under native conditions using a modification in the mutant E66 only the pharynx is decorated. We procedure to prepare this protein from insect muscle. conclude that the mini-titins of invertebrate muscles The proteins have an apparent molecular weight of defined earlier by ultrastructural criteria are very 600 000 and appear in oriented specimens as flexible likely to be twitchins, i.e. molecules necessary for thin rods with a length around 240-250 run. The normal muscle contraction. We discuss the molecular circular dichroism spectrum of the Ascaris protein is properties of the proteins in the light of the sequence dominated by /J-structure. The proteins react with established for twitchin. antibodies to insect mini-titin and also with antibodies raised against peptides contained in the sequence predicted for twitchin, the product of the Caenorhabditis elegans unc-22 gene. Antibodies to Key words: contraction, invertebrates, muscle, sarcomere, titin, insect mini-titin decorate the body musculature as twitchin.

Introduction nematodes Ascaris lumbricoides and Caenorhabditis elegans (Nave and Weber, 1990). The giant protein titin is the major component of the Recent cloning of the unc-22 gene of Caenorhabditis elastic filaments of sarcomeric muscles from vertebrates elegans predicts a body muscle protein of molecular weight (for reviews see Wang, 1985; Mamyama, 1986). Immuno- 668520 built from repetitive domains related to the electron microscopy using a bank of different monoclonal immunoglobulin superfamily. Mutants lacking the pro- antibodies has shown that the titin molecule extends from tein, which has been called twitchin, do not develop the Z-band into the M-band (Furst etol. 1988,1989). While normal body muscle contraction and small regions of the the native titin molecule is still difficult to obtain, a myofilament organization in individual cells contract defined proteolytic fragment TH, which spans the distance transiently in the absence of contraction in adjacent from the Ni line into the M line, is readily purified regions (Benian etal. 1989). In addition, a sizeable fraction (Maruyama et al. 1984; Trinick et al. 1984; Wang et al. of rabbit titin has been established by cDNA cloning 1984; Nave et al. 1989). Til seems to be a single (Labeit et al. 1990). The two cloning studies establish that polypeptide of apparent molecular weight 2.1 xlO6 to twitchin and titin belong to the expanding family of 2.4X106 (Kurzban and Wang, 1988; Nave et al. 1989). proteins that covers the immunoglobulins, cell adhesion When oriented on mica by centrifugal force, subsequent molecules and other muscle proteins such as C-protein, the metal shadowing demonstrates uniformly thin rods with a 86K protein (Einheber and Fischman, 1990), and myosin diameter of 3-4 run and a length around 900 run (Nave et light-chain kinase (Olson et al. 1990). Such molecules are al. 1989). Thus the parent titin TI probably has the same built from repetitive 100-residue domains of two distinct length as a half-sarcomere. types, designated motifs I and II. Among the collection of When flight and leg muscles of Locusta migratoria and proteins in this superfamily, titin and twitchin show other insects are subjected to a titin purification scheme, a enhanced sequence homology. protein of apparent molecular weight 0.6 x 106 is obtained. To relate the insect mini-titins characterized by their It resembles vertebrate titin in many physical-chemical ultrastructure and their counterparts denned immuno- properties, but has a length of only 260 run. In line with its logically in nematodes (Nave and Weber, 1990) with the reduced length, immunoelectron microscopy with rabbit twitchin molecule of C. elegans predicted by cDNA cloning antibodies locates the mini-titin at the I-band and the (Benian et al. 1989), we have used purification schemes for adjacent part of the A-band. Western blots and immuno- titin and mini-titin (Nave et al. 1989; Nave and Weber, fluorescence microscopy show that mini-titin is present in 1990) on the nematodes C. elegans and Ascaris. We obtain the muscles of various invertebrates, including the flexible and thin rods with a length of around 245 run, Journal of Cell Science 98, 491-496 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 491 which react with antibodies to insect mini-titin as well as Electron microscopy of single molecules with antibodies raised against two peptides from the Specimens of isolated molecules were oriented on the mica by predicted twitchin sequence. The combined results suggest centrifugation as described in detail (Nave et al. 1989). Rotary that the mini-titins of invertebrates defined ultrastructur- shadowing with tantalum/tungsten at an elevation angle of 5° ally correspond to the twitching defined functionally. and carbon at 90° was as described. Replicas were viewed with a Philips CM12 electron microscope at 80 kV. Materials and methods Results Nematodes Ascaris was obtained from pigs at the local slaughterhouse. Wild- Purification of nematode mini-titin type C. elegans (strain N2) and the unc-22-deficient mutants When the myofibrillar residue of insect muscles is (strain E66) were kindly provided by R. Schnabel (Max Planck extracted with high salt buffer, mini-titin and myosin are Institute for Developmental Biology, Tubingen, FRG) and grown as described (Brenner, 1974). solubilized. Dialysis against 80 mM KC1 leads to the precipitation of most of the myosin, and a subsequent Purification of mini-titin from body wall muscle chromatography step on DE52 provides mini-titin free of of Ascaris myosin in the flow-through fraction. Further gel-per- Body musculature of Ascaris was dissected. Tissue homogeniz- meation chromatography on TSK 6000 PW yields the pure ation, washing of myofibrils and subsequent extraction steps were protein (Nave and Weber, 1990). When the same protocol the same as those used for the isolation of minititin from Locusta is used on Ascaris body muscle, the protein obtained from migratona flight muscle (Nave and Weber, 1990). The final the DE52 column is heavily contaminated by myosin. This extract was dialyzed extensively at 4°C against buffer T (50 mM problem is overcome by substituting Q-Sepharose for Tris-HCl, pH7.9, 2mM EGTA, lmM 2-mercaptoethanol, lmM DE52. Mini-titin eluted from the column around 150 mM NaN3) containing 70 mM KC1 and clarified by centrifugation KC1 in the buffer specified in Materials and methods, (100000g, lh). The supernatant was applied to a column (1.6cmx5cm for 5g tissue) of Q-Sepharose (fast flow, Pharmacia while myosin eluted around 180 mM (Fig. 1). A number of LKB, Uppsala, Sweden) equilibrated in buffer T plus 70 mM KC1. small polypeptides present in the fractions containing The column was washed with several volumes of the same buffer, mini-titin are removed by the subsequent gel-permeation and then with buffer T containing 150 mM KC1. Fractions step on the TSK column. Here mini-titin elutes as a containing mini-titin were pooled, dialyzed against buffer T narrow peak at about 17 ml (Fig. 1, lane g), which containing 500 mM KC1 and subjected to high-resolution gel corresponds to a molecular weight of about 600 000 (for permeation chromatography (GPC) using a TSK 6000 PW column column calibration and viscosity radius calculation see (7.5mmx600mm, LKB) equilibrated with the same buffer. The column was developed at room temperature at a flow rate of Nave et al. 1989; Nave and Weber, 1990). Gel electrophor- 12mlh~1. Fractions containing pure mini-titin were pooled. The esis (Fig. 1) indicates a single polypeptide of apparent 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. mt - Antibodies Rabbit antibodies to mini-titin from Locusta migraioria 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-m- 205 - maleimidobenzoyl-/V-hydroxy8ulfosuccinimide 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 1 16 - activated by addition of 8.2 mg MBS dissolved in 300 /A water (i.e. 97 • a 40-fold molar excess of MBS over ovalbumin). After stirring for 30 min at room temperature, activated ovalbumin was separated 66 • 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 I adjusted to 7.5 with 1M K2HPO4. The samples were stirred at room temperature for 3 h and then dialyzed against PBS. A 200 ng sample of conjugated peptide was injected on days 0,14, 28,42 and a b c d e f g 56. Antipeptide antisera were affinity purified on peptide material conjugated to CNBr-activated Sepharose. Fig. 1. Purification of mini-titin from Ascaris body wall muscle monitored by gel electrophoresis (2 % to 12 % linear polyacrylamide gradient gels). Molecular weight standards Gel electrophoresis and immunological methods (205, 116, 97 and 66(xlOS), shown in lane a) and the position Gel electrophoresis on SDS-polyacrylamide gradient gels (2 % to of mini-titin (mt) are indicated on the left. A total extract of 12 % acrylamide, 0.5 % bisacrylamide) and immunoblotting were Ascaris muscle with SDS is shown in lane b. The clarified as described (Filrst et al. 1988). Samples solubilized in SDS were extract applied to the Q-Sepharose column (lane c) and the incubated at 50 °C for 15 min prior to electrophoresis. Immunoflu- fractions eluted from that column at 150 mM KC1 (lane d), orescence microscopy on frozen sections was as described (Furst et 180 mM KC1 (lane e) and 500 mM KC1 (lane f) were monitored al. 1988). In the case of C. elegans, crushed samples of wild-type by gel electrophoresis. Lane g shows mini-titin (mt) obtained and mutant animals were used. by gel permeation chromatography on a TSK 6000 PW column.

492 R. Nave et al. r

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Fig. 2. Electron-microscopic appearance of mini-titin molecules purified from Ascaris (A, C) and C. elegans (B). Molecules were oriented by centrifugation prior to rotary shadowing (see Materials and methods). Note the similar morphology of the mini-titin molecules from Ascaris (A) and C. elegans (B). Some molecules isolated from Ascaris are shown at higher magnification (C). Bars, 200 nm. 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. TW same procedure can be used on C. elegans. Here the animals were homogenized in toto in liquid nitrogen using a pestle and mortar. Although the final yield of C. elegans 5- 5- mini-titin varied, about 30 //g can be obtained from 1 g of pelleted nematodes.

£ 3 - infrastructure of single molecules and the circular dichroism spectrum of the Ascaris protein 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 180 200 220 240 260 280 300 2nm, we calculated a molecular diameter of 3-4 nm. The Length (nm) length distribution of the Ascaris protein obtained from a histogram of 400 molecules was rather narrow, yielding an Fig. 3. Contour length of the rod of miTii-t.it.in molecules average value of 245±25nm (Fig. 3). Corresponding purified from Ascaris. A histogram shows the length of individual stretched molecules obtained from 400 molecules in molecules from C. elegans showed a slightly more a field similar to that shown in Fig. 2A. The histogram yields heterogeneous appearance (Fig. 2) but, owing to the low an average length of 245±25nm. amount of protein available, no detailed analysis was made. The contour length of the molecules gave a value around 240 nm. experiment was not determined, we cannot give the As the Ascaris protein was obtained at sufficient amount of /3-structure in absolute terms. concentration, we also analyzed its circular dichroism spectrum. Fig. 4 shows that this spectrum is dominated by Immunological results relate mini-titin and twitchin /S-structure. Since the precise protein concentration in this Fig. 5 shows that the two nematode proteins react in

Characterization of nematode mini-titins 493 B

200 220 240 A (nm)

Fig. 4. Comparative circular dichroism (c.d.) spectra of Ascaris mini-titin (A) and chicken breast muscle titin Til (B). Samples were at a concentration of 0.05 mg ml"1 (A) and 0.2mgml~ (B), respectively, in 80 mM potassium phosphate buffer, pH7.9. The spectra were recorded at ambient temperature. [6] is the mean residue ellipticity, assuming a residue weight of 115.

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mt Fig. 6. Immunofluorescence and corresponding phase-contrast micrographs of frozen sections of the wild-type C. elegans (A, B) and the unc-22 gene-deficient mutant (C, D) stained with rabbit antibodies against Locusta mini-titin followed by fluorescently labeled goat anti-rabbit antibodies. Note the specific staining of the body wall musculature of C. elegans (A, B) with the epidermis and the cuticle being unstained. In the mutant, the antibody stained only the pharynx (C, D) and not the body muscle cells. Bar, 10/on.

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 a b c d e f acid sequence of C. elegans twitchin (Benian et al. 1989) to raise rabbit antibodies against defined peptides. Peptide Fig. 5. Cross-reactivity of polyclonal antibodies to mini-titin Pi with the sequence DLKWKPPADDGGAPIE is a from Locusta flight muscle with mini-titin from nematodes. consensus sequence present in various type I motifs. Lane a presents a total extract of Ascaris body wall muscle on Peptide P2 with the sequence DIWKQYYPQPVEIKHD a 2 % to 12 % gradient polyacrylamide gel. The corresponding covers residues 5130 to 5145 and lies in the putative blot with the rabbit antibodies to Locusta mini-titin is shown myosin-kinase domain (for sequences and nomenclature, in lane b. The other lanes show the reaction of the antibodies see Benian et al. 1989). Fig. 7 shows that both peptide on purified mini-titin from nematodes. Ponceau red-stained antibodies react in Western blotting experiments with the blots of the isolated protein from Ascaris are given in lane c, and from C. elegans in lane e. The corresponding blots are purified proteins from Ascaris body muscle and Locusta shown in lanes d and f, respectively. Note that in both cases a flight muscle. Interestingly, antibody Pi also detects the polypeptide doublet was detected by the antibodies. The vertebrate titin polypeptides T^ and T2 in myofibrils from position of mini-titin (mt) is marked on the left and chicken breast muscle. Antibody P2, on the other hand, did immunoblots are indicated by open circles at the top. not react with chicken titin.

494 R. Nave et al. tural aspect, which we can now use for the isolated nematode molecules. A single Ig domain of approximately i - 100 residues resembles, as shown by X-ray crystallo- graphic analysis, an ellipsoid with axes of 4nmx2.5nmx2.8nm. The polypeptide chain folds into two sheets formed by eight strands. About 50% of all residues are involved in antiparallel /S-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 /3-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 de 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 a b c as well as insect mini-titin. The occasional electron micrographs indicating local unravelling of titin TnA Fig. 7. Reactivity of antibodies to predicted sequences of C. molecules (Nave et al. 1989; see there for namenclature) elegans twitchin with mini-titin and titin. Lane a is a total could simply indicate breaks within the segment where extract of C. elegans with SDS on a 2 % to 12 % polyacrylamide the single polypeptide is displayed in eight strands (see gradient gel. Lanes b and c present the corresponding blots above). using rabbit antibodies against peptide Pi and peptide P2, respectively. Note that both antibodies specifically react with the mini-titin band (arrow). Lane d presents a total extract of The immunofluorescence microscopical results on wild- chicken breast muscle with SDS, and lane e shows the type C. elegans and the wnc-22-deficient mutant indicate corresponding immunoblot using the rabbit antibody against that the reaction of antibodies to mini-titin, although peptide Pi. Note that the antibody recognizes exclusively the abolished in the body muscle cells of the mutant, is titin doublet TI and Til (arrowhead). 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 Discussion polypeptide but not a total loss of reactivity. Preliminary experiments indicate that the reactivity in the 700000 Our results suggest that the long and flexible rod-like molecular weight range is about 10- to 20-fold reduced molecules isolated from various invertebrate muscles, (data not shown). This result is in line with the earlier which we have described in ultrastructural terms as mini- observation that a rabbit antibody raised against an unc- titins to emphasize their reduced length versus the giant 22-^S-galactosidase fusion polypeptide showed 'drastically vertebrate titins (see Results; and Nave and Weber, 1990), reduced staining of mutant animals in the body wall while are probably twitchins as defined by the cloning of the C. the pharyngeal signal is identical to wild-type' (Moerman elegans unc-22 gene (Benian et al. 1989). The mini-titins et al. 1988). Differential gene expression of structural have the circular dichroism spectrum predicted for a proteins in body wall muscle and pharynx seems a general protein rich in /3-structure (for the ^-spectrum of titin, see feature of C. elegans. Thus, for instance, of the four known also Maruyama et al. 1986). More importantly, body myosin heavy chains, two are specific for the body muscle muscle cells of the unc-22-deficient mutant of C. elegans and two restricted to the pharynx (Dibb et al. 1989). are not decorated by antibodies raised to the insect muscle After this study was completed, Lakey et al. (1990) protein. Finally, antibodies Pi and P2 raised against two reported a short stretch of amino acid sequence for a short sequences taken from the predicted protein sequence protein with an apparent molecular weight of 800 000 from of C. elegans twitchin are general reagents that detect Lethocerus muscle. The sequence clearly shows the mini-titins of various invertebrates (e.g. Ascaris and ^-structural features established for twitchin and titin. Locusta migratoria). Antibody Px reacts also with vertebrate titin. The latter result conforms nicely with the recently established moderate sequence homology of C. References elegans twitchin and rabbit titin (Labeit et al. 1990) and BENIAN, G. M , KDT, J. E., NECKELMANN, N., MOERMAN, D. G. AND the report that a monoclonal antibody to chicken breast WATERSTON, R. H. (1989). Sequence of an unusually large protein muscle titin decorates the myofibrils of Ascaris in implicated in the regulation of myosin activity in C. elegans. Nature immunoelectron microscopy (Matsuno et al. 1989). 342, 46-50. BRENNER, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, The sequence relation between twitchin and titin and 91-94. the IgG superfamily has introduced an important struc- DIBB, N. J., MARUYAMA, I. N., KBAUSE, M. AND KARN, J. (1989).

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