©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloned Rat Cardiac Titin Class I and Class II Motifs
EXPRESSION, PURIFICATION, CHARACTERIZATION, AND INTERACTION WITH F-ACTIN (*)

(Received for publication, November 16, 1994; and in revised form, January 4, 1995)

Jian-Ping Jin (§)

From the Department of Medical Biochemistry, University of Calgary Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Titin (connectin) is a giant protein that forms a single-molecule elastic filament extending from the M-line to the Z-line in the striated muscle sarcomere. The sequence of titin consists mainly of repeats of two types of 100-amino acid motifs (class I and class II that show homology to the fibronectin type III and immunoglobulin-C2 domains, respectively). To investigate the functions of the two classes of titin motifs as the basic units of this large sarcomere organizer molecule, titin cDNA segments encoding single class I or class II or linked class I-II motifs were cloned by polymerase chain reaction from a rat cardiac cDNA library into the T7 RNA polymerase-based pAED4 vector to express non-fusion titin fragments in Escherichia coli. High level expression of the three titin fragments was achieved, and effective rapid purification procedures were developed. The purified titin fragments were verified by their amino acid composition, apparent molecular mass, and charge. Antibodies raised against the genetically expressed titin motifs specifically recognized intact rat cardiac and skeletal muscle titins in Western blotting and immunofluorescence microscopy, confirming the authenticity of the cloned fragments. High beta-sheet contents of these titin motifs indicate a folding state very similar to that of intact native titin. Solid-phase protein-binding assays demonstrated that a single class I motif was able to bind both myosin and F-actin. In comparison, a single class II motif had weaker binding to only F-actin but the fragment containing linked class I and class II motifs showed significantly stronger interactions with both myosin and F-actin. The binding of titin motifs to myosin supports the proposed association of A-band titin with the thick filament, and the novel titin-F-actin interaction was confirmed by F-actin cosedimentation assays, suggesting that titin may also be involved in the structure and/or function of the thin filament.


INTRODUCTION

Titin, also named connectin, is the largest polypeptide so far discovered with a molecular mass of 3,000 kDa and is the third most abundant protein in both skeletal and cardiac muscle sarcomeres (for reviews, see Wang(1985), Maruyama(1986), Trinick(1991, 1992), and Fulton and Issaacs(1991)). Titin has been shown by immunoelectron microscopy to be a single-molecule filament extending from the M-line to the Z-line of the sarcomere. The titin filaments appeared elastic in the I-band portion, in contrast to the A-band portion, which was shown to be inextensibly associated with the thick filaments (Maruyama et al., 1985; Fürst et al., 1988; Wang and Wright, 1988; Wang et al., 1991; Trombitas and Pollack, 1993). It is proposed that titin and nebulin (another giant myofibrillar protein) form the third and fourth filamentous structures, in addition to the myosin and actin filaments, in the skeletal muscle sarcomere functioning as molecular templates or rulers. The extremely large titin and nebulin polypeptides both consist mainly of multiple repetitive sequence motifs (Labeit et al., 1990; Jin and Wang, 1991b), that may have evolved through an adaptation in which the single-molecule titin or nebulin filament performs corresponding roles together with the polymer myosin and actin filaments in the sarcomere. cDNA cloning and sequencing revealed that rabbit skeletal muscle titin contains mainly two types of 100-residue motifs (class I and class II, which are related to fibronectin type III and immunoglobulin C2 domains, respectively) (Labeit et al., 1990, 1992). This is also true for avian (Tan et al., 1993) and mammalian (Fritz et al., 1993; Gautel et al., 1993) cardiac muscle titin sequences. These two types of sequence motifs have also been identified in several other muscle proteins, for example, twitchin (Benian et al., 1989), the insect muscle mini-titin (Nave and Weber, 1990), projectin (Ayme-Southgate et al., 1991), C-protein (Einheber and Fischman, 1990), myosin light chain kinase (Olson et al., 1990), and skelemin (Price and Gomer, 1993). The two types of repeating sequence motifs are thought to be the functional units for titin's myosin filament association and elastic appearance (Labeit et al., 1992; Soteriou et al., 1993a). However, in contrast to the titin filament, consisting of two different types of motifs, both thick and thin filaments are known to be uniformly assembled by myosin or actin and their associated proteins. In addition, the nebulin polypeptide chain, which is proposed to function as a template for the actin filament, contains only a single type of repeat motifs (35 amino acids; Jin and Wang (1991b)). Therefore, in order to fully understand titin's biological function, it is necessary to characterize and distinguish the functions of the two classes of titin motifs of rather different sequences. Since cardiac muscle also contains titin filaments but does not have nebulin to associate with the thin filament (Wang, 1985; Wang and Wright, 1988), its sarcomeric assembly and contractile mechanism may differ from that of skeletal muscle and the structure-function relationships of cardiac titin may be analyzed without the complexing by nebulin. Isolation of native intact cardiac titin has been achieved recently (Pan et al., 1994), but direct testing of the function of titin by protein interaction studies remains to be established due to the large size and limited solubility of the full-length protein. The large number of similar sequence motifs in a titin molecule also makes it difficult to chemically prepare titin fragments containing defined single or combinations of motifs for functional characterization. The present study applies molecular cloning and genetic expression to prepare small and soluble rat cardiac titin motif fragments for structure-function characterization. cDNA templates encoding single or linked rat cardiac titin class I and class II motifs were cloned to express titin fragments in Escherichia coli, and effective purification methods were developed to provide materials for studying the interactions of the titin motifs with muscle myosin and F-actin.


MATERIALS AND METHODS

The basic recombinant DNA techniques used have been described previously (Jin and Lin, 1989; Jin and Wang, 1991b; Jin et al., 1992) or can be found in Current Protocols in Molecular Biology (Ausubel et al., 1987) and Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989).

PCR Cloning of cDNA Fragments Encoding Rat Cardiac Titin Sequence Motifs and Construction of Expression Plasmids

To obtain cDNA templates encoding the titin class I and class II motifs, polymerase chain reaction (PCR) (^1)was applied to amplify titin cDNA fragments from aliquots of a gt11 rat cardiac cDNA library (Jin and Lin, 1989). As illustrated in Fig. 1, the PCR primers flanking a class I or/and a class II motif coding sequences were designed according to the previous rat cardiac titin cDNA cloning/sequencing results (from a segment of 2 kilobases). (^2)The PCR products of the expected sizes (348 bp for a motif I, 326 bp for a motif II, and 643 bp for a segment of these two motifs linked together) were identified by agarose gel electrophoresis (Fig. 1).


Figure 1: Cloning of rat cardiac titin cDNA fragments into the expression vector. Three pairs (four individuals) of synthetic oligonucleotide primers were designed for the PCR cloning of titin cDNA coding templates from a rat cardiac library. These primers were engineered to contain a minimal base pair mismatch (indicated as the boldletters) for the introduction of translation initiation or termination codons (marked by the arrowheads indicating the translational direction) as well as the appropriate restriction endonuclease sites (outlined on the sequences) for a unidirectional in-frame insertion of the amplified cDNA fragments into the expression vector. Three cDNA fragments encoding a single class I (Ti I) or class II (Ti II) or these two motifs linked together (Ti I-II) were obtained (shown in the inset picture of 0.9% agarose gel) and cloned into the pAED4 plasmid. I, the class I motif; II, the class II motif; 10-S/D, coupled T7 promoter and Shine-Dalgarno ribosomal binding sequence; T, transcription terminator; amp^R, ampicillin-resistant.



To express nonfusion titin motifs in E. coli, three recombinant plasmids (pTi I, pTi II, and pTi I-II) were constructed using the T7 polymerase-based (Studier et al., 1990) ampicillin-resistant pAED4 expression vector (generously provided by Dr. D. S. Doering, Whitehead Institute). For unidirectional insertion of the rat cardiac titin cDNA templates, the vector- and PCR-amplified cDNA fragments were double-digested by NdeI and EcoRI or NdeI and HindIII (Fig. 1) and purified by agarose gel electrophoresis (recovered by the Bio-Rad Prep-A-Gene DNA purification matrix according to the manufacturer's protocol). Recombinant plasmids were then constructed using T4 DNA ligase and used to transform JM109 E. coli cells. Ampicillin-resistant colonies were mini-cultured and extracted to identify the insert-bearing recombinant plasmids with a shifted mobility in agarose gel electrophoresis. To verify the orientation, translation reading frame and full sequence of the coding template, the recombinant plasmids were sequenced on both strands by the dideoxy chain termination method using a T7 polymerase DNA sequencing kit (Pharmacia Biotech Inc.).

Protein Gel Electrophoresis

The following gel electrophoresis was performed using Bio-Rad minigel apparatuses.

Laemmli Gels

As described previously (Jin and Smillie, 1994), 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an acrylamide:bisacrylamide ratio of 30:1 was used to examine the expression of rat cardiac titin fragments in E. coli.

Small-pore Gels

To monitor the purification and to analyze gel mobility of the genetically expressed small titin fragments, small-pore SDS-PAGE using Tris-Tricine buffer was modified from that described by Schagger and von Jagow(1987). Concentrations of the resolving and stacking gels were 14% and 4%, respectively. The acrylamide:bisacrylamide ratio was 20:1, and SDS was omitted from the resolving gel.

Large-pore Gradient Gels

For resolving the very high molecular weight intact titin in muscle tissues, large-pore low cross linker gradient (2-12%) polyacrylamide SDS-gels containing 0.25% low melting point agarose in the Fairbanks' continuous buffer system (modified from Wang(1986)) were prepared in a multigel caster with a linear gradient maker.

Two-dimensional Gel Electrophoresis

The purified titin fragments were analyzed on the first dimension non-equilibrium pH gradient gel electrophoresis (NEPHGE; O'Farrell et al., 1977) with tube gels contained pH 3.5-10 Ampholine (Pharmacia Biotech Inc.). After electrophoresis at 200 V for 10 min and 350 V for 2.5 h, the gel pieces were equilibrated in SDS-PAGE sample buffer for 15 min and transferred onto the blank top of a small-pore gel for the second dimension SDS-PAGE. 10 min after the dye front ran off the bottom edge, the gel was stained with Coomassie Blue R250 to reveal the resolved protein spots (Jin and Lin, 1988).

Expression of Rat Cardiac Titin Motifs in E. coli

Chloramphenicol-resistant BL21(DE3)pLysS E. coli cells (Studier et al., 1990) were transformed with the recombinant pTi plasmids, and ampicillin-chloramphenicol dual resistant colonies were selected. The transformed host cells were cultured in Luria broth containing 100 µg/ml ampicillin and 25 µg/ml chloramphenicol at 37 °C with vigorous shaking. When the A of the culture reached 0.2 to 0.5, isopropyl-1-thio-beta-D-galactopyranoside (IPTG) was added to the culture to 0.4 mM. After an additional 2.5 h, the bacterial cells were pelleted and analyzed by SDS-PAGE for the IPTG-induced expression of rat cardiac titin fragments.

Purification of the Bacterially Expressed Titin Fragments

Although intact titin is known to have a limited solubility, the short titin fragments produced in this study are recovered in the supernatant of the IPTG-induced bacterial lysates. The following rapid large scale purification procedures were developed according to the salting-out property and predicted isoelectric point (pI) of each individual titin fragment. All steps were performed at 4 °C unless specified otherwise, and the indicated volumes were based on a preparation from 4 liters of bacterial culture.

Purification of the Basic Fragments Ti II and Ti I-II

The pIs of these two rat cardiac titin fragments as calculated from their sequences are 9.35 and 8.45, respectively, and they were purified by the same procedure as follows. The induced bacterial cells were collected by centrifugation at 3,000 times g for 10 min and resuspended in 40 ml of 50 mM Tris-HCl, pH 8.0, containing 5 mM EDTA. The cells were disrupted by three passages through a French cell press at 800-1,000 p.s.i. The lysate was diluted to 200 ml with the same buffer and ammonium sulfate fractionation was carried out. The fraction of 50-80% saturation was dialyzed against two changes of 4 liters of 0.1 mM EDTA, pH 8.0, and diluted with the dialysis buffer to 90 ml. After adding sodium acetate, pH 4.5, to 10 mM and adjusting the pH to 4.5 with acetic acid, the solution was centrifuged at 10,000 rpm in a JA14 rotor for 20 min to remove the precipitated bacterial proteins. The supernatant was adjusted to 100 ml and loaded on a CM-52 cation-exchange column (2.5 times 10 cm) equilibrated in 10 mM sodium acetate, pH 4.5, containing 0.1 mM EDTA. After washing with 100 ml of the equilibration buffer, the column was eluted at 1 ml/min with a linear gradient of 0-300 mM KCl in the same buffer (300 ml total). Column fractions containing the purified Ti II or Ti I-II fragment, as revealed by SDS-PAGE of the A peaks, were collected.

Purification of the Acidic Ti I Fragment

Since the Ti I fragment is soluble in a high percentage of ammonium sulfate and is more acidic than the other two titin fragments (pI = 6.64), a different strategy was developed for its purification. The bacterial lysate was diluted to 190 ml with double distilled water and heated to 56 °C in a water bath with gentle shaking. After cooling on ice for 1 h, the lysate was adjusted to pH 9.0 with 1 M Tris base and centrifuged at 10,000 rpm in a JA14 rotor for 30 min to remove the insoluble material. The supernatant was fractionated on a DE-52 anion-exchange column (2.5 times 30 cm) equilibrated in 10 mM Tris-HCl, pH 9.0, containing 0.1 mM EDTA and eluted at 1 ml/min with a linear gradient of 0-300 mM KCl in the same buffer (500 ml total). The 280 nm absorbency peaks were analyzed by SDS-PAGE, and fractions containing purified Ti I protein were combined. Since the large scale lysate was loaded on the ion-exchange column without dialysis, the high salt content in the bacterial cytoplasm was carried over and might decrease or abolish the binding of Ti I protein to the matrix. Therefore, conductivity of the column load was measured and a dilution made if necessary with 10 mM Tris-HCl, pH 9.0, to reduce it to <2 millisiemens/cm.

Further Purification by Gel Filtration Chromatography

To remove the small amount of high molecular weight contaminants from the three titin fragments obtained from the ion-exchange columns, the fractions collected were concentrated by lyophilization and redissolved in 5-10 ml of 10 mM imidazole-HCl, pH 7.0, 15 mM beta-mercaptoethanol containing 6 M urea for further purification on a G75 column (2.5 times 120 cm) equilibrated in 6 M urea, 10 mM imidazole-HCl, pH 7.0, 0.1 mM EDTA, and 6 mM beta-mercaptoethanol. The column was eluted with the equilibration buffer at 0.5 ml/min. A peak fractions were analyzed by SDS-PAGE and fractions containing highly purified rat cardiac titin fragments were collected. After dialysis against three 2-liter changes of 1% formic acid (Pearlstone et al., 1976; Pearlstone and Smillie, 1977), the proteins were lyophilized and stored at -20 °C. The titin fragment stocks used for the ELISA and F-actin cosedimentation binding assays as well as the CD measurement were quantified by amino acid analysis (performed by the Protein Sequencing Facility, University of Calgary Faculty of Medicine).

Analytical Gel Filtration Chromatography

30 µg each of the purified Ti I and Ti I-II, and 5 µg of Ti II fragments were mixed together in 50 µl of 0.2 M NaCl, 50 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 0.02% NaN(3), 15 mM beta-mercaptoethanol and analyzed on an 1 times 30-cm Superose 12 column equilibrated in the same buffer. The column was run by a Pharmacia Biotech Inc. FPLC system at 0.1 ml/min to examine the apparent molecular mass of the titin motifs. Fractions (0.2 ml) were collected and the A peak fractions were analyzed by small-pore SDS-PAGE to identify the positions of each titin fragment on the molecular weight standard curve obtained under the same conditions.

Circular Dichroism Measurement

The purified Ti I, Ti II, and Ti I-II proteins were dialyzed at 4 °C against three changes of 500 volumes of 0.1 M KCl, 50 mM Tris-HCl, pH 7.5, 6 mM beta-mercaptoethanol. Circular dichroism (CD) measurements were carried out at 25 °C on a Jasco J-720 spectropolarimeter (Jasco Inc., Easton, MD). The instrument was calibrated with ammonium d(+)-10-camphor sulfonate at 290.5 nm and 192 nm, and with d(-)-pantoyllactone at 219 nm. The cell used was 0.02 cm, and protein concentration was 0.25-1 mg/ml. Each sample was scanned 10 times, and noise reduction was applied to remove the high frequency before calculating molar ellipticities. As calculated from their sequences, the mean residue weights for Ti I, Ti II, and Ti I-II were 110.8, 107.4, and 109.2, respectively.

Immunological Methods

Anti-titin Motif Antibody Preparation

An antiserum (MATi) against the Ti I-II fragment was produced in BALB/c mice. The immunization was carried out by an initial intraperitoneal injection of 50 µg of the purified antigen in Freund's complete adjuvant followed by a boost of the same amount of antigen 3 weeks later. The mouse sera were tested for the specific anti-titin antibody titer by indirect enzyme-linked immunosorbent assay (ELISA, performed as described by Jin and Lin(1988)). Using the same immunization plan, two hybridoma monoclonal antibodies (mAb) against the Ti I-II immunogen were prepared as described previously (Jin et al., 1990). The antiserum and mAb ascites fluid were stored in 50% glycerol at -20 °C.

Western Blotting

The purified rat cardiac titin motifs were resolved by 14% small-pore SDS-PAGE and transferred to nitrocellulose membrane (0.22 µm) using a Bio-Rad semidry electrotransfer apparatus. The total proteins of rat cardiac or skeletal muscle myofibril (prepared as described by Jin et al.(1990)) were dissolved in SDS-PAGE sample buffer and resolved by 2-12% large-pore gradient SDS-PAGE. The semidry transfer of resolved myofibril protein bands onto nitrocellulose membrane (0.45 µm) was carried out with the three-buffer procedure (Jin and Wang, 1991a). The following blocking, incubations with MATi at 1:4,000 dilution or anti-Ti II mAb Ti104 hybridoma culture supernatant at 1:100 dilution and I-labeled sheep anti-mouse IgG second antibody (0.5-1 µCi/ml, from ICN Biomedicals), and autoradiography were carried out as described previously (Jin and Lin, 1988; Jin and Smillie, 1994).

Immunofluorescence Microscopy

The rat cardiac and skeletal muscle myofibril preparation and indirect immunofluorescence microscopy were carried out as described previously (Jin et al., 1990). Briefly, the myofibrils were sedimented on glass slides and fixed in 3.7% formaldehyde for 15 min. After blocking in 1% bovine serum albumin (BSA), the slides were incubated with 1:100 dilutions of the mouse anti-rat cardiac titin motif antiserum MATi, mAb Ti102 ascites fluid, or normal mouse serum controls at room temperature for 2 h. It was followed by washing and incubation with tetramethylrhodamine isothiocyanate-conjugated anti-mouse IgG second antibody (Sigma) at room temperature for 1 h. After three final washes, the slide was mounted with a coverslip and viewed under a Nikon Optiphot X-2 phase contrast-epifluorescence microscope. A CF phase Fluor DL-100X objective lens (oil, numerical aperture 1.30) was used for the photography of both phase-contrast and fluorescence images with a Nikon Microflex UFX-DX automated photographic system.

ELISA-mediated Solid-phase Protein-binding Assay

ELISA-mediated titin fragment-binding assays were established using the specific antibody against titin motifs. Microtiter plates (Falcon 3915) were coated with 100 µl/well purified rabbit skeletal muscle F-actin (30 µg/ml, prepared from acetone powder as described previously by Jin and Lin(1988)) or myosin (50 µg/ml, prepared by the method of Heeley et al.(1989)) in 10 mM Tris-HCl, pH 8.0, 100 mM KCl, 3 mM MgCl(2) (Buffer A) at 4 °C overnight. After washing once with Buffer A plus 0.05% Tween-20 (Buffer T), the plate were blocked with 1% BSA in Buffer T (100 µl/well) at room temperature for 2 h. After three washes with Buffer T and blot-drying the wells, the plates were incubated with serial dilutions of purified Ti I, Ti II (2 times 10M to 6.4 times 10M), or Ti I-II (1 times 10M to 3.2 times 10M) titin fragments in Buffer A at room temperature (22 °C) for 2 h. The plates were then washed with Buffer T three times and incubated with the MATi antiserum at 1:4,000 dilution, 100 µl/well in buffer T plus 0.1% BSA (Buffer B) at room temperature for 1 h. Following three washes with Buffer T, horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin second antibody (Sigma) at 1:1,500 dilution in Buffer B was added to the plate (100 µl/well) and incubated at room temperature for 45 min. The plates were finally washed three times with Buffer T and H(2)O(2)-ABTS substrate was added (100 µl/well) for the color development at room temperature. The A of each assay well was monitored at a series of time points (3-30 min) by a Bio-Rad model 3550UV automated microplate reader and the data from a time point within the linear range of the color development were chosen for each set of binding experiments.

F-actin Cosedimentation Assays

To examine the interactions between F-actin and the titin motifs in solution, rabbit skeletal muscle F-actin (32 µg, prepared as described by Jin and Lin(1988)) was incubated with serial dilutions of the titin fragments Ti I, Ti II, or Ti I-II in 100 µl of an actin binding buffer (100 mM KCl, 3 mM MgCl(2), 20 mM Tris-HCl, pH 8.0). After incubation at room temperature (22 °C) for 1 h, the reaction mixtures, together with appropriate protein controls, were centrifuged in a Beckman Airfuge at 26 p.s.i. at room temperature for 30 min. Supernatants and pellets were analyzed by small-pore SDS-PAGE and quantitation of Coomassie Blue-stained protein bands was carried out by transmission scanning on a laser scanning densitometer (LKB 2202, scanning at 20 mm/min).


RESULTS

Cloning of cDNA Templates Encoding Rat Cardiac Titin Class I and Class II Sequence Motifs and Construction of Recombinant Expression Plasmids

Three overlapping titin cDNA coding templates were obtained from the rat cardiac library by the PCR amplification and cloned into the pAED4 vector (Fig. 1). DNA sequencing showed that cDNA inserts (334, 314, and 629 bp) in the three recombinant plasmids (pTi I, pTi II, and pTi I-II) encode titin fragments Ti I (a class I motif, 108 residues), Ti II (a class II motif, 102 residues), and Ti I-II (a fragment containing the two motifs, 207 residues), respectively. The two titin motifs cloned and expressed are the first rat titin sequence so far characterized. Sequences of the T I and Ti II motifs show extensive homology to the rabbit skeletal (Labeit et al., 1990) and chicken (Tan et al., 1993), rabbit (Fritz et al., 1993), and human (Gautel et al., 1993) cardiac titin class I and class II motif sequences (80% or 75% identity was found between the Ti I or Ti II and the motif consensus sequences, respectively). Position of the cloned segment in the whole rat cardiac titin polypeptide chain remains to be revealed by more extensive cDNA sequencing data. All three titin fragments begin with a methionine residue at the NdeI-cut sites (Fig. 1). The initial Met in both Ti I and Ti I-II is from the natural sequence and only the initiating Met in Ti II is artificially introduced for the genetic expression of a protein internal fragment.

Expression and Purification of Rat Cardiac Titin Fragments from Bacterial Culture

In the recombinant pTi plasmid-transformed BL21(DE3)pLysS E. coli culture, the three rat cardiac titin fragments were successfully expressed upon IPTG induction (Fig. 2). Although toxicity of various titin fragments to the host bacterial cell has been observed during expression, (^3)we have achieved a high level expression of these single or linked titin motif fragments. When carrying out small or large scale (multiple 1-liter) cultures for the titin fragment expression, it was important to start the culture from a freshly transformed positive bacterial colony, to use a rich medium, to induce with IPTG at early log phase (A of 0.2-0.5) of the bacterial growth and to culture for a limited time (2.5-3 h) after induction. The purification methods developed for the three titin fragments proved very effective and highly purified Ti I, Ti II, and Ti I-II titin motifs (Fig. 3) were obtained at large quantities.


Figure 2: Expression of rat cardiac titin motifs in E. coli. The recombinant pTi plasmid-transformed BL21(DE3)pLysS E. coli was cultured in liquid media and induced by IPTG (+). The bacteria were then lysed in SDS-PAGE sample buffer to analyze the total cellular protein on SDS-PAGE (15% Laemmli gel). The uninduced aliquot from the same cultures was processed as control(-). The results show a high level expression of the single class I, class II, and the linked class I-II rat cardiac titin motifs (as indicated by the whitediamonds) upon IPTG induction.




Figure 3: Gel mobility of the cloned rat cardiac titin fragments. The purified rat cardiac titin fragments Ti I, Ti II, and Ti I-II were analyzed by 14% small-pore SDS-PAGE (left) and two-dimensional gel electrophoresis (right), in which NEPHGE with pH 3.5-10 Ampholine was used as the first dimension and 14% small-pore SDS-PAGE as the second dimension.



Characterization of the Genetically Expressed Titin Fragments

Amino Acid Composition

The experimentally determined amino acid compositions of all three purified Ti I, Ti II, and Ti I-II titin fragments showed a very close match of residue molar ratio to that calculated from the cDNA-derived protein sequences (data not shown). This is a good indication of the accurate cloning and expression of the nonfusion titin fragments in E. coli and the high efficiency of the purification methods.

Two-dimensional Gel Mobility

The bacterially expressed rat cardiac titin fragments consisting of a class I motif (Ti I), a class II motif (Ti II), or the two motifs linked together (Ti I-II) were analyzed by one-dimensional SDS-PAGE and two-dimensional gel electrophoresis. According to their gel mobilities demonstrated in Fig. 3, the experimentally determined apparent molecular masses and charge variations of the three titin fragments under the SDS- or/and urea-denaturing condition corresponded with their calculated molecular weight (11,972 for the 108-residue Ti I, 10,954 for the 102-residue Ti II, and 22,609 for the 207-residue Ti I-II) and pI (6.64 for Ti I, 9.35 for Ti II, and 8.45 for Ti I-II), respectively. These results confirm the accurate cloning and expression of these rat cardiac titin fragments.

Apparent Molecular Mass at the Native State

Fig. 4shows the gel filtration chromatographic analysis of the three rat titin motifs by the Superose 12 column. According to the molecular weight standard curve, the apparent molecular masses of native Ti I-II, Ti I, and Ti II motifs were calculated as 29.5, 20, and 13.7 kDa, respectively. The gel filtration chromatography also demonstrated that the Ti I fragment tended to form dimers when the sulfhydryl reductant was less effective. The small amount of Ti I dimer formed in the column buffer containing 15 mM beta-mercaptoethanol was eluted at the position corresponding to an apparent molecular mass of 33-kDa (indicated by the arrowhead on the SDS gel inset in Fig. 4). The Ti I dimers formed under the benign conditions were reversible in the SDS-PAGE sample buffer containing 150 mM fresh beta-mercaptoethanol and no corresponding high molecular weight bands found on the SDS-gel (Fig. 4). Similar to the 2% cysteine content found in the intact bovine cardiac titin (Pan et al., 1994), there are 2 cysteine residues present in the Ti I fragment and 1 cysteine in the Ti II fragment. In the presence of 15 mM beta-mercaptoethanol, there was no cross-linked Ti I polymer nor Ti II dimer found but Ti I would gradually form irreversible dimers under the SDS-denaturing condition when the sulfhydryl reductant aged (the 24-kDa band shown in Fig. 6A and Fig. 9). The formation of intermolecular disulfide bonds may have caused the Ti I dimerization and the two sulfhydryl groups in the Ti I motif may both be exposed in the presence of SDS contributing together to the irreversible dimer formation. Similar to that found with intact titin (Pan et al., 1994), very high concentrations (50-200 mM) of fresh dithiothreitol were unable to convert the Ti I dimers that had formed in the SDS-PAGE sample buffer back to monomers (data not shown). Therefore, to carefully maintain titin preparations at the native state to minimize the sulfhydryl groups exposed (i.e. 45% according to Pan et al.(1994)), together with effective sulfhydryl reductant, may also help to limit the irreversible aggregation observed with the intact titin (Pan et al., 1994).


Figure 4: Apparent molecular masses of the cloned titin motifs determined by gel filtration chromatography. The purified Ti I-II, Ti I, and Ti II fragments were analyzed on a Superose 12 FPLC column in 0.2 M NaCl, 50 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 0.02% NaN(3), 15 mM beta-mercaptoethanol. The A peak fractions were analyzed by 14% small-pore SDS-PAGE to reveal the positions of each titin fragment (as indicated in the inset gel) on the standard curve plotted by the Macintosh Cricket Graph program from molecular weight markers run under the same conditions. The arrowhead indicates the position of the Ti I dimer peak (see ``Apparent Molecular Mass at the Native State'').




Figure 6: Western blots of the cloned titin fragments and intact titin using the anti-titin motif antibodies. Mouse antiserum MATi and mAb Ti104 against the Ti I-II fragment were used to stain nitrocellulose blots of the Ti I/Ti II protein (A) or the total protein extracted from rat cardiac and skeletal myofibrils (B) via I-labeled sheep anti-mouse IgG second antibody. The stained SDS-gel and autoradiographs of each blot are shown side by side, and the positions of the major sarcomere proteins are indicated (T, titin; N, nebulin; M, myosin heavy chain; A, actin). The results show that the MATi recognized both Ti I and Ti II besides the immunogen Ti I-II (A). MATi and the anti-Ti II Ti104 also specifically recognized intact titin in both cardiac and skeletal myofibrils (B). Small amounts of Ti I dimer (see ``Apparent Molecular Mass at the Native State'') were detected by the MATi antibody (A).




Figure 9: F-actin cosedimentation assays. Purified Ti I, Ti II, and Ti I-II fragments at serial dilutions (40 µM to 1.25 µM as indicated above the lanes) and muscle F-actin (7 µM of the actin monomer) were incubated together or separately and centrifuged in a Beckman Airfuge. The supernatants (lanesS) and pellets (lanesP) were separated and analyzed by 14% small-pore SDS-PAGE. Similar to that observed in Fig. 6A, there were Ti I dimers showed on the SDS gel (the 24-kDa band in the samples from the Actin-Ti I binding experiments) caused by an irreversible dimerization after the SDS denaturation (see ``Apparent Molecular Mass at the Native State'').



CD Spectra

Fig. 5shows the CD spectra of Ti I, Ti II, and Ti I-II titin motifs. The results demonstrate that the spectra of Ti I, Ti II, and Ti I-II had negative troughs at 199, 216, and 212 nm with mean residue ellipticity of 6,006, 4,047, and 4,319 degrees cm^2 dmol, respectively. The secondary structure contents for the Ti I (class I) motif, Ti II (class II) motif, and Ti I-II (the two motifs linked together) as estimated by the method of Provencher and Glöckner(1981) are summarized in Table 1. The CD Spectra and 50% beta-sheet contents of all three fragments correspond well with the crystal or solution structures determined for the fibronectin type III domain (Dickinson et al., 1994; Main et al., 1992) and immunoglobulin C2 domain (Holden et al., 1992). The data on the titin motif folding states also agree with that determined from native intact bovine cardiac titin (53% beta-sheet, Pan et al., 1994), considering that the class I motif forms a major portion of the titin polypeptide chain. Data from the CD measurement also demonstrated that the Ti II preparations before or after exposure to 6 M urea and 1% formic acid had no significant difference in spectra nor the secondary structure contents (data not shown).


Figure 5: Circular dichroism spectra of the cloned rat cardiac titin motifs. The CD spectra of Ti I, Ti II, and Ti I-II in 0.1 M KCl, 50 mM Tris-HCl, pH 7.5, 6 mM beta-mercaptoethanol at 25 °C revealed high beta-sheet contents for all of the three proteins (Table 1).





Authenticity of the Cloned Titin Motifs

The mouse polyclonal antiserum (MATi) raised using the Ti I-II fragment as immunogen recognized both purified Ti I and Ti II fragments in Western blots (Fig. 6A). This immunological reactivity pattern among the three cloned titin fragments reflects their overlapping sequence relationships. Reaction of this polyclonal antibody to Ti II motif is comparable with that to the immunogen Ti I-II but is much stronger than the reaction to Ti I motif. This may imply a stronger antigenicity of the class II motif (Ti II is a rather basic protein fragment) than that of the Ti I motif. A trace amount of Ti I dimers was also recognized by the MATi antiserum (Fig. 6A). To verify the authenticity of the rat titin fragments cloned, Western blotting demonstrated that both the polyclonal anti-Ti I-II antiserum and an anti-Ti-II mAb Ti104 specifically recognized intact titin from rat myofibrils of both cardiac and skeletal muscles (Fig. 6B). These data demonstrate not only that the Ti I-II sequence cloned is a titin fragment but also that this sequence forms epitopes found in both cardiac and skeletal muscle titin molecules.

Indirect immunofluorescence microscopy further confirmed the presence of epitopes recognized by the mouse anti-Ti I-II antibodies in both rat cardiac and skeletal muscle myofibrils (Fig. 7). Both the anti-Ti II mAb Ti102 and the antiserum MATi, which also reacted mainly to the Ti II motif (Fig. 6A), labeled a wide region of the I-band of the cardiac sarcomere. The results indicate a presence of multiple Ti II homologous epitopes and suggest that the class II motif may form a major portion of the I-band titin as also revealed by the sequence data (Maruyama et al., 1993). The results also indicate that mAb Ti102 recognized multiple similar, instead of site-specific, I-band epitope(s) exposed in the rat cardiac myofibril samples examined. The Z-lines (indicated by the arrowheads) were not labeled by the antibodies. Differently, both MATi and Ti102 labeled the sarcomere I-A junction on the skeletal myofibril with a weak stain on the entire A-band. Similar difference was also observed on the bovine cardiac and skeletal myofibrils (Pan et al., 1994). The difference in the anti-titin motif antibody immunofluorescence staining patterns on the cardiac and skeletal myofibrils suggests that the cardiac and skeletal muscle titins are different in their motif organization and/or assembly into the sarcomeric structure. It was reported that there were structural differences between cardiac and skeletal muscle titins detected by mAbs in Western blotting (Hill and Weber, 1986), although only a single titin gene was identified (Labeit et al., 1990; Müller-Seitz et al., 1993).


Figure 7: Immunofluorescence microscopic localization of Ti I-II epitopes in the sarcomere. Rat cardiac and skeletal myofibrils were examined by indirect immunofluorescence microscopy for the staining of anti-Ti I-II serum MATi and anti-Ti II mAb Ti102 via rhodamine-conjugated anti-mouse IgG second antibody. The phase-contrast (upper) and fluorescence (lower) micrographs are aligned for each image with the Z-lines indicated by the arrowheads. Both MATi and Ti102 gave wide I-band staining in the cardiac sarcomere and A-I junction labeling (together with weak A-band staining) in the skeletal muscle sarcomere.



Interaction of the Cloned Titin Motifs with Muscle Myosin and F-actin in Solid-phase Binding Assays

The ELISA-mediated solid-phase binding experiments demonstrated a high affinity interaction of rat titin fragments Ti I-II and Ti I with immobilized rabbit muscle myosin or F-actin. The results showed that the two-motif Ti I-II fragment and the single class I motif (Ti I) had very similar saturable binding curves to both myosin and F-actin (Fig. 8, A and B, respectively). There were only slight differences between the concentrations of either fragment required for the 50% maximal binding (B) to myosin and F-actin (5 times 10M and 3 times 10M for Ti I-II; 5 times 10M and 2.5 times 10M for Ti I, respectively) although Ti I-II was 10-fold higher in binding affinity than Ti I to both myosin and F-actin. In contrast, the single class II motif (Ti II) bound significantly weaker to F-actin (the B was 1.9 times 10M) and no specific binding was detected between Ti II and myosin (Fig. 8C), although the anti-Ti I-II antibody (MATi) used in the assay showed a better detection of Ti II than that of Ti I (Fig. 6A).


Figure 8: Interaction of the cloned titin motifs with muscle myosin and actin. Serial dilutions of purified Ti I-II (A), Ti I (B), or Ti II (C) were incubated with myosin, F-actin, or BSA coated on microtiter plates. The amount of Ti fragments bound to the immobilized proteins was determined by the ELISA procedure via the mouse anti-Ti I-II antiserum, horseradish peroxidase-conjugated rabbit anti-mouse total immunoglobulin second antibody, and H(2)O(2)-ABTS substrate (see ``ELISA-mediated Solid-phase Protein-binding Assay''). The assay was performed in triplicate wells and titration curves were constructed by the average Aversus molar concentrations of the Ti fragments.



Interaction of the Titin Motifs with F-actin in Co-sedimentation Assays

The ELISA-mediated solid-phase protein-binding assay is a rapid, sensitive, and quantitative method to examine the interaction of titin motifs with muscle myosin and F-actin. It is especially useful for an effective screening of a large number of experimental conditions. To confirm the titin motifs' actin binding activity in a physiological solution, the novel titin-F-actin interaction detected by the solid-phase binding assays was further investigated by the traditional F-actin co-sedimentation assay (Laki et al., 1962; Hitchcock et al., 1973; Eaton et al., 1975). The results (Fig. 9) demonstrated that the Ti fragments cosedimentated with F-actin under conditions in which, alone or combined, they were not pelleted. Similar to the results from the solid-phase binding experiments, binding of the two-motif fragment Ti I-II to F-actin in solution is apparently stronger than that of the single class I motif fragment Ti I. Since the co-sedimentation assay was done in the absence of sulfhydryl reductant, a small part of the Ti I fragments was likely in the dimer form (the same protein stock was used in the analytical gel filtration chromatography experiment shown in Fig. 4). The amount of Ti I dimers had increased significantly after SDS denaturation and became irreversible as the 24-kDa band appeared on the SDS gel (Fig. 9), as this band was not detectable when the Ti I samples were applied to electrophoresis immediately after a brief treatment in the SDS-gel sample buffer containing fresh beta-mercaptoethanol ( Fig. 3and unpublished F-actin cosedimentation gel data). The stoichiometries for Ti I (at a total concentration of 40 µM) and Ti I-II (at a total concentration of 10 µM) binding to F-actin (at 7 µM monomer concentration), as calculated from SDS-gel densitometry, are 1:3.9 (calculated from both the Ti I monomer and dimer bands) and 1:2.6, respectively. Under the experimental conditions, no significant interaction was observed between Ti II (at 13 µM) and F-actin (the Ti II:actin ratio was 1:30).


DISCUSSION

Cloning and Genetic Expression in E. coli of Single or Linked Titin Motifs for Functional Characterization

For the complexity associated with the solubilization and characterization of intact native titin, molecular cloning and genetic expression of its fragments becomes a practical way to study the structure-function relationships of this extremely large protein. It is estimated that 300 repeating sequence motifs exist in a single titin molecule and whole picture of the motif organization remains to be revealed by the full-length sequence. To focus on the two different types of titin motifs proposed as the basic functional units, single class I (Ti I), class II (Ti II), and linked class I and class II motifs (Ti I-II) of rat cardiac titin were designed and cloned in this study. To obtain non-fusion titin fragments for more precise functional characterization, the T7 polymerase-based pAED4 vector was utilized (Fig. 1) and a high level of titin motif expression in E. coli culture was achieved (Fig. 2). Accurate cloning and authenticity of these titin fragments was verified by amino acid analysis, two-dimensional gel mobility (Fig. 3), and immunological assays in which the anti-Ti I-II antibodies specifically recognized intact titin extracted from muscle tissues (Fig. 6) and located the antigenic epitopes in the sarcomere (Fig. 7). In addition to homologies to the titin motif consensus sequences (Labeit et al., 1990), it has been shown that the anti-Ti I-II antibodies recognized both cardiac and skeletal muscle titins of many other species (mouse, cow, chicken, and toad) in the Western blots (data not shown). Although further characterization of titin motifs from multiple regions is needed to conclude the characterization of the two classes of sequence motifs, the rat cardiac titin motifs cloned in this study have provided materials for a dissective examination of the functional property of and difference between the class I and class II motifs.

Effective Purification of the Genetically Engineered Nonfusion Rat Cardiac Titin Fragments from Bacterial Culture

Good yield, high degree of purity, appropriate folding, and biological activity of a cloned protein genetically expressed and isolated from bacterial culture are essential for its use in functional characterization. The expression of a protein without any fusion peptide will directly provide materials for structure-function studies but requires the development of a specific purification method. Using conventional biochemical procedures, two effective methods for the purification of three titin fragments from E. coli lysate were developed in this study. Without sacrificing yield and purity, both methods can be completed within 3-4 days. The rapid purification procedures may limit the proteolytic degradation of the product and, therefore, no expensive protease inhibitors were necessary. These purification methods are based on the biochemical properties of the recombinant proteins and may be adopted for the purification of other genetically expressed proteins, especially an engineered protein fragment whose physical property has been altered from that of the intact protein. The titin fragments purified by our methods further demonstrated the expected folding feature (Fig. 5) and binding activity to muscle myosin and F-actin ( Fig. 8and Fig. 9).

Interaction between Titin Motifs and Muscle Myosin

Ultrastructural studies have shown an inextensible association of the A-band titin with the thick filament (Maruyama et al., 1985; Fürst et al., 1988; Wang and Wright, 1988; Wang et al., 1991). To support titin's association with thick filaments, a binding of titin to myosin rod (light meromyosin) was demonstrated (Labeit et al., 1992; Soteriou et al., 1993b). Our present study has quantitatively examined the interaction of titin with myosin using the cloned motifs. The results show that the bindings of single class I (Ti I) or linked class I-II (Ti I-II) titin motifs to myosin are both saturable (Fig. 8, A and B). Although there was no detectable binding of Ti II to immobilized myosin (Fig. 8C), link of the same class II motif to the class I motif in the Ti I-II fragment resulted in a 10-fold higher binding affinity to myosin than that of the Ti I fragment. The data suggest that class I motifs are able to interact with the myosin filament and the intercalation of class II motifs in the titin filament may facilitate the interaction.

Interaction between the Titin Motifs and Muscle F-actin

The interaction of titin motifs with F-actin demonstrated by both solid-phase binding assay (Fig. 8) and F-actin co-sedimentation experiments (Fig. 9) was an inquisitive finding. The titin-actin interaction observed in this study may depend on the native conformation of F-actin since very little binding was detected between titin and the actin bound to nitrocellulose membrane (Soteriou et al., 1993b). The interactions of single or linked titin motifs with myosin and actin demonstrated here also confirmed the early reports that the T2 titin fragment (beta-connectin) interacted with myosin and actin under physiological conditions (Kimura and Maruyama, 1983; Kimura et al., 1984).

Similar to their binding to myosin, Ti I-II is 10-fold stronger than Ti I in the binding to F-actin. The binding stoichiometries determined for the Ti I-II and Ti I fragments versus actin are in the same range (1:2.6 and 1:3.9, respectively) and binding of either Ti I or Ti I-II to both myosin and F-actin showed very similar affinities. The Ti II fragment that did not bind to myosin bound weakly to F-actin in the solid-phase binding assay (Fig. 8C) and cosedimentation assay (Fig. 9). These data suggest that the class II motif functions differently from Ti I that composes the major portion of the titin molecule (at least in the A-band region). The 2.6-3.9 ratio between actin monomer versus one class I motif may suggest that two or three class I titin motifs interact with each 7-actin (troponin repeat) unit (Fürst et al., 1989). This model will also fit the (I-I-II-I-I-I-II-I-I-I-II)(n) super-repeat pattern found in the titin sequence (Labeit et al., 1990, 1992) in which clusters of two or three class I motifs are intercalated by single class II motifs. Facilitated by the adjacent class II motif(s), each of the class I motif cluster might form a functional module of the titin filament to interact with the myosin/actin filaments. It has been found that a long segment consisting of continuous class II motifs presents in the I-band titin (Maruyama et al., 1993). The weak class II motif-actin interactions may, therefore, combine to produce a higher avidity in regions containing multiple class II motifs. Further engineering of more combinations of the titin motifs will be useful to investigate functions of the two classes of motifs, and it will also be interesting to clone another single class I motif that does not form dimers under the assay conditions to verify its reactivity to myosin and actin.

The biological significance of the titin-F-actin interaction remains to be established. It is known that titin's A-band portion is inextensible, while its I-band region appears elastic (Maruyama et al., 1985; Fürst et al., 1988; Wang and Wright, 1988; Trombitas and Pollack, 1993). The possible different motif organization of I-band and A-band titin (Labeit et al., 1992; Maruyama et al., 1993) and the functional difference between the two types of titin motifs need to be investigated. The observation of titin-actin interaction suggests that the I-band portion of titin is not a ``naked'' filament or a ``free spring,'' especially in the cardiac muscle sarcomere where no nebulin filaments associate with the actin filaments (Wang, 1985; Wang and Wright, 1988). The interaction between titin and the actin filament might also contribute to the resting tension of myofibril (Wang et al., 1991, 1993; Horowits, 1992), or the titin molecule might play a role in the assembly of actin filaments in the sarcomere. The titin-F-actin interaction may even explain the elasticity of I-band titin. Unlike the titin motif secondary structure unfolding hypothesis (Soteriou et al., 1993a), a reversible ``uneven zipping'' between actin filament and titin polypeptide chain during contraction or stretching might be a mechanism responsible for the titin filament's elastic behavior. These hypotheses invite further experimental studies toward a better understanding of muscle contraction.


FOOTNOTES

*
This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Alberta. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L38717[GenBank].

§
Recipient of a research scholarship from the Heart and Stroke Foundation of Canada. To whom correspondence should be addressed: Dept. of Medical Biochemistry, University of Calgary Faculty of Medicine, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-8861; Fax: 403-270-2211; jjin{at}acs.ucalgary.ca.

(^1)
The abbreviations are: PCR, polymerase chain reaction; ABTS, 2,2`-azinobis-(3-ethybenzthiazolinesulfonic acid); B, 50% maximal binding; bp, base pair(s); BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; mAb, monoclonal antibody; NEPHGE, non-equilibrium pH gradient gel electrophoresis; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

(^2)
J.-P. Jin and K. Wang, unpublished results.

(^3)
J.-P. Jin, unpublished results.


ACKNOWLEDGEMENTS

I thank Mary Resek for technical assistance, Dr. Don Doering for the pAED4 expression vector, Drs. Bruce Allen and Michael Walsh for FPLC gel filtration analysis, Kim Oikawa and Dr. Cyril Kay for CD measurement, and Dr. Michael Walsh for critical reading of this manuscript.


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