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 nematodesAscaris lumbricoides and Caenorhabditis elegansunder native conditions using a modification in theprocedure to prepare this protein from insect muscle.The proteins have an apparent molecular weight of600 000 and appear in oriented specimens as flexiblethin rods with a length around 240-250 run. Thecircular dichroism spectrum of the Ascaris protein isdominated by /J-structure. The proteins react withantibodies to insect mini-titin and also with anti-bodies raised against peptides contained in thesequence predicted for twitchin, the product of theCaenorhabditis elegans unc-22 gene. Antibodies toinsect mini-titin decorate the body musculature as

well as the pharynx of wild-type C. elegans inimmunofluorescence microscopy. In the twitchinmutant E66 only the pharynx is decorated. Weconclude that the mini-titins of invertebrate musclesdefined earlier by ultrastructural criteria are verylikely to be twitchins, i.e. molecules necessary fornormal muscle contraction. We discuss the molecularproperties of the proteins in the light of the sequenceestablished for twitchin.

Key words: contraction, invertebrates, muscle, sarcomere, titin,twitchin.

Introduction

The giant protein titin is the major component of theelastic filaments of sarcomeric muscles from vertebrates(for reviews see Wang, 1985; Mamyama, 1986). Immuno-electron microscopy using a bank of different monoclonalantibodies has shown that the titin molecule extends fromthe Z-band into the M-band (Furst etol. 1988,1989). Whilethe native titin molecule is still difficult to obtain, adefined proteolytic fragment TH, which spans the distancefrom the Ni line into the M line, is readily purified(Maruyama et al. 1984; Trinick et al. 1984; Wang et al.1984; Nave et al. 1989). Til seems to be a singlepolypeptide of apparent molecular weight 2.1 xlO6 to2.4X106 (Kurzban and Wang, 1988; Nave et al. 1989).When oriented on mica by centrifugal force, subsequentmetal shadowing demonstrates uniformly thin rods with adiameter of 3-4 run and a length around 900 run (Nave etal. 1989). Thus the parent titin TI probably has the samelength as a half-sarcomere.

When flight and leg muscles of Locusta migratoria andother insects are subjected to a titin purification scheme, aprotein of apparent molecular weight 0.6 x 106 is obtained.It resembles vertebrate titin in many physical-chemicalproperties, but has a length of only 260 run. In line with itsreduced length, immunoelectron microscopy with rabbitantibodies locates the mini-titin at the I-band and theadjacent part of the A-band. Western blots and immuno-fluorescence microscopy show that mini-titin is present inthe muscles of various invertebrates, including theJournal of Cell Science 98, 491-496 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

nematodes Ascaris lumbricoides and Caenorhabditiselegans (Nave and Weber, 1990).

Recent cloning of the unc-22 gene of Caenorhabditiselegans predicts a body muscle protein of molecular weight668520 built from repetitive domains related to theimmunoglobulin superfamily. Mutants lacking the pro-tein, which has been called twitchin, do not developnormal body muscle contraction and small regions of themyofilament organization in individual cells contracttransiently in the absence of contraction in adjacentregions (Benian etal. 1989). In addition, a sizeable fractionof rabbit titin has been established by cDNA cloning(Labeit et al. 1990). The two cloning studies establish thattwitchin and titin belong to the expanding family ofproteins that covers the immunoglobulins, cell adhesionmolecules and other muscle proteins such as C-protein, the86K protein (Einheber and Fischman, 1990), and myosinlight-chain kinase (Olson et al. 1990). Such molecules arebuilt from repetitive 100-residue domains of two distincttypes, designated motifs I and II. Among the collection ofproteins in this superfamily, titin and twitchin showenhanced sequence homology.

To relate the insect mini-titins characterized by theirultrastructure and their counterparts denned immuno-logically in nematodes (Nave and Weber, 1990) with thetwitchin molecule of C. elegans predicted by cDNA cloning(Benian et al. 1989), we have used purification schemes fortitin and mini-titin (Nave et al. 1989; Nave and Weber,1990) on the nematodes C. elegans and Ascaris. We obtainflexible and thin rods with a length of around 245 run,

491

which react with antibodies to insect mini-titin as well aswith antibodies raised against two peptides from thepredicted twitchin sequence. The combined results suggestthat the mini-titins of invertebrates defined ultrastructur-ally correspond to the twitching defined functionally.

Materials and methods

NematodesAscaris was obtained from pigs at the local slaughterhouse. Wild-type C. elegans (strain N2) and the unc-22-deficient mutants(strain E66) were kindly provided by R. Schnabel (Max PlanckInstitute for Developmental Biology, Tubingen, FRG) and grownas described (Brenner, 1974).

Purification of mini-titin from body wall muscleof AscarisBody musculature of Ascaris was dissected. Tissue homogeniz-ation, washing of myofibrils and subsequent extraction steps werethe same as those used for the isolation of minititin from Locustamigratona flight muscle (Nave and Weber, 1990). The finalextract was dialyzed extensively at 4°C against buffer T (50 mMTris-HCl, pH7.9, 2mM EGTA, lmM 2-mercaptoethanol, lmMNaN3) containing 70 mM KC1 and clarified by centrifugation(100000g, lh). The supernatant was applied to a column(1.6cmx5cm for 5g tissue) of Q-Sepharose (fast flow, PharmaciaLKB, 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. Fractionscontaining mini-titin were pooled, dialyzed against buffer Tcontaining 500 mM KC1 and subjected to high-resolution gelpermeation chromatography (GPC) using a TSK 6000 PW column(7.5mmx600mm, LKB) equilibrated with the same buffer. Thecolumn was developed at room temperature at a flow rate of12mlh~1. Fractions containing pure mini-titin were pooled. Thesame procedure was also used to isolate mini-titin from the smallnematode C. elegans. Animals ( lg kindly provided by DrSchnabel) were homogenized in toto in liquid nitrogen using apestle and mortar. Subsequent steps were as above.

AntibodiesRabbit antibodies to mini-titin from Locusta migraioria have beendescribed (Nave and Weber, 1990). Rabbit antibodies to twodifferent peptides in the published sequence of twitchin (Benian etal. 1989) were also raised. The peptides were synthesized andpurified by HPLC. They were conjugated to ovalbumin (chickenegg, Sigma A-5503) via an N-terminal cysteine with sulfo-m-maleimidobenzoyl-/V-hydroxy8ulfosuccinimide ester (sulfo-MBS,Pierce no. 22312) as crosslinker (Liu et al. 1979). The modifiedprocedure of Green et al. (1982) was used. Briefly, 22 mg ofovalbumin, dissolved in 1 ml phosphate-buffered saline (PBS) wasactivated by addition of 8.2 mg MBS dissolved in 300 /A water (i.e.a 40-fold molar excess of MBS over ovalbumin). After stirring for30 min at room temperature, activated ovalbumin was separatedfrom free MBS by gel filtration on a PD10 column (PharmaciaLKB, Uppsala, Sweden) equilibrated in 50 mM potassium phos-phate, pH 6.0. Peptides dissolved in PBS were added in a 40-foldmolar excess over ovalbumin and the pH of the solution wasadjusted to 7.5 with 1 M K2HPO4. The samples were stirred atroom temperature for 3 h and then dialyzed against PBS. A 200 ngsample of conjugated peptide was injected on days 0,14, 28,42 and56. Antipeptide antisera were affinity purified on peptidematerial conjugated to CNBr-activated Sepharose.

Gel electrophoresis and immunological methodsGel electrophoresis on SDS-polyacrylamide gradient gels (2 % to12 % acrylamide, 0.5 % bisacrylamide) and immunoblotting wereas described (Filrst et al. 1988). Samples solubilized in SDS wereincubated at 50 °C for 15 min prior to electrophoresis. Immunoflu-orescence microscopy on frozen sections was as described (Furst etal. 1988). In the case of C. elegans, crushed samples of wild-typeand mutant animals were used.

Electron microscopy of single moleculesSpecimens of isolated molecules were oriented on the mica bycentrifugation as described in detail (Nave et al. 1989). Rotaryshadowing with tantalum/tungsten at an elevation angle of 5°and carbon at 90° was as described. Replicas were viewed with aPhilips CM12 electron microscope at 80 kV.

Results

Purification of nematode mini-titinWhen the myofibrillar residue of insect muscles isextracted with high salt buffer, mini-titin and myosin aresolubilized. Dialysis against 80 mM KC1 leads to theprecipitation of most of the myosin, and a subsequentchromatography step on DE52 provides mini-titin free ofmyosin in the flow-through fraction. Further gel-per-meation chromatography on TSK 6000 PW yields the pureprotein (Nave and Weber, 1990). When the same protocolis used on Ascaris body muscle, the protein obtained fromthe DE52 column is heavily contaminated by myosin. Thisproblem is overcome by substituting Q-Sepharose forDE52. Mini-titin eluted from the column around 150 mMKC1 in the buffer specified in Materials and methods,while myosin eluted around 180 mM (Fig. 1). A number ofsmall polypeptides present in the fractions containingmini-titin are removed by the subsequent gel-permeationstep on the TSK column. Here mini-titin elutes as anarrow peak at about 17 ml (Fig. 1, lane g), whichcorresponds to a molecular weight of about 600 000 (forcolumn calibration and viscosity radius calculation seeNave et al. 1989; Nave and Weber, 1990). Gel electrophor-esis (Fig. 1) indicates a single polypeptide of apparent

mt -

205 -

1 16 -97 •

6 6 • Ia b c d e f g

Fig. 1. Purification of mini-titin from Ascaris body wallmuscle monitored by gel electrophoresis (2 % to 12 % linearpolyacrylamide gradient gels). Molecular weight standards(205, 116, 97 and 66(xlOS), shown in lane a) and the positionof mini-titin (mt) are indicated on the left. A total extract ofAscaris muscle with SDS is shown in lane b. The clarifiedextract applied to the Q-Sepharose column (lane c) and thefractions eluted from that column at 150 mM KC1 (lane d),180 mM KC1 (lane e) and 500 mM KC1 (lane f) were monitoredby gel electrophoresis. Lane g shows mini-titin (mt) obtainedby gel permeation chromatography on a TSK 6000 PW column.

492 R. Nave et al.

r

. : ( ' . : • • .

2A *i>&f

. * • : • f

Fig. 2. Electron-microscopic appearance of mini-titin molecules purified from Ascaris (A, C) and C. elegans (B). Molecules wereoriented by centrifugation prior to rotary shadowing (see Materials and methods). Note the similar morphology of the mini-titinmolecules 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 Westernblots in Fig. 1 of Nave and Weber, 1990). Up to 0.6 mg ofpure protein can be obtained from 1 g of body muscle. TWsame procedure can be used on C. elegans. Here theanimals were homogenized in toto in liquid nitrogen usinga pestle and mortar. Although the final yield of C. elegansmini-titin varied, about 30 //g can be obtained from 1 g ofpelleted nematodes.

5- 5 -

£ 3 -

infrastructure of single molecules and the circulardichroism spectrum of the Ascaris proteinThe nematode proteins were applied to mica, oriented bycentrifugation (Nave et al. 1989) and subjected to metalshadowing. Fig. 2 shows that the individual molecules arevisualized as long flexible rods of a uniform diameter.Using the known diameter of the myosin rod domain of2nm, we calculated a molecular diameter of 3-4 nm. Thelength distribution of the Ascaris protein obtained from ahistogram of 400 molecules was rather narrow, yielding anaverage value of 245±25nm (Fig. 3). Correspondingmolecules from C. elegans showed a slightly moreheterogeneous appearance (Fig. 2) but, owing to the lowamount of protein available, no detailed analysis wasmade. The contour length of the molecules gave a valuearound 240 nm.

As the Ascaris protein was obtained at sufficientconcentration, we also analyzed its circular dichroismspectrum. Fig. 4 shows that this spectrum is dominated by/S-structure. Since the precise protein concentration in this

180 200 220 240 260 280 300Length (nm)

Fig. 3. Contour length of the rod of miTii-t.it.in moleculespurified from Ascaris. A histogram shows the length ofindividual stretched molecules obtained from 400 molecules ina field similar to that shown in Fig. 2A. The histogram yieldsan average length of 245±25nm.

experiment was not determined, we cannot give theamount of /3-structure in absolute terms.

Immunological results relate mini-titin and twitchinFig. 5 shows that the two nematode proteins react in

Characterization of nematode mini-titins 493

200 220 240A (nm)

Fig. 4. Comparative circular dichroism (c.d.) spectra of Ascarismini-titin (A) and chicken breast muscle titin Til (B). Sampleswere 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 themean residue ellipticity, assuming a residue weight of 115.

o o o

mt

B

Fig. 6. Immunofluorescence and corresponding phase-contrastmicrographs of frozen sections of the wild-type C. elegans (A,B) and the unc-22 gene-deficient mutant (C, D) stained withrabbit antibodies against Locusta mini-titin followed byfluorescently labeled goat anti-rabbit antibodies. Note thespecific staining of the body wall musculature of C. elegans (A,B) with the epidermis and the cuticle being unstained. In themutant, the antibody stained only the pharynx (C, D) and notthe body muscle cells. Bar, 10/on.

•a b c d e f

Fig. 5. Cross-reactivity of polyclonal antibodies to mini-titinfrom Locusta flight muscle with mini-titin from nematodes.Lane a presents a total extract of Ascaris body wall muscle ona 2 % to 12 % gradient polyacrylamide gel. The correspondingblot with the rabbit antibodies to Locusta mini-titin is shownin lane b. The other lanes show the reaction of the antibodieson purified mini-titin from nematodes. Ponceau red-stainedblots of the isolated protein from Ascaris are given in lane c,and from C. elegans in lane e. The corresponding blots areshown in lanes d and f, respectively. Note that in both cases apolypeptide doublet was detected by the antibodies. Theposition of mini-titin (mt) is marked on the left andimmunoblots are indicated by open circles at the top.

Western blotting experiments with the rabbit antibodiesagainst insect mini-titin that were previously character-ized (Nave and Weber, 1990). In immunofluorescencemicroscopy these antibodies decorated both the bodymusculature and the pharynx of wild-type C. elegans(Fig. 6). In contrast, the unc-22-deficient mutant (strainE66) lacked reactivity of the body muscle cells, while thepharynx retained decoration.

Since the combined results are in line with thepossibility that mini-titin and twitchin are the same orvery similar molecules, we have used the predicted aminoacid sequence of C. elegans twitchin (Benian et al. 1989) toraise rabbit antibodies against defined peptides. PeptidePi with the sequence DLKWKPPADDGGAPIE is aconsensus sequence present in various type I motifs.Peptide P2 with the sequence DIWKQYYPQPVEIKHDcovers residues 5130 to 5145 and lies in the putativemyosin-kinase domain (for sequences and nomenclature,see Benian et al. 1989). Fig. 7 shows that both peptideantibodies react in Western blotting experiments with thepurified proteins from Ascaris body muscle and Locustaflight muscle. Interestingly, antibody Pi also detects thevertebrate titin polypeptides T^ and T2 in myofibrils fromchicken breast muscle. Antibody P2, on the other hand, didnot react with chicken titin.

494 R. Nave et al.

i -

de

a b cFig. 7. Reactivity of antibodies to predicted sequences of C.elegans twitchin with mini-titin and titin. Lane a is a totalextract of C. elegans with SDS on a 2 % to 12 % polyacrylamidegradient gel. Lanes b and c present the corresponding blotsusing rabbit antibodies against peptide Pi and peptide P2,respectively. Note that both antibodies specifically react withthe mini-titin band (arrow). Lane d presents a total extract ofchicken breast muscle with SDS, and lane e shows thecorresponding immunoblot using the rabbit antibody againstpeptide Pi. Note that the antibody recognizes exclusively thetitin doublet TI and Til (arrowhead).

Discussion

Our results suggest that the long and flexible rod-likemolecules isolated from various invertebrate muscles,which we have described in ultrastructural terms as mini-titins to emphasize their reduced length versus the giantvertebrate 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-titinshave the circular dichroism spectrum predicted for aprotein rich in /3-structure (for the ^-spectrum of titin, seealso Maruyama et al. 1986). More importantly, bodymuscle cells of the unc-22-deficient mutant of C. elegansare not decorated by antibodies raised to the insect muscleprotein. Finally, antibodies Pi and P2 raised against twoshort sequences taken from the predicted protein sequenceof C. elegans twitchin are general reagents that detectmini-titins of various invertebrates (e.g. Ascaris andLocusta migratoria). Antibody Px reacts also with ver-tebrate titin. The latter result conforms nicely with therecently established moderate sequence homology of C.elegans twitchin and rabbit titin (Labeit et al. 1990) andthe report that a monoclonal antibody to chicken breastmuscle titin decorates the myofibrils of Ascaris inimmunoelectron microscopy (Matsuno et al. 1989).

The sequence relation between twitchin and titin andthe IgG superfamily has introduced an important struc-

tural aspect, which we can now use for the isolatednematode molecules. A single Ig domain of approximately100 residues resembles, as shown by X-ray crystallo-graphic analysis, an ellipsoid with axes of4nmx2.5nmx2.8nm. The polypeptide chain folds intotwo sheets formed by eight strands. About 50% of allresidues are involved in antiparallel /S-pleated sheetformation (see, for instance, Epp et al. 1974). Since N and Ctermini of each domain are located at opposite ends of thelong axis, repetitive segments can easily be envisaged asforming a long rod. With 31 copies of motif I and 26 copiesof motif II in the C. elegans twitchin sequence (Benian etal. 1989), one expects a length of at least 230 nm, since thisvalue does not include the interspersed region resemblingthe catalytic domain of myosin light-chain kinase. Sincethis region of some 600 residues is again dominated by/3-structure, our measured molecular length values of240-245 nm for nematode twitchins are in good agreementwith possible predictions from the sequence. Themeasured thickness of the rods with a value of 3-4 nm isbased on corrections necessary for metal decoration. Thesehave been done using the myosin rod as standard (see alsoNave et al. 1990). Given the dimension of the repeating100-residue domains (see above), the molecular diametersupports the argument for single monomers rather than adimeric character for the isolated molecule. This is in linewith previous hydrodynamic measurements made on titinas well as insect mini-titin. The occasional electronmicrographs indicating local unravelling of titin TnAmolecules (Nave et al. 1989; see there for namenclature)could simply indicate breaks within the segment wherethe single polypeptide is displayed in eight strands (seeabove).

The immunofluorescence microscopical results on wild-type C. elegans and the wnc-22-deficient mutant indicatethat the reaction of antibodies to mini-titin, althoughabolished in the body muscle cells of the mutant, isretained in the pharynx muscle cells. In line with thisresult we find, from immunoblotting studies of full mutantorganisms, a strong reduction in the antibody-reactivepolypeptide but not a total loss of reactivity. Preliminaryexperiments indicate that the reactivity in the 700000molecular weight range is about 10- to 20-fold reduced(data not shown). This result is in line with the earlierobservation that a rabbit antibody raised against an unc-22-^S-galactosidase fusion polypeptide showed 'drasticallyreduced staining of mutant animals in the body wall whilethe pharyngeal signal is identical to wild-type' (Moermanet al. 1988). Differential gene expression of structuralproteins in body wall muscle and pharynx seems a generalfeature of C. elegans. Thus, for instance, of the four knownmyosin heavy chains, two are specific for the body muscleand 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 aprotein with an apparent molecular weight of 800 000 fromLethocerus muscle. The sequence clearly shows the^-structural features established for twitchin and titin.

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(Received 23 November 1990 - Accepted 16 January 1991)

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