From the Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970
Received for publication, June 12, 2002, and in revised form, November 20, 2002
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ABSTRACT |
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Nebulin (600-900 kDa) and nebulette (107-109
kDa) are two homologous thin filament-associated proteins in skeletal
and cardiac muscles, respectively. Both proteins are capped with a
unique region at the amino terminus as well as a serine-rich linker
domain and SH3 domains at the COOH terminus. Their significant size
difference is attributed to the length of the central region wherein
both proteins are primarily composed of ~35 amino acid repeats termed nebulin-like repeats or motifs. These motifs are marked by a conserved SXXXY sequence and high affinity binding to F-actin.
To further characterize the effects that nebulin-like proteins may have
on the striated muscle thin filament, we have cloned, expressed, and
purified a five-motif chicken nebulette fragment and tested its
interaction with the thin filament regulatory proteins. Both tropomyosin and troponin T individually bound the nebulette fragment, although the affinity of this interaction was significantly increased when tropomyosin-troponin T was tested as a binary complex. The addition of troponin I to the tropomyosin-troponin T complex decreased the binding to the nebulette fragment, indicating an involvement of the
conserved T2 region of troponin T in this interaction. F-actin
cosedimentation demonstrated that the nebulette fragment was able to
significantly increase the affinity of the tropomyosin-troponin assembly for F-actin. The relationships provide a means for
nebulin-like motifs to participate in the allosteric regulation of
striated muscle contraction.
Nebulin (600-900 kDa) and nebulette (107-109 kDa) are two
homologous thin filament-associated proteins in skeletal and cardiac muscles, respectively (1, 2). cDNA sequences of nebulin and
nebulette have shown that both proteins are primarily composed of ~35
amino acid repeats termed nebulin-like repeats or motifs, each marked
by a conserved SXXXY sequence (2, 3). Both nebulin and
nebulette are capped by a unique region at the amino terminus in
addition to serine-rich linker and SH3 domains at the carboxyl terminus
(2-6). Immunoelectron microscopy has demonstrated that a single
nebulin molecule extends from the Z-line (COOH terminus of the protein)
to the pointed ends of the thin filament at its NH2
terminus (7). Nebulette is similarly anchored to the Z-line through its
COOH terminus, but because of its relatively smaller size, it does not
extend as far as the pointed ends of the actin filament. Experiments
using in vitro systems have formed a consensus that
nebulin-like motifs from both nebulin and nebulette associate with
F-actin (4, 6, 8). Additional experiments have extended this
observation to demonstrate that nebulin-like motifs may interact directly with tropomyosin
(Tm)1 and troponin (Tn) (4)
along the striated muscle thin filament.
At the Z-line, the nebulin/nebulette interactions are probably mediated
by contributions from the linker domain as well as interactions of the
SH3 domains with Although the majority of experiments to date have focused on the binary
interactions between nebulin/nebulette and other sarcomeric proteins,
there is preliminary evidence to suggest that nebulin-like motifs may
serve a role in the regulation of striated muscle contraction. These
investigations have concentrated on the role that nebulin may play in
regulating interactions within the framework of the isolated acto-S1
myosin system. Consistent with this hypothesis, it has been
demonstrated that nebulin fragments of varying lengths are able to
inhibit the acto-S1 myosin ATPase, an inhibition that can be reversed
by calmodulin (12). Subsequent experiments by this group have suggested
that nebulin fragments affect the acto-S1 myosin complex by optimizing
the alignment of actomyosin interactions (13). To further clarify the
interactions that nebulin-like repeats may have along the thin
filament, we have characterized the binding of a chicken nebulette
fragment to Tm and the Tn subunits. Our data suggest that Tm and
troponin T (TnT) both contain interaction sites for the nebulin-like
motifs. These interactions were strengthened when Tm and TnT were
present as a binary complex and weakened by the addition of troponin I
(TnI). Furthermore, the nebulette fragment was able to significantly
increase the affinity of Tm·Tn for F-actin, demonstrating that
nebulin-like motifs contribute to the assembly and allosteric property
of the striated muscle thin filament.
Reverse Transcription (RT)-PCR Cloning of Mouse Nebulin and
Chicken Nebulette cDNA Fragments--
To clone mouse nebulin (MSN)
and chicken nebulette (CN5) cDNAs, a partial sequence of nebulin
cDNA from BALB/c strain mice (14) and a partial cDNA
sequence from chicken nebulette (2) were used to design oligonucleotide
primers suitable for RT-coupled PCR. Total RNA was prepared from 129 Sv/J mouse gastrocnemius muscle or white Leghorn chicken heart
by the TRIzol reagent (Invitrogen) according to the
manufacturer's instructions. Using 5 µg of total RNA as template and
20 pmol of a reverse primer (mouse: Neb-R, 5'-
CGGCATACTATGCCTTATACTGATTATCAC-3', and chicken: CCN-R2, 5'-CCTTTGAATTCTCAAAATCTTAATGGTA-3'
(mismatches are in bold to introduce restriction enzyme recognition
sites and translation stop codons)), 20 units of Moloney murine
leukemia virus reverse transcriptase was used for an RT reaction that
was incubated overnight at 37 °C. Subsequently, a PCR was set
up using one-tenth of the RT products and 20 pmol each of two pairs of synthetic oligonucleotides, Neb-F/Neb-R and CCN-F/CCN-R2 (Neb-F, 5'-TCTCCAGAGTAAACCAGATCCATATGAGTG-3', and CCN-F,
5'-AGCACTCATATGAACATC-3' (mismatches are in bold
to introduce restriction enzyme recognition sites and translation
initiation codons)). The PCR was done with a program of 35 cycles at
94 °C for 1 min, 48 °C for 1 min, and 72 °C for 3 min.
Aliquots of the PCR products were analyzed by agarose gel
electrophoresis to confirm amplified cDNA fragments at the expected
sizes of 470 bp (MSN) and 540 bp (CN5). The amplified mouse nebulin
cDNA was precipitated with ethanol and cloned into the
SmaI site of pBluescript II SK vector (Stratagene). The
amplified chicken nebulette cDNA was precipitated, digested by
NdeI and EcoRI endonucleases, and resolved by
0.7% agarose gel electrophoresis with Tris acetate-EDTA buffer (0.04 M Tris acetate, pH 8.0, 1 mM EDTA). The DNA
band of interest was excised from the agarose gel and recovered using
the Prep-A-Gene DNA recovery kit (Bio-Rad). The recovered cDNA was
cloned into NdeI- and EcoRI-digested pAED4 expression vector (15). The insert sequences of the two constructs as
well as the translation reading frame of the pAED4-CN5 construct were
confirmed by dideoxy chain termination sequencing (15).
Isolation of a
The positive phage DNA was prepared in large quantity using the Qiagen
Lambda Midi Kit according to the manufacturer's instructions (Qiagen).
An ~15-kb mouse genomic DNA insert was excised from the
Protein Expression and Purification--
Escherichia
coli BL21(DE3)pLysS cells were transformed with the T7
polymerase-based pAED4-CN5 chicken nebulette expression construct and
allowed to grow until the colonies were just visible. Four fresh
colonies were randomly picked to inoculate 4 liters of NZ broth
(10 g/liter casein hydrolysate, and 5 g/liter NaCl) containing 100 µg/ml ampicillin and 25 µg/ml chloramphenicol. The
cultures were incubated overnight at 37 °C with shaking until A600 = 0.8 at which time protein
expression was induced by the addition of
isopropyl-1-thio-
The purification protocols for TnC, TnI, TnT, and Generation of a Specific Antiserum and Monoclonal Antibody
against CN5--
Using the purified chicken nebulette CN5 protein, a
female BALB/c mouse was immunized intraperitoneally to generate
a specific antiserum. For the primary immunization, 200 µg of the CN5
antigen was dissolved in phosphate-buffered saline and mixed into a
water-in-oil emulsion using an equal volume of Freund's complete
adjuvant. Subsequent boosts using 100 µg of the purified CN5 protein
were given at 3-week intervals using Freund's incomplete adjuvant. To
collect the anti-CN5 antiserum, the mouse was sacrificed for terminal
bleeding and the blood was recovered and allowed to clot at 4 °C
overnight. The serum was separated by centrifugation (2000 × g for 5 min), an equal volume of glycerol was added, and the antiserum was stored at
As described previously (19), a short term immunization procedure was
applied to develop a specific anti-CN5 monoclonal antibody (mAb). A
7-week-old female BALB/c mouse was injected intraperitoneally
and intramuscularly with a total of 50 µg of the purified CN5 antigen
in 100 µl of phosphate-buffered saline mixed with an equal volume of
Freund's complete adjuvant. Ten days later, the mouse was
intraperitoneally boosted twice with 200 µg each of the CN5 antigen
in 100 µl of phosphate-buffered saline without adjuvant on two
consecutive days. Two days following the last boost, spleen cells were
harvested from the immunized mouse to fuse with SP2/0-Ag14 mouse
myeloma cells using 50% polyethyleneglycol4000 containing
7.5% dimethyl sulfoxide as described previously (20). Hybridomas were
selected by HAT (0.1 mM hypoxanthine, 0.4 µM
aminopterin, and 16 µM thymidine) media containing
20% fetal bovine serum and screened by indirect enzyme-linked
immunosorbent assay against coated CN5 using horseradish
peroxidase-labeled goat anti-mouse total immunoglobulin (Sigma) as the
second antibody. The positive hybridoma culture supernatants were
verified by Western blotting against the CN5 protein using alkaline
phosphatase-labeled goat anti-mouse total immunoglobulin second
antibody (Sigma) as described previously (17). The anti-CN5
antibody-secreting hybridomas were subcloned three times by limiting
the dilution using young BALB/c mouse spleen cells as a feeder
to establish stable cell lines. A high affinity anti-CN5 mAb (Ne-110)
was characterized. Immunoglobulin subclass typing by sandwich
enzyme-linked immunosorbent assay using a reagent kit from BD
Biosciences determined that this mAb is mouse IgG1 Western Blot Identification of Nebulette--
To monitor
nebulette expression, Western blots were done using either the anti-CN5
antiserum or CN5 mAb Ne-110 on fresh SDS extracts of cardiac muscle
from various species. The SDS-PAGE samples were prepared in the
presence of 1 mM phenylmethanesulfonyl fluoride and 20 µM leupeptin (Sigma) to ensure protein integrity. To
better resolve large proteins (>100 kDa), 6% SDS-PAGE with an
acrylamide:bisacrylamide ratio of 29:1 was used. However, in this
system, the resolving gel does not retain proteins <60 kDa in
molecular mass. To ensure that no proteolyic products <60 kDa in molecular mass were present, 14% (180:1) SDS-PAGE was used to
present a complete SDS extract profile. After resolving total cardiac
muscle extracts by SDS-PAGE, a three-buffer system was used to
efficiently transfer the resolved protein bands onto nitrocellulose membranes (21). Following the transfer, the nitrocellulose replicas were blocked by incubation at room temperature for 3 h with
Tris-buffered saline (150 mM NaCl, 2.5 mM KCl,
and 25 mM Tris-HCl, pH 7.5) containing 1% bovine serum
albumin. The blocked membrane was then incubated at 4 °C overnight
with either the anti-CN5 antiserum or Ne-110 mAb diluted in
Tris-buffered saline containing 0.1% bovine serum albumin. The Western
blots were developed via alkaline phosphatase-labeled second antibody
as described previously (17).
Nebulette Affinity Chromatography--
The relative binding
strengths of F-actin Cosedimentation--
To determine the binding affinity
of the CN5 nebulette fragment to F-actin and the interactions between
thin filament regulatory proteins and CN5 in the presence of F-actin, a
series of cosedimentation experiments were performed. Purified rabbit
skeletal muscle F-actin (0.13 nmol) was incubated with serial
concentrations of CN5, Tm, TnT, TnI, or whole troponin in either a 30- or 50-µl volume for 45 min at 4 °C. The cosedimentation buffer
contained 10 mM potassium phosphate buffer, pH 7.4, 65 mM KCl, 2.5 mM MgCl2, 0.1 mM K2EGTA, and 1 mM dithiothreitol.
In experiments in which CN5 was present during Tm or Tn subunit
titrations, a constant concentration of 1.52 µM CN5 was
used. Following incubation, F-actin was sedimented by centrifugation at
100,000 × g (45,000 rpm) for 30 min at 4 °C in a
Beckman Optima TL Ultracentrifuge using a TLA100.1 rotor. The
supernatant and pellet were separated, and 15 µl of 1× SDS-PAGE sample buffer was used to solubilize the pellet. The supernatant and
pellet were examined by either 14% (180:1) or 12% (29:1) SDS-PAGE. Resolved gels were silver-stained as described before and analyzed by
gel densitometry. The titration curve was constructed from results of
three experiments, and the data are presented as the mean ± S.D.
To estimate the binding affinity, the total [CN5] required for 50%
maximum binding was obtained by averaging the data calculated from the
fits for each individual experiment using single exponential fits.
Exon Organization of the Nebulin Gene--
A mouse nebulin
cDNA fragment was cloned by RT-PCR according to a published
cDNA sequence (14). This cDNA was used as a probe to isolate an
~15-kb mouse nebulin genomic DNA fragment from a phage library, and
an ~3.2-kb EcoRI fragment of the genomic DNA was subcloned
and sequenced. The nucleotide sequence has been submitted to the
GenBank Data Base (GenBankTM accession number AF440239).
Two consecutive exons of the nebulin gene were found in the genomic DNA
fragment encoding 69 amino acids and 35 amino acids, which correspond
to two and one of the ~35 amino acid-repeating motifs, respectively
(Fig. 1). Both exons had splice
boundaries within the conserved pentapeptide, specifically at
SX/XXY, indicating this
sequence to be at the boundary between the ~35 amino acid motifs.
Partial sequencing of other EcoRI subclones of the ~15-kb
genomic DNA showed similar exon boundaries (data not shown). Because
exon organization reflects the evolutionary and potentially functional
units of multi-domain proteins, the genomic structure of the mouse
nebulin gene indicates that the single ~35 amino acid motif (Fig. 1,
exon B) represents the fundamental functional unit of
nebulin-like proteins.
Cloning, Expression, and Purification of the CN5 Nebulette
Fragment--
Using oligonucleotide primers designed according to a
published partial cDNA sequence (2), a chicken nebulette cDNA
encoding a polypeptide beginning and ending with the conserved
SXXXY pentapeptide was amplified. This frame coincides with
the exon boundaries of the nebulin gene (Fig. 1). The chicken nebulette
cDNA encodes a 168-amino acid protein spanning five nebulin-like
motifs with a theoretical Mr of 19398 and an
isoelectric point of 9.73 and is shown to be aligned to the area of
highest homology within the repeating domain of human nebulette (Fig.
2) (5, 6). Similar to the human nebulin
fragments as reported previously (8), CN5 expressed in E. coli at high levels. The CN5 protein was readily purified by
standard biochemical procedures. Purified CN5 migrated as a single band
in SDS-PAGE (Fig. 3) and was found to be
relatively soluble under non-denaturing conditions (solubility >0.5
mM). This was in contrast to the MSN fragment, which was confined to the inclusion bodies upon large scale expression in E. coli (data not shown), similar to previously published
data on mammalian nebulin fragments (6, 8, 24). A mouse polyclonal antiserum was generated using the purified CN5 protein as the immunogen. As shown in Fig. 3, the polyclonal antiserum was
specifically identified the CN5 protein in Western blot. This finding
was also recapitulated by the anti-CN5 mAb Ne-110 (data not shown).
Expression of Nebulette in Cardiac Muscle and during
Development--
As shown in Fig. 4, the
anti-CN5 mAb strongly identified nebulette proteins from chicken and
bovine heart extracts. The monoclonal antibody did not strongly
recognize nebulette in mouse, sheep, or rabbit cardiac muscle extracts,
implying epitope diversity of nebulette proteins across species.
Initially, 14% (180:1) SDS-PAGE was used to resolve the isoforms to
ensure that no proteolytic fragments were present. To further increase
the resolution of isoforms, 6% (29:1) SDS-PAGE was used. Although the
chicken nebulette isoforms were identified as two very closely
migrating bands under 14% (180:1) SDS-PAGE, 6% (29:1) SDS-PAGE was
better able to resolve the isoform diversity. Using the chicken as a
developmental model (total extracts from embryonic days 14 to 20),
neonatal and adult chicken hearts were analyzed for nebulette
expression (Fig. 5). Multiple bands were
identified by the anti-CN5 mAb, suggesting nebulette isoform diversity
(5). A higher Mr isoform appeared to be
predominant throughout heart development but was down-regulated by 6 weeks post-hatch. This developmental pattern of high to low Mr isoform expression is reflective of the
regulation of TnT expression with development (23), suggesting that
this type of isoform switch is fundamentally conserved in the
myofilament proteins of striated muscle. Two closely migrating
isoforms, a predominant lower Mr isoform and a
less expressed higher Mr isoform, were identified in extracts from bovine heart chambers, suggesting that
isoform diversity was not confined to the avian heart. It was also
noted that the ratio of the isoforms varied between chambers with the
lower Mr isoform being predominant in the left
ventricle. The primary sequence differences between the nebulette
isoforms are of interest but remain to be determined.
TnT Interaction with CN5 Is Strengthened by Tm and Weakened by
TnI--
Using affinity chromatography and F-actin cosedimentation,
the interactions between CN5 and the thin filament proteins F-actin, TnT, TnI, and Tm were tested. Consistent with data previously demonstrated for other nebulin fragments, CN5 specifically bound to
F-actin in cosedimentation experiments with half-maximal binding occurring at 2.09 ± 0.6 µM CN5 (Fig.
6). The maximum stoichiometry of CN5
binding to undecorated F-actin was ~0.4 mol CN5/mol actin or 2.8 mol
of the 5-unit CN5 protein/7 actin monomers that defines the striated
muscle thin filament regulatory unit. This finding suggests that in the
absence of regulatory proteins on the actin filament, the CN5 protein
binds at approximately twice the stoichiometry that would be expected
if each of the five nebulin motifs bound one actin monomer (1.4 mol
CN5/7 mol actin). Assuming that the ~35-amino acid sequence motif
represents an actin-binding unit, this 2:1 binding ratio to naked actin
filaments may be a reflection of the multiple binding sites available
for nebulin on each actin monomer as well as additional
nonphysiological binding modes that nebulin fragments may undertake
under these conditions (25). It should also be noted that the
interaction between two or three of the nebulin-like motifs with
adjacent actin monomers may be sufficient to sustain a stable
association between CN5 and F-actin in vitro, allowing an
overestimation of the stoichiometry with the unregulated actin
filament. This is supported by the observation that a 3-4-unit nebulin
fragment can cross-link adjacent F-actin strands (25). Although the
binding between CN5 and F-actin was analyzed simply as a one-step
reaction, it remains possible that the binding of nebulin and nebulette
fragments containing multiple actin binding motifs to F-actin may be a
corporative multiple-step process.
To test the direct interactions between CN5 and TnT or Tm, a CN5
affinity chromatography column was used. Purified
Cosedimentation experiments verified the affinity chromatography
results within the framework of the actin filament. In the presence of
serial concentrations of the thin filament regulatory proteins and a
fixed concentration of CN5, two likely classes of interactions were
reflected by the cosedimentation experiments. Because the thin filament
regulatory proteins bind CN5 with various affinities, the free proteins
during the cosedimentation experiments may compete with F-actin for CN5
binding. In addition, the F-actin-bound Tm and/or Tn subunits may
affect the interaction between CN5 and F-actin, either through steric
blocking, an increase in the number of available CN5 binding sites
beyond those on F-actin alone, or conformational modulation of the
CN5-F-actin interaction. The results in Fig.
8 show that the binding of CN5 to F-actin
decreases with increasing concentrations of Tm and/or Tn subunits.
However, CN5 binding remained largely unchanged once the physiological stoichiometric decoration of F-actin (7:1) by the regulatory protein(s) was reached. This finding suggests that the free Tm and/or Tn subunits
were not able to compete with F-actin and eliminate CN5 binding. As
demonstrated in Fig. 8, the decoration of F-actin with Tm reduced
concurrent nebulette binding, possibly because of Tm constraining CN5
to binding at its physiological sites on F-actin while limiting
non-physiological interactions between CN5 and the actin filament (25).
In contrast, TnT was more efficient in reducing the binding of
nebulette to F-actin, suggesting that the strong interaction between
nebulette and TnT weakened nebulette-actin binding. However, the
Tm·TnT binary complex resulted in only a moderate effect on nebulette
binding to F-actin (Fig. 8B) and provided an approximate
stoichiometry of 0.7 CN5/7 actin regulatory units, or half of the
theoretical stoichiometry expected if each of the five nebulin motifs
bound an actin monomer (1.4 CN5/regulatory unit). This lower
stoichiometry may be explained by the hypothesis that in contrast to
the head-tail-registered binding of Tm to F-actin, the unorganized
binding of nebulin fragments would not fully decorate the actin
filament. These experiments demonstrated that the Tm·TnT complex and
nebulette were able to decorate F-actin simultaneously. The higher
amounts of CN5 binding to Tm·TnT-decorated F-actin versus
TnT-decorated F-actin suggest relative increase in the CN5-actin
affinity. This effect was confirmed by assays using the Tm·TnT·TnI
complex. The higher levels of CN5 binding to F-actin in the presence of
Tm·TnT or Tm·TnT·TnI rather than in the presence of TnT alone
exclude the possibility that steric blocking by TnT competes with the
CN5-actin interaction. Because the binding of CN5 to TnT is stronger
than that observed for Tm (Fig. 7, A and B), the
difference between CN5 interaction with F-actin-Tm versus
F-actin-TnT is not likely due to the number of binding sites on these
decorated actin filaments since it would predict stronger binding to
F-actin-TnT, which was not observed. Therefore, the results support the
hypothesis that the Tm·Tn complex and nebulin have an allosteric
interactive relationship dictating the interaction of nebulin-like
motifs with the thin filament.
Additional cosedimentation experiments tested the effect of CN5 on the
affinity of Tm·Tn assembly onto the actin filament. Tropomyosin-troponin concentration was titrated in the presence or
absence of a fixed concentration (1.52 µM) of CN5. The
CN5 fragment decreased the concentration of Tm·Tn required to achieve half-maximal binding to F-actin (147.4 ± 32.1 nM
without CN5 versus 70.8 ± 4.9 nM with CN5;
p = 0.05 by Student's t test) (Fig.
9), further suggesting that nebulin-like
motifs participate in the striated muscle thin filament assembly.
The role of nebulin-like proteins in the sarcomere of striated
muscles has been reported to be primarily a structural one. Evidence
has been mounted by studies showing the interaction of nebulin or
nebulette fragments with F-actin (6, 8), protein constituents of the
Z-line (6, 9), and with tropomodulin at the pointed ends of the thin
filaments (11). Correlations between nebulin/nebulette expression and
Z-line thickness as well as nebulin protein size and actin filament
length in skeletal muscle sarcomeres (5, 26) have all contributed to
the large body of evidence in support of a structural role for
nebulin/nebulette.
Through genomic DNA cloning, we showed in this study that the exons of
the nebulin gene are arranged in a manner reflective of the ~35-amino
acid nebulin-like motifs that serve as the basic functional units. The
partial genomic DNA sequence data suggest that the conserved
SXXXY sequence straddles the junction between adjacent exons
(Fig. 1) as also suggested by Millevoi et al. (5), providing
a compelling case for the SXXXY sequence serving as the boundary between the repeating nebulin-like motifs. Although the
use of intact proteins remains desirable, the insolubility of intact
nebulin and nebulette in physiological buffers necessitates the use of
protein fragments during functional characterizations (6, 8, 24). This
afforded the rationale in designing a five-motif nebulette fragment
defined by the conserved pentapeptide flanking each motif (Fig. 2).
Similar to published observations with other nebulin fragments
(4, 8, 24), we found that the nebulette CN5 fragment bound F-actin with
affinity in the low micromolar range (Fig. 6). Although unsurprising
given the homology between nebulin and nebulette, this finding
confirmed that F-actin binding is a conserved function of the
nebulin-like motifs. For the CN5 protein, F-actin binding is relevant
because this region of nebulette localizes to actin filaments when
transfected into primary cultures of cardiomyocytes (6).
We observed that the binding of CN5 and In contrast to Tm, the interaction between the CN5 fragment and TnT was
stronger with the peak of elution occurring at 280-360 mM
NaCl by affinity chromatography (Fig. 7B). Troponin T
complexed to Tm showed even higher affinity binding to CN5 (peak at
480-560 mM NaCl) (Fig. 7C). Three possible
mechanisms may underlie this observation: (a) higher avidity
from the sum of two independent CN5 binding sites on Tm and TnT;
(b) Tm and TnT as a binary complex possibly presenting a
unique interaction pocket with increased affinity for nebulin-like
motifs; or (c) the binding of Tm to TnT resulting in a
conformational change in TnT, consequently increasing the binding
affinity of the protein for CN5. F-actin cosedimentation results
further supported the formation of a Tm·TnT binding pocket for the
nebulin-like motifs. Although TnT alone caused a large decrease in the
binding of nebulette to F-actin (Fig. 8A), the addition of
Tm moderated the effect and allowed the assembly of a thin filament
accommodating both Tm·TnT and CN5 (Fig. 8B). Additional
affinity chromatography experiments demonstrated that TnI decreased the
affinity of Tm·TnT for the nebulette fragment (Fig. 7D),
suggesting that the interaction pocket formed by the Tm·TnT complex
is either directly competed by TnI or altered by a change in the
conformation of TnT upon TnI binding. In either case, these results
underscore the role of the conserved COOH-terminal T2
region of TnT in mediating the interaction with CN5. These findings
were verified by cosedimentation experiments in which Tm·TnT·TnI
was also found to modulate CN5 binding to F-actin, similar to the
results using Tm·TnT (Fig. 8B). Therefore, the association
of CN5 with an F-actin-Tm·TnT filament demonstrated both a Tm·TnT
and an F-actin binding component with the former being challenged
moderately in the presence of TnI. The interaction between CN5 and the
thin filament was extended by additional cosedimentation experiments
demonstrating that the nebulette fragment was able to increase the
affinity of Tm·Tn for F-actin (Fig. 9). This increase suggests that
the documented interactions, particularly between nebulin-like motifs
and Tm·TnT, provided a tangible means of increasing the affinity of
the thin filament assembly for F-actin. This observation also supports the hypothesis that nebulin-like motifs are able to interact with and
contribute to the regulation of the Tm·Tn assembly on the actin filament.
The data collected indicate that during the allosteric
control of striated muscle contraction, a reversible interaction may occur along the thin filament between nebulin and the Tm·Tn
regulatory system, affecting its interaction with F-actin. The binding
between Tm·TnT and nebulin-like motifs may be the pivotal association underlying this observation and offers a role for regulation by TnC or
an analogous Ca2+-binding protein such as calmodulin (12).
Experiments by Root and Wang (13) detailing changes in actomyosin in
the presence of nebulin-like motifs may be a preliminary account of how
these interactions integrate within the actin filament. The data
presented in Figs. 8 and 9 necessitate future experiments in testing
the acto-S1 interaction in an intact regulated filament. We propose that allosteric changes during muscle contraction due to
Ca2+ binding to TnC and/or strong cross-bridge attachment
may contribute to the cooperativity of striated muscle, possibly by
allowing a reversible nebulin/nebulette interaction with the Tm·Tn
complex and in turn facilitating a more pronounced propagation of the transition from the blocked to the closed or from the closed to the
open states of the thin filament (29). We hypothesize that the putative
effect of the nebulin-like motifs on the coordinated regulation of the
thin filament may contribute to the higher cooperativity of skeletal
versus cardiac muscles (30-32), a possibility that will be
tested most effectively in the framework of an integrated muscle fiber.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin (6, 9). However, results from Ojima and
coworkers (10) suggest that neither the serine-rich linker domain nor
the SH3 domain are obligatory elements for the incorporation of nebulin
and presumably nebulette into the Z-line of striated muscle sarcomeres.
Although both proteins are anchored at the Z-line in vivo,
the primary sequence regions responsible for this interaction remain
unclear. Recent experiments have also shown an association between the
NH2 terminus of nebulin and tropomodulin, a protein known
to cap actin-Tm filaments (11). Therefore, emerging data demonstrate
that the NH2- and COOH-terminal domains of nebulin and
nebulette form unique interactions at both the pointed end of the thin
filament and the Z-line of sarcomeres, complementing the established
actin binding properties of these proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-DASHII Phage Containing Mouse Nebulin Genomic
DNA--
To isolate nebulin genomic DNA, a
-DASHII 129 SvJ mouse
genomic DNA phage library was screened by radioactive probe
hybridization (16). The MSN cDNA was cut from the pBluescript II SK
plasmid by BamHI and EcoRI restriction enzyme
digestion, resolved by agarose gel electrophoresis, and recovered as
described above. The purified DNA fragment was labeled with
[
-32P]dCTP by random priming synthesis for use as a
radioactive probe in screening nylon filter replicas of the phage
library plated on agar plates. Based on the average insert size of the
mouse genomic DNA library (12-15 kb pair), an aliquot of phage
harboring inserts totaling 1.5× the mouse genome (~4.5 × 109 bp) was plated out on twelve 150-mm agar plates. The
nylon replicas were prehybridized in 100 ml of 30% (v/v) formamide,
5× SSPE (0.9 M NaCl, 0.05 M
NaH2PO4, 5 mM EDTA, pH 7.7), 5×
Denhardt's solution, and 100 µg/ml denatured salmon sperm DNA at
50 °C for 6 h after which time the heat-denatured radioactively
labeled probe was added and incubated at 50 °C for 16 h.
Following the hybridization period, the filters were washed
successively at room temperature with 1× SSPE, 0.5× SSPE, and 0.1×
SSPE followed by washing with 0.1 × SSPE at 50 °C. The filters were
exposed to x-ray film overnight to reveal hybridization signals. A
small agar plug was recovered from the plate corresponding to the area
of each putative positive signal. The hybridization screening was then
used in plaque purification to isolate the positive phage clones.
-phage arms by EcoRI digestion, and the EcoRI
fragments were subcloned into the EcoRI site of pBluescript
SK II vector. One subclone containing a 3.2-kb mouse genomic DNA
fragment was sequenced by preparing a set of serial deletions using an
exonuclease III-based nested deletion kit according to the
manufacturer's instructions (Promega).
-D-galactopyranoside to 0.4 mM. Following 3 h of growth, the bacteria were
harvested by centrifugation and lysed in 20 mM imidazole,
pH 7.0, and 1 mM EDTA by three passes through a French
press at 1000 p.s.i. Successive ammonium sulfate cuts from 0 to
30% saturation and from 30 to 50% saturation were taken from the
cleared bacterial lysate. The pH of the solution was maintained at 7 throughout the ammonium sulfate precipitation procedure. The 30-50%
ammonium sulfate precipitation pellet was resuspended in 30 ml of 20 mM imidazole, pH 7.0, 1 mM EDTA, and 6 mM
-mercaptoethanol and dialyzed at 4 °C against 4 liters of 20 mM imidazole, pH 7.0, 1 mM EDTA,
and 6 mM
-mercaptoethanol for two changes. To the
dialyzed fraction, urea powder was added to 6 M, the pH was
adjusted to 7, and the solution was clarified by centrifugation. Urea
was used to minimize protein-protein interactions and maximize
separation during ion-exchange chromatography. The supernatant was
loaded onto a 100-ml (2.5 × 20 cm) CM52 column equilibrated with
6 M urea, 20 mM imidazole, pH 7.0, 1 mM EDTA, and 15 mM
-mercaptoethanol. A
linear KCl gradient of 0-400 mM in the equilibration
buffer was used to elute the CN5 protein from the column. The fractions
from the CM52 column were analyzed by 12% SDS-polyacrylamide gel
electrophoresis with an acrylamide to bisacrylamide ratio of 29 to 1, and fractions containing the protein of interest were collected,
dialyzed against 4 liters of 0.1% formic acid, a volatile agent, for
three changes, and lyophilized. Amino acid analysis of the purified CN5
protein verified the authenticity of cDNA expression and the
effectiveness of protein purification (Molecular Biology Core
Laboratory, Case Western Reserve University, Cleveland, OH).
/
-Tm from
chicken skeletal muscle have been described previously, and the purity
of these preparations have been demonstrated (17, 18). Concentrations
for all protein preparations were determined by the Nanoorange protein
quantitation kit (Molecular Probes, Eugene, OR) with the exception of
TnC, which was determined by absorption, assuming an extinction
coefficient of 0.132 at 259 nm for a 1-mg/ml solution.
20 °C.
. The hybridoma
cells were then cultured to produce high titer supernatant and
introduced into 2,6,10,14-tetramethyl pentadecane (Pristane, Sigma)
primed peritoneal cavity of BALB/c mice to produce mAb-enriched
ascites fluids. The hybridoma culture supernatant was used in this study.
/
-Tm and Tn subunits to the chicken nebulette
fragment CN5 were determined using affinity chromatography. The
purified nebulette fragment was coupled to CNBr-activated Sepharose 4B
gel (Amersham Biosciences) according to the manufacturer's
instructions using 9 mg of CN5 protein/0.5 ml of gel for coupling. A
0.4-ml column was packed and equilibrated with 100 mM NaCl,
15 mM PIPES, pH 7.0, 3 mM MgCl2, 1 mM K2EGTA, and 0.1 mM
tris(carboxyethyl) phosphine. Proteins to be analyzed on the column
were dissolved in 3 ml of the column buffer and dialyzed overnight at
4 °C against 2 liters of the column buffer to ensure homogenous
buffer conditions and complex formation. For binding experiments that
test the interaction of
/
-Tm or TnT with the CN5 affinity column,
0.4 nmol of protein was typically used. For experiments involving
complexes of Tm with Tn subunits, 0.4 nmol of Tm dimer was used and the
Tn subunits were mixed in a 1:1 molar ratio. After loading the dialyzed
protein mixtures, the column was washed with 8 bed volumes of the
equilibration buffer and eluted by a step gradient of 120-680
mM NaCl in the equilibration buffer. All of the steps were
carried out at room temperature. The fractions were examined by 12%
(29:1) SDS-PAGE and visualized by silver staining to determine the
protein peaks (22). A control experiment was done by mixing 0.4 nmol of
TnT and bovine serum albumin (New England Biolabs) and loading onto the
CN5 affinity column. Only the TnT bound specifically to the column
since the bovine serum albumin was eluted in the flow-through and wash
fractions only, indicating that nonspecific interactions in this
setting were minimal (data not shown).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Exon organization in a 3.2-kb fragment of the
mouse nebulin gene. A map of the cloned ~3.2-kb mouse nebulin
genomic DNA fragment is shown with the positions of two consecutive
exons outlined. The amino acid sequences encoded by the
exons are shown below containing two and one of the ~35-amino acid
repeats, respectively. The boundaries of both exons span the conserved
SXXXY pentapeptide. The exon organization suggests that the
~35-amino acid nebulin-like motif as represented by the exon
B-encoded repeat is the basic structural and functional unit of nebulin
and nebulin-like proteins.
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Fig. 2.
A cloned chicken nebulette cDNA and its
homology to human nebulette. A, the cloned chicken
nebulette CN5 fragment is shown aligned to its area of highest
similarity to the human nebulette protein (5). B, the CN5
fragment contains 5 of the ~35 amino acid nebulin-like motifs.
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Fig. 3.
Expression and purification of
the chicken nebulette fragment. The cloned chicken nebulette CN5
fragment was expressed in E. coli at a high level upon
induction shown in the total bacterial protein extracts by Coomassie
Blue R250 staining of 12% (29:1) SDS-PAGE (left panel). The
protein was purified effectively and used to raise a specific
antiserum. A Western blot using the antiserum identified the CN5
protein in both the induced bacterial lysate and purified protein lanes
(right panel).
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Fig. 4.
Expression of nebulette in cardiac
muscle. Using the anti-nebulette mAb, SDS homogenates of fresh
adult heart muscles from various species were examined by Western blot
for nebulette expression. Left, the SDS extracts of cardiac
muscles from various species were resolved by either 14% (180:1)
SDS-PAGE (A) or 6% (29:1) SDS-PAGE (B) and
stained by Coomassie Blue R250. The anti-CN5 mAb Ne-10 strongly
recognized nebulette bands in chicken and bovine left ventricular
homogenates and suggested isoform diversity in nebulette
expression.
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Fig. 5.
Expression of nebulette isoforms during heart
development and in different chambers. Left, the expression
of nebulette isoforms was determined during chicken heart development
by Western blots of total SDS homogenates of embryonic through
6-week-old chick hearts; top, 6% (29:1) SDS-PAGE gel shows
integrity of the muscle samples; bottom, Western blots using
the anti-CN5 mAb Ne-110 detected multiple closely migrating nebulette
bands in the chicken cardiac muscle, demonstrating a high to lower
Mr transition during development;
right, the top panel shows SDS extracts of bovine
heart chambers samples resolved by 14% (180:1) SDS-PAGE. The
bottom panel Western blot shows nebulette expression in the
various chambers of the bovine heart as detected by the Ne-110 mAb,
demonstrating a predominance of the lower Mr
isoform in the left ventricle.
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Fig. 6.
Cosedimentation analysis of the binding of
CN5 to F-actin. F-actin cosedimentation experiments were done with
0.13 nmol of F-actin and a series of CN5 concentrations. The binding of
CN5 to F-actin was analyzed by SDS-PAGE and gel densitometry. The
results plotted are presented as the pooled average ± S.D. from
three experiments (solid line). To estimate the binding
affinity, the total [CN5] required for 50% maximum binding was
obtained by averaging the data calculated from each individual
experiment using single exponential fits by the equation
y = yo + Ae( x/
), where y is the mol CN5
bound per mol actin, yo is the maximum mol CN5 bound
per mol actin, A is a constant, x is the total
concentration of CN5, and
is the time constant of the fit. For a
representative illustration, the averaged data were fit by the same
equation in the figure (dotted line).
/
-Tm dimers
only moderately interacted with the nebulette fragment as evidenced by
the flow-through and subsequent rapid elution from the CN5 affinity
column between 120 and 160 mM NaCl (Fig. 7A). In contrast, TnT alone
interacted strongly with the nebulette fragment and was eluted with a
primary peak at 280-360 mM NaCl (Fig. 7B).
Combining TnT and Tm in a 1:1 molar ratio resulted in both proteins
binding to the column and a noticeable shift to higher affinity as
compared with Tm or TnT alone (Fig. 7C). The main peak of
the Tm·TnT complex was eluted at 480-560 mM NaCl. Early
elution of some
/
-Tm alone was observed in Fig. 7C,
corresponding to the elution of uncomplexed
/
-Tm as seen in Fig.
7A. However, the overlapping elution profiles suggest that
/
-Tm bound TnT as expected, forming a binary complex that
interacted with the CN5 fragment, and remained resistant to higher salt
concentrations. Troponin I significantly reduced the binding affinity
between CN5 and the Tm·TnT complex, resulting in peak elution of
Tm·TnT·TnI between 280 and 400 mM NaCl (Fig.
7D) well below the elution range for Tm·TnT. The complete
overlap of the Tm, TnT, and TnI peaks under this condition demonstrates
that TnI stabilized the interaction between Tm and TnT as expected to
form a Tm·TnT·TnI complex and subsequently minimized the early
elution of Tm seen in Fig. 7C. The flow-through of some
Tm·TnT·TnI complexes from the CN5 affinity column was observed in
Fig. 7D, indicative of TnI affecting the Tm-TnT-CN5
interaction. When TnC was included to form a Tm·Tn complex, no
binding to the CN5 column was detected (data not shown).
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Fig. 7.
Binding of Tm, TnT, or TnI to a CN5 affinity
column. The interaction of Tm and troponin subunits, alone or in
combination, with the CN5 fragment was tested by CN5 affinity
chromatography. The column profiles were analyzed by SDS-PAGE.
A, /
-Tm alone showed weak binding to the immobilized
CN5 fragment, demonstrated by Tm in the flow-through
(F/T) as well as an early elution peak at
120-160 mM NaCl. B, fast TnT (fTnT)
alone bound strongly to the CN5 column, because it was absent in the
flow-through and showed a primary peak eluting at 280-360
mM NaCl. C, the Tm·TnT complex bound more
effectively to the CN5 column, resulting in a main Tm·TnT peak
eluting at 480-560 mM NaCl significantly higher than that
observed for Tm or TnT alone. D, addition of fast TnI
(fTnI) weakened the binding of Tm·TnT with the immobilized
CN5 protein as evidenced by the elution of Tm·TnT·TnI from the
affinity column between 280 and 400 mM NaCl. All SDS-PAGE
was 12% gel with an acrylamide:bisacrylamide ratio of 29:1 and was
silver-stained to visualize the resolved proteins.
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Fig. 8.
Effects of thin filament regulatory proteins
on the binding of CN5 to F-actin. The effect of TnT, Tm, and TnI
binding on CN5 interaction with F-actin was demonstrated by F-actin
cosedimentation experiments titrating Tm or Tn subunits in the presence
of a constant concentration of CN5 (1.52 µM). In each
panel, the precipitates from ultracentrifugation were resolved by
SDS-PAGE and silver-stained. The molar ratio of F-actin to the titrated
protein during the cosedimentation is indicated under each lane. Bound
fractions were assessed by densitometry of silver-stained gels, and the
binding of CN5 was plotted against total concentration of the thin
filament regulatory proteins. Tm moderately reduced CN5 binding to
F-actin in contrast to TnT, which more effectively decreased the
binding of CN5 to F-actin (A). The addition of Tm·TnT
binary complexes to the titration also demonstrated a saturable
inhibitory effect. (B) addition of TnI to the Tm·TnT
binary complex slightly increased the inhibitory effect on CN5 binding
to F-actin.
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Fig. 9.
Binding of Tm·Tn to F-actin in the presence
and absence of CN5. F-actin cosedimentation experiments were done
using a constant concentration of F-actin (2.66 µM) and
serial concentrations of Tm·Tn in the presence ( ) or absence (
)
of a fixed concentration of CN5 (1.52 µM). The binding of
Tm·Tn to F-actin was analyzed by SDS-PAGE and gel densitometry on
silver-stained gels. The data are plotted as the bound Tm·Tn
versus the total Tm·Tn used in the experiments. The
results demonstrate that inclusion of the nebulette fragment increased
the affinity of Tm·Tn for F-actin as determined by the concentration
of Tm·Tn required for half-maximal saturation (147.4 ± 32.1 nM without CN5; 70.8 ± 4.9 nM with CN5).
The results presented are the pooled average ± S.D. from three
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
-Tm to F-actin (Fig.
8A) was not mutually exclusive, although there was apparent competition consistent with both proteins binding to subdomain 1 on the
actin monomer (25). However, it was evident that CN5 binding to
undecorated F-actin was at approximately twice the stoichiometry when
compared with Tm, Tm·TnT, or Tm·TnT·TnI-decorated F-actin (Fig.
8). This finding suggests that decoration of naked actin filaments with
nebulin-like proteins is not fully indicative of their physiological
position on F-actin and that the regulatory Tm·Tn filament on F-actin
is able to further constrain nebulin binding sites. In addition, the
results demonstrate that both nebulin and Tm have binding domains on
F-actin that are not mutually exclusive, although the cosedimentation
experiments did demonstrate some overlap (Fig. 8). This recapitulates
an in vivo setting in which nebulin-like motifs and Tm
dimers run parallel along the F-actin filament. Using affinity
chromatography, we found a weak but reproducible association between
the five-motif nebulette fragment and
/
-Tm (Fig. 7A).
Although this interaction was a relatively low affinity, it opened the
possibility that head-to-tail-linked Tm dimers and repeating
nebulin-like motifs running parallel along the actin filament may
provide a higher total avidity of interaction. Because the allosteric
regulation of striated muscle contraction is known to involve shifts of
the position of Tm on F-actin (27, 28), nebulin-like motifs and Tm may
come closer into contact or distance themselves depending on the
dynamic position of Tm on the actin filament, thus allowing the
nebulin-Tm association to contribute reversibly to thin filament
dynamics. This model agrees with previously published data (25)
demonstrating that one site of nebulin-actin interaction lies at
subdomain 1 of the actin, a position believed to be occupied by
tropomyosin during the blocked or resting state of the striated muscle
thin filament.
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FOOTNOTES |
---|
* This study was supported in part by a grant-in-aid from the Heart and Stroke Foundation of Canada (to J.-P. J.) and Postdoctoral Award 0120359B from the American Heart Association, Ohio Valley affiliate (to O. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EBI Data Bank with accession number(s) AF440239.
To whom correspondence should be addressed. Tel.: 216-368-5525;
Fax: 216-368-3952; E-mail: jxj12@po.cwru.edu.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M205853200
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ABBREVIATIONS |
---|
The abbreviations used are: Tm, tropomyosin; CN5, 5-motif chicken nebulette fragment; mAb, monoclonal antibody; MSN, 4-motif mouse nebulin fragment; RT, reverse transcription; Tn, troponin; TnC, troponin C; TnI, troponin I; TnT, troponin T; PIPES, piperazine-N,N'- bis(2-ethanesulfonic acid); kb, kilobase; SSPE, saline/sodium phosphate/EDTA.
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REFERENCES |
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