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) (
)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). (
)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
,
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-
-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
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
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
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
-mercaptoethanol
containing 6 M urea for further purification on a G75 column
(2.5
120 cm) equilibrated in 6 M urea, 10 mM imidazole-HCl, pH 7.0, 0.1 mM EDTA, and 6 mM
-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
, 15 mM
-mercaptoethanol
and analyzed on an 1
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
-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
(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
10
M to 6.4
10
M), or
Ti I-II (1
10
M to 3.2
10
M) 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
O
-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
, 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, (
)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
-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
-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
-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
, 15 mM
-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
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%
-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%
-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
-mercaptoethanol at 25 °C revealed high
-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
10
M and 3
10
M for Ti I-II; 5
10
M and 2.5
10
M 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
10
M) 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
O
-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 A
versus 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
-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 (
-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)
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.