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Journal of Cell Science 112, 69-79 (1999)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS0137
Identification of the A-band localization domain of myosin binding proteins C
and H (MyBP-C, MyBP-H) in skeletal muscle
Rénald Gilbert*, Julie A. Cohen, Sherly Pardo, Amartya Basu and Donald A. Fischman‡
Department of Cell Biology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, USA
*Present address: Montreal Neurological Institute, 3801 University Street, Montreal, QC, Canada H3A 2B4
‡Author for correspondence (e-mail: [email protected])
Accepted 26 October; published on WWW 8 December 1998
SUMMARY
Although major constituents of the thick filaments of
vertebrate striated muscles, the myosin binding proteins
(MyBP-C and MyBP-H) are still of uncertain function.
Distributed in the cross-bridge bearing zone of the A-bands
of myofibrils, in a series of transverse 43 nm stripes, the
proteins are constructed of a tandem series of small
globular domains, each composed of ~90-100 amino acids,
which have sequence similarities to either the C2-set of
immunoglobulins (IgC2) and the fibronectin type III
(FnIII) motifs. MyBP-C is composed of ten globular
domains (~130 kDa) whereas MyBP-H is smaller (~58 kDa)
and consists of a unique N-terminal segment followed by
four globular domains, the order of which is identical to
that of MyBP-C (FnIII-IgC2-FnIII-IgC2). To improve our
understanding of this protein family we have characterized
the domains in each of these two proteins which are
required for targeting the proteins to their native site(s) in
the sarcomere during myogenesis. Cultures of skeletal
muscle myoblasts were transfected with expression
plasmids encoding mutant constructs of the MyBPs
bearing an N-terminal myc epitope, and their localization
to the A-band examined by immunofluorescence
microscopy. Based on the clarity and intensity of the myc
A-band signals we concluded that constructs encoding the
four C-terminal motifs of MyBP-C and MyBP-H (~360
amino acids) were all that was necessary to efficiently
localize each of these peptides to the A-band. Truncation
mutants lacking one of these 4 domains were less efficiently
targeted to the C-zone of the sarcomere. Deletion of the last
C-terminal motif of MyBP-H, its myosin binding domain,
abolished all localization to the A-band. A chimeric
construct, HU-3C10, in which the C-terminal motif of
MyBP-H was replaced by the myosin binding domain of
MyBP-C, efficiently localized to the A-band. Taken
together, these observations indicate that MyBP-C and
MyBP-H are localized to the A-band by the same Cterminal domain, composed of two IgC2 and two FnIII
motifs. A model has been proposed for the interaction and
positioning of the MyBPs in the thick filament through a
ternary complex of the four C-terminal motifs with the
myosin rods and titin.
INTRODUCTION
isoforms have been reported (Takano-Ohmuro et al., 1989). In
contrast, only one isoform of MyBP-H has been isolated to date
(Starr and Offer, 1983; Bahler et al., 1985a; Vaughan et al.,
1993a,b). In skeletal muscle, MyBP-H is principally associated
with fibers of fast twitch muscles (Bahler et al., 1985b; Bennett
et al., 1986; Vaughan et al., 1993a). In cardiac tissue it is
restricted to the myofibrils of Purkinje fibers (Alyonycheva et
al., 1997a). The MyBPs are predicted to be composed of a
series of globular motifs, each 90-100 amino acids in length,
which bear resemblance to the C2-set of the immunoglobulin
superfamily (IgC2) and the fibronectin type III motif (FnIII).
Depending on the isoform, MyBP-C is composed of ten or
eleven motifs (skeletal muscle isoforms have 10 motifs, cardiac
muscle isoforms 11) whereas MyBP-H is smaller and contains
only four motifs (Fig. 1). Except for its unique N-terminal
sequence of 131 amino acids, MyBP-H is quite homologous to
Two closely related members of the family of myosin binding
proteins (MyBPs), myosin binding protein C (MyBP-C) and
myosin protein H (MyBP-H), also known as C-protein and Hprotein, respectively, are significant myofibrillar constituents of
vertebrate skeletal and cardiac muscles. Located in the A-band
of the myofibrils in close association with the thick filaments,
they are restricted to a series of transverse stripes, 43 nm apart,
in the central cross-bridge bearing region (Craig and Offer,
1976; Dennis et al., 1984; Bahler et al., 1985b; Bennett et al.,
1986). Three isoforms of MyBP-C (skeletal fast, skeletal slow
and cardiac) have been characterized in adult muscles
(Einheber and Fischman, 1990; Fürst et al., 1992; Weber et
al., 1993; Okagaki et al., 1993; Kasahara et al., 1994; Gautel
et al., 1995; Yasuda et al., 1995) and additional embryonic
Key words: C-protein, H-protein, A-band, Thick filament,
Sarcomere, Muscle protein, Development, Familial hypertrophic
cardiomyopathy, Titin
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R. Gilbert and others
MyBP-C and the arrangement of its four globular motifs is
identical to the last four C-terminal motifs of MyBP-C (Fig.
1). The unique N-terminal region of MyBP-H, composed of
short repetitive sequences enriched in prolines and alanines, is
responsible for the abnormally slow mobility of MyBP-H upon
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
(Vaughan et al., 1993a).
One of the best characterized properties of the MyBPs is
their relatively strong affinity for myosin (Offer et al., 1973;
Moos et al., 1975; Yamamoto, 1984). Using an in vitro binding
assay it has been shown that the principle myosin binding
domain of MyBP-C resides within the C-terminal IgC2 motif,
a region that is highly conserved in all MyBPs (Okagaki et al.,
1993). MyBP-C also binds to titin and weakly to actin (Moos
et al., 1978; Yamamoto, 1986; Fürst et al., 1992; Koretz et al.,
1993). Its titin binding domain has been mapped to the last
three or four C-terminal motifs (Freiburg and Gautel, 1996). It
has been reported that mammalian MyBP-H does not bind to
titin (Soteriou et al., 1993) but avian MyBP-H appears to do
so (D. A. Fischman and M. Gautel, unpublished observations).
The precise functions of the MyBPs are uncertain. In the
case of MyBP-C, evidence exists for a role in myofibril
assembly and the regulation of contraction. When purified
myosin is polymerized in vitro at physiological stoichiometries
with MyBP-C, the resulting thick filaments are slightly longer
and more regular than in the absence of MyBP-C (Koretz,
1979; Davis, 1988). The presence of MyBP-C reduces the
critical concentration required for myosin polymerization
suggesting that it may regulate thick filament assembly (Davis,
1988). This is consistent with the observation that coexpression of the MyBPs with myosin heavy chain in COS
cells promotes the formation of long cable-like co-polymers of
both proteins (Seiler et al., 1996). Deletion of the C-terminal
IgC2 motif from MyBP-H or MyBP-C prevents such cable
formation. In addition, we have observed that the expression
of truncated forms of MyBP-C lacking the myosin binding
domain in muscle cultures inhibit myofibrillogenesis (Gilbert
et al., 1996). It has recently been shown that mutations of the
MyBP-C cause familial hypertrophic cardiomyopathy
(Watkins et al., 1995; Bonné et al., 1995). Most but not all of
these mutations generate truncated forms of MyBP-C that alter
or lack the myosin or the myosin and titin binding domains
(Watkins et al., 1995; Carrier et al., 1997; Yu et al., 1998). It
is not clear how expression of these truncated proteins cause
this disease, but our work in muscle cultures suggests that they
may interfere with the assembly of the myofibrils.
The observation that MyBP-C inhibits actin-activated
skeletal muscle myosin ATPase but stimulates actin-activated
cardiac muscle myosin ATPase (Offer et al., 1973; Watkins,
1998; Yamamoto and Moos, 1983; Hartzell, 1985) suggests a
potential role in the regulation of contraction. The cardiac
isoform of MyBP-C is phosphorylated by two sets of kinases:
one that is calcium-calmodulin regulated (Gautel et al., 1995)
the other by a cAMP dependent kinase (PKA); and this
phosphorylation correlates with the rate of twitch relaxation
(Hartzell and Titus, 1982; Hartzell, 1984). The reactive serine
residues have been identified and are located at the N terminus
within the linker between motifs I and II of the cardiac isoform
of MyBP-C (Gautel et al., 1995). Recent studies indicate that
phosphorylation of cardiac MyBP-C by PKA increases the
orientation of crossbridges and thereby affects crossbridge
cycling (Weisberg and Winegrad, 1996, 1998). Partial
extraction of MyBP-C from myofibrils enhances tension
generation at submaximal concentrations of Ca2+ and
accelerates the contractile velocity at low levels of Ca2+
activation, again supporting a role in the regulation of
contraction (Hofmann et al., 1991a,b). MyBP-H may also be
involved in the regulation of contraction since it inhibits actinactivated skeletal muscle myosin ATPase in vitro (Yamamoto,
1984).
We have previously demonstrated that the C terminus of
MyBP-C encodes the information that specifies its targeting to
the A-bands of myofibrils (Gilbert et al., 1996). However, in
that study it was not certain if this information was encoded by
the last four or by the last three C-terminal motifs. In addition,
the motifs of MyBP-H that interact with the in vivo thick
filament, and those essential for its accurate localization to the
A-band had not been identified. To address these questions, we
have analyzed the expression and distribution of a series of
truncation mutants of the MyBPs in muscle cultures. We show
that both MyBP-C and MyBP-H are targeted to the A-band by
the same domain composed of two IgC2 and two FnIII motifs
at their C-termini. We also demonstrate that the C-terminal
IgC2 motif of MyBP-C (its myosin binding domain) can
replace the C-terminal IgC2 motif of MyBP-H without
affecting its localization to the A-band, indicating that these
motifs share similar targeting properties. The data suggest a
topological model in which the MyBPs are associated with the
thick filament through the same four globular motifs at their
C-termini.
MATERIALS AND METHODS
Vector constructs
Recombinant DNA procedures followed standard methods (Sambrook
et al., 1989). The polymerase chain reaction (PCR) products and the
junctions of the spliced regions of the various constructs were all
confirmed by DNA sequencing, which was accomplished by the DNA
Services facility of Cornell University (Ithaca, NY). Unless otherwise
specified, the linkers and enzymes were purchased from New England
Biolabs. For PCR, the primers were synthesized by the DNA Services
facility of Cornell University (Ithaca, NY) and the PCR reactions were
accomplished using the GeneAmp PCR Reagent Kit with AmpliTaq
DNA Polymerase (Perkin Elmer) according to the manufacturer’s
recommendations.
Mutant C7-10 was generated by PCR using pMC (Gilbert et al.,
1996) as the template and the following two primers: 5′ATTCTGCAGTCCGACGAGCGAACCGACCCACGTG-3′ and 5′GACTCTAGAACCCAAAACGCCGCTC-3′. The first primer
generated a PstI site in front of proline 728 in the protein sequence.
The amplified DNA fragment was digested with PstI and NotI and
cloned into the PstI and NotI sites of pBSmyc (Gilbert et al., 1996).
This reaction added the linker encoding the myc tag at the 5′ end of
C7-10. The insert was then excised by digestion with HindIII and NotI
and cloned into the HindIII and NotI sites of pMC. Mutant C8-10,
was generated by PCR using pMC as the template and the following
two primers: 5′-ATTATCTGCAGTCCGCGGCACCTCCGCCAG-3′
and 5′-GACTCTAGAACCCAAAACGCCGCTC-3′. The first primer
generated a PstI site in front of proline 835 in the protein sequence.
The amplified DNA fragment was digested with PstI and NotI and
cloned into the PstI and NotI sites of pBSmyc. This reaction added
the linker encoding the myc tag at the 5′ end of C8-10. The insert was
then excised by digestion with HindIII and NotI and cloned into the
Myosin binding proteins
HindIII and NotI sites of pMC. The plasmid expressing MyBP-H of
chicken skeletal muscle with a myc tag at the N terminus (pMH) was
generated according to the following protocol: a plasmid encoding the
cDNA of MyBP-H with a NdeI site engineered at its initiation codon
(Vaughan et al., 1993a), was digested with NdeI and a double stranded
linker (5′-TATGTGAATTCACA-3′) encoding two NdeI and one
EcoRI sites was ligated at this site. MyBP-H cDNA was then removed
by digestion with EcoRI and cloned into the EcoRI site of pBSmyc.
Myc-tagged MyBP-H cDNA was then removed by digestion with
HindIII and BalI, the ends were blunted with Klenow, it was then
cloned into the SmaI site of pJDp (Mikawa et al., 1992). To construct
HU, a PCR reaction was done using pMH as the template and the
following
two
primers:
5′-AGTCTACCATATGCCCAAAGAAGAGCCGCCCAG-3′ and 5′-CTGTGTTGCGGATGTTGACC-3′.
The first primer generated an NdeI site in front of proline 130 in the
protein sequence. The PCR product was digested with NdeI and
BstEII and cloned into the NdeI and BstEII sites of pMH. To construct
HU-1, pMH was digested with BamHI and the fragment encoding
motifs II, III and IV was isolated, blunted with Klenow and cloned
into the SmaI site of pBSmyc. The insert was removed by digestion
with NcoI and XbaI and cloned into the NcoI and XbaI sites of pMH.
Mutant H4 was generated by PCR using pMH as the template and the
following two primers: 5′-GCTGTGAACCTGCTGATCC-3′ and 5′CTACGTCTAGAGGGATCTTCTGTGGCTGG-3′. The second
primer generated an XbaI site after proline 438 in the protein
sequence. The PCR product was digested with BstEII and XbaI and
cloned into the BstEII and XbaI sites of pMH. The resulting plasmid
was called pMH4X. A termination codon was generated after proline
438 by inserting into the XbaI site of pMH4X a NheI linker (5′CTAGCTAGCTAG-3′) that encodes a termination codon in any
reading frame. To generate the chimera HU-3C10, the C-terminal Ig
C2 motif of MyBP-C was amplified by PCR using pMC as the
template
and
the
following
two
primers:
5′ATCTATCTAGAACGCGACCTCCGCGCCGCCCCAC-3′ and 5′CGTCGAAGCTTACCCCAAAACGCCGCTCTG-3′.
The
first
primer provided an XbaI site before glutamic acid 1032 in the protein
sequence and the second primer generated an HindIII site after the
termination codon. The PCR product was digested with XbaI and
HindIII and cloned into the XbaI and HindIII sites of pMH4X.
Western blots
Canine fibroblasts (D17) and primary cultures of 11-day-old
embryonic chicken pectoralis myoblasts were cultured, transfected
with a mixture of DNA and lyposomes, and processed for western
blotting as described previously (Gilbert et al., 1996). One day after
the beginning of transfection of chicken myoblasts, the fetal bovine
serum of the culture medium was replaced with 10% heat inactivated
horse serum (Hyclone). The fibroblasts and the myoblasts were
analyzed one day, or three and four days after the beginning of
transfection, respectively. To improve the transfer of positively
charged proteins, such as HU-1, the proteins were transferred to
nitrocellulose membranes in 25 mM 3-N-cyclohexylamino-2
hydroxypropanesulfonic acid (CAPSO, Sigma), pH 10, 20% methanol
(Szewczyk and Kozloff, 1985). The blots were incubated with
monoclonal antibodies (mAbs) specific for the fast isoform of chicken
skeletal MyBP-C (MF1) (Reinach et al., 1982) or for the myc epitope
(9E10) (Evan et al., 1985), followed by horseradish peroxidaselabeled affinity purified goat anti-mouse antibodies (Sigma). Blots
were also incubated with an affinity purified anti-H antibody (see
below), followed by horseradish peroxidase-labeled affinity purified
goat anti-rabbit antibodies (Sigma). The antibody complex was
visualized by chemiluminescence (Dupont).
Myoblast cultures and immunofluorescence microscopy
Myoblasts were isolated, grown and transfected as described above.
The cells were processed for indirect immunofluorescence three to
four days after transfection as described previously (Gilbert et al.,
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1996). To study the distribution of the recombinant proteins and
myosin, the cells were incubated with mAb 9E10, followed by
Texas Red-conjugated affinity purified rabbit anti-mouse IgG1
antibodies (American Qualex). They were then incubated with F59,
a mAb specific for the S1 fragment of chicken skeletal myosin
heavy chain, isotype IgG1 (Miller et al., 1989) which was
conjugated to biotin (Harlow and Lane, 1988), followed by FITCconjugated avidin (Cappel). To study the distribution of the
endogenous MyBP-H and myosin, the cells were incubated with the
affinity purified anti-H antibody (see below), followed by FITCconjugated affinity purified goat anti-rabbit antibodies (Cappel).
They were then incubated with F59, followed by Texas Redconjugated affinity purified goat anti-mouse IgG antibodies
(Jackson). Samples were examined with a Nikon Microphot SA
upright epifluorescence microscope and photographs taken with
Kodak TMAX 400 black and white film.
Production and purification of antibodies against MyBP-H
Native MyBP-H from chicken pectoralis muscle was purified
according to the method of Okagaki et al. (1993). The purified protein
was resolved in 7% polyacrylamide gel, the 86 kDa band was excised,
the protein eluted electrophoretically from the gel, and used as an
immunogen. Two female New Zealand white rabbits were injected
subcutaneously with 100 µg of MyBP-H mixed with an equal volume
of complete Freud’s adjuvant. The rabbits were boosted four times
with 50 µg of MyBP-H mixed with an equal volume of incomplete
Freud’s adjuvant. The antibody against MyBP-H (anti-H antibody)
was affinity purified using a column made with recombinant MyBPH produced in Escherichia coli. Briefly, the cDNA of MyBP-H was
excised from pMH by digestion with EcoRI and SalI, cloned in frame
into the EcoRI and XhoI sites of the GST-fusion vector pGEX-4T-3
(Pharmacia), and transformed into BL21(DE3)pLys cells. The
transformed cells were grown to an OD600 of 0.3 in Luriat Bertani
(LB) medium supplemented with 50 µg/ml carbenicillin and 34 µg/ml
chloramphenicol. The bacteria were then induced with 0.1 mM
isopropyl-1-thio-β-D-galactopyranoside (Sigma) for 4 hours at 37°C.
The induced GST-MyBP-H was purified from soluble bacterial extract
by affinity chromatography using a glutathione-Sepharose 4B column
(Pharmacia) according to the manufacturer’s recommendations. The
purified GST-MyBP-H was dialyzed extensively against PBS and was
coupled to CNBr-activated Sepharose 4B (Sigma) as described by the
instructions of the manufacturer. The immunoglobulins of the anti-H
serum were fractionated and concentrated by ammonium sulfate
precipitation (Harlow and Lane, 1988). They were resuspended in
PBS, dialyzed against PBS and passed twice through the MyBP-Hconjugated Sepharose column. The bound antibodies were eluted with
0.1 M glycine-HCl, pH 3.0, and neutralized with 3 M Tris-HCl, pH
8.0. Fractions containing purified immunoglobulins were pooled and
dialyzed against PBS.
RESULTS
Description of the mutants of MyBP-C
In a previous study (Gilbert et al., 1996), we showed that fulllength recombinant MyBP-C was correctly and efficiently
incorporated into the A-bands of myofibrils following
expression of its cDNA plasmids in cultured myotubes. We
also demonstrated that the information specifying localization
of MyBP-C to the A-band is encoded by its C terminus.
However, it remained unclear if this information was encoded
by the last four or the last three C-terminal motifs of MyBPC. To clarify this point, mutants C7-10 and C8-10 were
constructed and their distribution in cultured myotubes
investigated by immunofluorescence microscopy. C7-10 and
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R. Gilbert and others
Fig. 2. Evidence for the expression of C7-10 and C8-10 in cultured
muscle. Myoblasts transfected with plasmids without insert (-), or
coding for C7-10, or C8-10 were analyzed by SDS-PAGE after
differentiation. The proteins were then transferred to a nitrocellulose
membrane and subsequently reacted with mAbs specific for the myc
epitope. Numbers at the left of each gel indicate the relative
mobilities of molecular mass markers (kDa).
Fig. 1. Description of the mutants used in the present study.
(A) Mutants of MyBP-C (white). (B) Mutants of MyBP-H (black).
The numbers above the proteins correspond to the first and last
amino acids, starting from the N terminus (left). The roman numerals
below indicate the position of each IgC2 and FnIII motif. The
position of the myc epitope at the N terminus is indicated. The
distribution of each protein was investigated in well differentiated
myotubes and their presence in the A-band is noted. Mutant HU3C10 was constructed by fusing the first 438 amino acids of MyBPH to amino acids 1032 to 1132 of MyBP-C.
C8-10 encode motifs VII to X and VIII to X, respectively (Fig.
1). A short linker encoding a myc epitope of 12 amino acids
was inserted at the 5′ end of all cDNAs and a mAb specific for
this epitope was used to study recombinant protein expression
in the muscle cells. We have shown previously that the
presence of the myc epitope does not interfere with the
localization of MyBP-C to the A-band (Gilbert et al., 1996).
Pectoralis myoblasts were isolated from day 11 chicken
embryos and transfected with plasmids encoding C7-10 and
C8-10 one day after plating in monolayer. Three to four days
later, the time required for the myoblasts to fuse and
differentiate into myotubes with robust cross-striated
myofibrils, the expression of these two constructs was analyzed
by western blots and by immunofluorescence microscopy.
Bands of 55 kDa and 40 kDa were observed in the lanes
containing lysates of myotubes expressing C7-10 and C8-10,
respectively (Fig. 2). The relative mobilities of these bands
correspond to the expected products of these two constructs.
An extra band with a relative mobility ~45 kDa was also
present in both lysates. This band was also observed when untransfected myotubes or myotubes transfected with plasmids
lacking an insert were analyzed (Fig. 2). Since the predicted
molecular mass of the chicken c-myc is 45 kDa (Shih et al.,
1984), this extra band most likely corresponds to endogenous
c-myc protein expressed in the fibroblasts and undifferentiated
myoblasts present in the culture. The products of C7-10 and
C8-10 were not recognized by mAb MF1 (Reinach et al.,
1982), an antibody specific for the fast isoform of MyBP-C
(data not shown). This is in agreement with a previous study
indicating that the epitope recognized by MF1 is located within
motifs III to VI of MyBP-C (Gilbert et al., 1996).
Motifs VII to X encode the A-band localization
domain of MyBP-C
The peptide encoded by mutant C7-10 was efficiently localized
to the A-bands of cross-striated myofibrils. Following
expression of its cDNA in cultured muscle, it co-localized with
myosin heavy chain (Fig. 3A,B). In the A-band, C7-10 was
distributed as a doublet, with one bright stripe on each side of
the M-line. At the level of the light microscope, the distribution
of this truncation mutant was identical to that of endogenous
MyBP-C or full-length recombinant MyBP-C. In addition, its
signal intensity in the A-band and the sharpness of the doublet
were comparable to that of recombinant full-length MyBP-C.
Mutant C8-10 also co-localized with myosin heavy chain in the
A-band (Fig. 3C,D). However, its signal intensity in the Abands was weak, sometimes blurred and most of the product
of C8-10 remained diffuse in the cytoplasm. At higher
magnification, a weak doublet with one faint band on each side
of the M-line was apparent. We have previously demonstrated
that larger deletions of the N terminus of MyBP-C, or deletion
of one or more of the C-terminal motifs, completely abolish
localization to the A-band (Gilbert et al., 1996). Taken
together, these data indicate that the A-band targeting domain
of MyBP-C is encoded by motifs VII to X. This contains the
minimal sequence allowing its efficient localization to the Aband, because removal of one more motif at the N- or C
terminus reduces or abolishes localization to this site.
Description of the mutants of MyBP-H
MyBP-H, which shares extensive homologies with MyBP-C
Myosin binding proteins
(Vaughan et al., 1993a), is another significant constituent of
the thick filaments of vertebrate striated muscle (see
Introduction). MyBP-H is smaller than MyBP-C and consists
of a unique N-terminal sequence of 131 amino acids followed
by two IgC2 and two FnIII motifs (Fig. 1B). In chicken muscle,
this protein is found in a series of 9 transverse stripes 43 nm
apart in the C-zone of each half A-band (Bahler et al., 1985b).
To define which motifs specify the precise localization of
MyBP-H to the A-band, a series of mutants was constructed
and their expression was investigated in muscle cultures by
western blot and by immunofluorescence microscopy. The
structures of the MyBP-H mutants used in the present study
are shown in Fig. 1B.
Mutant HU lacks the first 129 amino acids of MyBP-H
encoding its unique N-terminal sequence. Mutant HU-1 lacks
the first 237 amino acids of MyBP-H containing the unique
sequence and the first FnIII motif. The last 101 amino acids
are deleted in mutant H4. Therefore, this truncation mutant
lacks the C-terminal IgC2 motif (motif IV) of MyBP-H
containing its myosin binding domain (Alyonycheva et al.,
1997b). Mutant HU-3C10 was generated by replacing motif IV
of MyBP-H with the C-terminal IgC2 motif of MyBP-C, motif
X, containing its myosin binding domain. To distinguish the
recombinant proteins from endogenous MyBP-H normally
expressed in cultured muscle, a linker encoding a myc epitope
73
of 12 amino acids was inserted at the 5′ end of all cDNAs. The
full-length MyBP-H encoding the myc epitope is termed
MyBP-Hmyc.
To compare the distribution of MyBP-Hmyc with endogenous
MyBP-H, a polyclonal antibody was raised against MyBP-H.
The antigen consisted of a pure preparation of MyBP-H
isolated from the pectoralis muscle of adult White Leghorn
chickens. The antibody was affinity purified against full-length
recombinant MyBP-H synthesized in E. coli. The specificity of
the anti-H antibody was investigated by western blot using
lysates of un-transfected myotubes and lysates of fibroblasts
transfected with plasmids encoding MyBP-Hmyc (Fig. 4A). A
single product of ~85-86 kDa was detected in the lane
containing lysate of myotubes. This product migrated with the
predicted mobility of the chicken MyBP-H (Bahler et al.,
1985a; Vaughan et al., 1993a). When fibroblasts expressing
MyBP-Hmyc were analyzed, a single band was observed that
migrated with the same relative mobility as endogenous
MyBP-H. This band was absent in the lane containing
fibroblasts transfected with plasmids lacking an insert, thus
confirming the specificity of the anti-H antibody (Fig. 4A). The
mobility of MyBP-H in SDS-PAGE is aberrantly slow; its
cDNA encodes a protein with a predicted molecular mass of
54 kDa (Vaughan et al., 1993a). The unique N-terminal
sequence of MyBP-H is responsible for this aberrant mobility.
Fig. 3. Distribution of C7-10 and C8-10. Myoblasts transfected with plasmids coding for C7-10 (A,B), or C8-10 (C,D) were processed for
immunofluorescence after differentiation. The cells were double immunostained with mAbs specific for the myc epitope and sarcomeric
myosin. The left row shows the distribution of the recombinant proteins (Texas Red channel), the right row indicates the distribution of myosin
heavy chain in the same cells (FITC channel). C7-10 and C8-10 are localized to the A-bands of cross-striated myofibrils. Note that the signal of
C8-10 in the A-band is not as sharp as that of C7-10 and that a relatively large fraction of the protein remains diffuse in the cytoplasm. Insets:
higher magnifications illustrating that C7-10 and C8-10 are distributed as A-band doublets (arrows, A,C). Bars, 20 µm.
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R. Gilbert and others
When this sequence is removed, the resulting protein migrates
with a mobility that corresponds to the value deduced from its
amino acid sequence (Vaughan et al., 1993b).
Before studying the distribution of MyBP-Hmyc and its
mutants in cultured skeletal myoblasts, we examined the size
and antigenicity of the expressed proteins in these cells.
Chicken myoblasts were transfected one day after plating with
plasmids encoding MyBP-Hmyc, HU, HU-1, H4 and HU-3C10.
Three to four days later, the cells were lysed and analyzed by
western blots using a mAb against the myc epitope. Lysates of
myotubes transfected with MyBP-Hmyc and the four mutants
contained products that were recognized by the anti-myc
antibody and that migrated with the expected mobility (Fig.
4B). A band of 45 kDa was detected in all the lanes containing
myotube lysates (Fig. 4B). As mentioned above, the size of this
band and the fact that it is recognized by the anti-myc antibody
suggest it corresponds to endogenous chicken c-myc protein.
Fibroblast cultures expressing the various MyBP-H mutants
were also analyzed by western blot using the anti-H antibody.
Fig. 4. Specificity of the anti-H antibody and evidence for expression
of the MyBP-H mutants in cell culture. (A) Culture of myotubes
(myo), or fibroblasts transfected with plasmids without insert (-), or
coding for MyBP-Hmyc were analyzed by SDS-PAGE after
differentiation. The proteins were then transferred to a nitrocellulose
membrane and reacted with the anti-H antibody. (B) Myoblasts
transfected with plasmids without insert (-), or coding for MyBPHmyc, HU, H4, HU-3C10, or HU-1 were analyzed by SDS-PAGE
after differentiation. The proteins were then transferred to a
nitrocellulose membrane and reacted with mAbs specific for the myc
epitope. Numbers at the left of each gel indicate the relative
mobilities of molecular mass markers (kDa).
All of the constructs were recognized specifically by this
antibody except for HU-1 (data not shown). This observation
indicates that the epitopes recognized by the anti-H antibody
are mainly located within the unique sequence and motif I.
Motifs I to IV encode the A-band localization domain
of MyBP-H
To test if recombinant MyBP-H would incorporate into the Abands of the cross-striated myofibrils, myoblasts were isolated
from day-11 chicken embryos and transfected with plasmids
encoding MyBP-Hmyc. Three to four days after transfection,
the cells were fixed and processed for immunofluorescence
microscopy. The distribution of MyBP-Hmyc was assessed by
staining the cells with a mAb against the myc epitope and
compared with myosin by double-labeling the cells with mAbs
specific for myosin. MyBP-Hmyc distribution was also
compared with endogenous MyBP-H by double-staining the
cells with the anti-H antibody and mAbs specific for myosin.
In differentiated myotubes, MyBP-Hmyc staining was confined
to the A-band of myofibrils, co-staining this region of the
sarcomere with mAbs against myosin (Fig. 5A,B). At the
resolution of the light microscope, the distribution of MyBPHmyc was identical to that of endogenous MyBP-H (compare
Fig. 5A,C). Both proteins stained as doublets, one fluorescent
stripe in each half A-band (insets Fig. 5A,C).
To identify those domains of MyBP-H containing the
information required for localization of the protein to the Aband, myoblasts were transfected with the mutants of Fig. 1B.
In well differentiated myotubes, HU was identified in
fluorescent doublets (Fig. 6A,B), one stripe in each half Aband (inset, Fig. 6A). The distribution of HU and its signal
intensity in the A-band was indistinguishable from that of
MyBP-Hmyc. Thus, the unique N-terminal sequence of MyBPH is not required for efficient A-band localization of this
protein. In well differentiated myotubes, HU-1 was also colocalized with myosin in the A-band. However, its signal
intensity in the A-band was weaker than that of MyBP-Hmyc
and a large fraction of HU-1 remained diffuse in the cytoplasm
(Fig. 6C). At higher magnification, a faint and somewhat
blurred doublet was observed (Fig. 6C, inset). These data
indicate that HU-1 was incorporated less efficiently into the Aband than HU and that motifs II to IV do not compete very
efficiently with endogenous MyBP-H for A-band localization.
Therefore, motif I contains important targeting information and
is needed for efficient localization of MyBP-H to the A-band.
The results obtained with HU and HU-1 are similar to those
obtained with C7C10 and C8C10, respectively. Mutant H4,
which lacks motif IV at the C terminus, was not localized to
the A-band at all. Instead, it remained diffusely distributed in
the myotube sarcoplasm (Fig. 6E,F). Comparable results were
obtained by deleting motif X of MyBP-C where the resulting
mutant was not localized to the A-band (Gilbert et al., 1996).
These observations suggest that the A-band targeting domain
of these two MyBPs is similar and consists of the same four
analogous motifs. To further investigate this point, we asked if
motif X of MyBP-C could replace motif IV of MyBP-H. This
question was answered by studying the distribution of HU3C10. This chimera was efficiently localized to the A-band of
myofibrils (Fig. 6G,H). At higher magnification a clear doublet
was apparent (inset, Fig. 6G). The distribution of HU-3C10
appeared identical to that of MyBP-Hmyc. Its signal intensity
Myosin binding proteins
75
Fig. 5. Evidence for the incorporation of recombinant MyBP-H into A-bands. Myoblasts transfected with MyBP-Hmyc (A,B), or un-transfected
myoblasts (C,D) were processed for immunofluorescence after differentiation. The cells were double immunostained with mAbs specific for the
myc epitope and sarcomeric myosin (A,B), or with the anti-H antibody and mAbs specific for sarcomeric myosin (C,D). The left row of figures
indicates the distribution of the recombinant (A) and the endogenous MyBP-H (C). The right row shows the distribution of myosin in the same
cells. In mature myotubes, the recombinant and endogenous MyBP-H are localized to the A-bands of the cross-striated myofibrils. Insets:
higher magnifications illustrating that both recombinant and endogenous MyBP-H are distributed as doublets within each A-band (arrows,
A,C). Bars, 20 µm.
in the A-band and the sharpness of its doublet was comparable
to MyBP-Hmyc. This result indicates the C-terminal IgC2
motifs of both MyBP-C and MyBP-H possess comparable
targeting properties and can be interchanged with impunity, at
least in this assay.
DISCUSSION
In this report, we have identified the domains of MyBP-C and
MyBP-H that specify their intracellular targeting to the Abands of skeletal muscle myofibrils. These domains are
composed of motifs VII to X of MyBP-C and I to IV for
MyBP-H, the last four globular segments of both proteins. We
believe that these four motifs encode all of the targeting
information needed for faithful A-band incorporation in vivo
because mutants composed of these four motifs (mutants C710 and HU) were localized to the A-band in an identical
manner to the respective full-length proteins. The localization
domains of MyBP-C and MyBP-H possess very similar
characteristics: (i) their dimensions are the same (in both cases
the domains consist of two IgC2 and two FnIII motifs arranged
in alternating sequence); (ii) deletion of the upstream FnIII
motif in this domain reduces but does not abolish protein
targeting to the A-band (mutants C8-10 and HU-1); (iii)
deletion of the C-terminal motif in either domain completely
abolishes protein localization to the A-band (mutants H4 and
10 in Gilbert et al., 1996); and (iv) the C-terminal IgC2 motif
of MyBP-C can replace the C-terminal IgC2 motif of MyBPH without affecting localization of the chimeric protein to the
A-band (mutant HU-3C10). Based on these data, we have now
extended our previous model (Gilbert et al., 1996) concerning
the organization of the MyBPs in the thick filament to include
MyBP-H. We propose that both MyBP-C and MyBP-H make
contact with, and are positioned on, specific thick filament sites
by a localization domain consisting of two FnIII and two IgC2
motifs located at their C-termini (Fig. 7). In addition, because
the N-terminal moiety of these two proteins (motifs I to VI for
MyBP-C and the unique sequence of MyBP-H) are not
localized to the A-band when expressed in muscles (Gilbert et
al., 1996; Koshida et al., 1995), they may not be physically
part of the thick filament or are held on the thick filament by
the C-terminal domains of both proteins.
It is not known if MyBP-C and MyBP-H bind to the same
sites on the thick filament. Both proteins are localized at the
same stripes in the C-zone of the A-band in chicken pectoralis
76
R. Gilbert and others
Fig. 6. Distribution of the mutants of MyBP-H. Myoblasts transfected with mutants HU (A,B), HU-1 (C,D), H4 (E,F) and HU-3C10 (G,H)
were processed for immunofluorescence after differentiation. The cells were double immunostained with mAbs specific for the myc epitope and
sarcomeric myosin. The left row shows the distribution of the recombinant proteins (Texas Red channel), the right row indicates the distribution
of myosin in the same cells (FITC channel). Mutants HU, HU-1, and HU-3C10 were localized to the A-bands of cross-striated myofibrils (AD,G,H), whereas the product of H4 remains diffuse in the cytoplasm (E) and was not incorporated into the A-bands (F). Insets: higher
magnifications illustrating that HU, HU-1 and HU-3C10 are distributed as A-band doublets (arrows, A,C,G). Note that the signal of HU-1 in
the A-band is not as sharp as that for HU and HU-3C10 and that a relatively large fraction of the protein remains diffuse in the cytoplasm. Bars,
20 µm.
Myosin binding proteins
Fig. 7. Model indicating the tentative positioning of MyBP-C and
MyBP-H on the thick filament. The data present in this study suggest
that MyBP-C and MyBP-H are associated with the thick filament
through their last four C-terminal motifs. In this model, the Nterminal moiety of these two proteins does not bind to the thick or to
the thin filament in resting muscle. The C-termini of MyBP-C and
MyBP-H are depicted as lying on the surface of the thick filament,
but none of our data preclude deeper embedment of these proteins in
the thick filament. Roman numerals indicate the position of the IgC2
and FnIII motifs (see Fig. 1).
muscle (Bahler et al., 1985b). As demonstrated in the present
study, the last IgC2 motif of MyBP-C can replace the last
IgC2 motif of MyBP-H without affecting its localization to
the A-band. Furthermore, pure preparations of MyBP-H can
displace bound MyBP-C from synthetic myosin filaments
(Alyonycheva et al., 1997b). Taken together, these
observations suggest that MyBP-C and MyBP-H bind to the
same or closely associated sites. Subtle but specific binding
properties must exist that distinguish these two proteins since
MyBP-C and MyBP-H are distributed in the pectoralis
muscle of chicken in 7 or 9 stripes, respectively, in each half
A-band.
The C-terminal IgC2 motif is highly conserved among the
various members of the MyBP-C and MyBP-H families that
have been examined. More than 60 out of 100 amino acids are
conserved in birds, mouse and man. In addition, in vitro
binding studies have demonstrated that the major myosin
binding domain of MyBP-C and MyBP-H is located within the
C-terminal IgC2 motif (Okagaki et al., 1993; Alyonycheva et
al., 1997b). Because of these structural and functional
similarities, it is not surprising that the C-terminal motifs of
MyBP-C and -H can be interchanged without affecting the
localization of the resultant protein to the A-band. Electron
microscopy will be required to see if subtle differences exist
in the localization of the recombinant protein to specific stripes
in the C-zones of the A-bands.
The biochemical steps involved in the targeting of these
proteins to the A-band remain unknown. Using an in vitro
binding assay, we have demonstrated that the myosin binding
domains of MyBP-C and MyBP-H are located within the Cterminal IgC2 motif of both proteins (Okagaki et al., 1993;
Alyonycheva et al., 1997b). However, the myosin binding
domain of MyBP-C, although essential, is not sufficient for
protein targeting to the A-band, suggesting that other
biochemical interactions are required for this reaction (Gilbert
et al., 1996). MyBP-C binds to titin, and its titin binding
77
domain has been mapped to motifs VIII to X (Freiburg and
Gautel, 1996). A comparable titin binding domain has been
identified in chicken MyBP-H (D. A. Fischman and M. Gautel,
unpublished observations). The fact that the titin binding
motifs are part of the A-band localization domains of both
proteins suggests that an interaction with titin is as important
as an interaction with myosin for proper localization of either
protein to the A-band. Soteriou et al. (1993) have
demonstrated, using an overlay assay, that MyBP-H of rabbit
skeletal muscle does not bind to titin. Conceivably, there are
species-specific differences or technical differences in the
assays used. Future work will be required to iron out these
discordant data. However, it should be appreciated that MyBPH of the rabbit is present in only one stripe in each half A-band
(Bennett et al., 1986) whereas the chicken skeletal muscle
protein, used in the present study, is distributed in nine 43 nm
stripes in each half A-band (Bahler et al., 1985b). One of these
stripes, the one closest to the M-line, corresponds to the stripe
found in rabbit muscle. Conceivably, the incorporation of
rabbit MyBP-H to this single stripe does not require titin
binding, but association with titin may be required for the
localization of the chicken protein to the additional 8 stripes in
each half A-band avian muscle sarcomeres. Cross-species
studies comparing the incorporation of both proteins into the
corresponding muscle fibers now appear warranted.
Evidence has been presented that suggest the involvement
of MyBP-C in both myofibril assembly and the regulation of
contraction (see Introduction). These two functions are not
mutually exclusive and it is possible, as proposed earlier, that
different protein domains serve distinct functions (Gilbert et
al., 1996). It now seems clear that the last four C-terminal
motifs of MyBP-C are involved in myofibril assembly and it is
likely that these regions of the molecule are an integral part of
the thick filament (see Fig. 7). The observation that mutants of
MyBP-C with deletions at the C terminus inhibit
myofibrillogenesis supports this hypothesis (Gilbert et al.,
1996). The domains of MyBP-C involved in the regulation of
contraction may be located near the N terminus where it could
potentially interact with a neighboring thin filament or with an
adjacent cross-bridge. The cardiac isoform of MyBP-C is
phosphorylated, and there is a correlation between the extent
of such phosphorylation and the rate of cardiac relaxation
(Hartzell and Titus, 1982; Hartzell, 1984). Interestingly, the
phosphorylated amino acids are located in the linker joining
motifs I and II in this N-terminal region of MyBP-C where they
could participate in this regulation of contraction (Gautel et al.,
1995). Furthermore, phosphorylation of these residues by
phosphokinase A increases the apparent order of crossbridges
in isolated cardiac thick filaments, possibly affecting the
regulation of crossbridge cycling during cardiac muscle
contraction (Weisberg and Winegrad, 1996, 1998).
The function of MyBP-H is unclear. It appears that the last
four motifs of this protein serve the same function as the
comparable motifs in MyBP-C, since both are necessary and
sufficient for A-band targeting of the respective proteins. In
support of this hypothesis is the observation that co-expression
of MyBP-H or MyBP-C with sarcomeric myosin heavy chain
in COS cells promotes the formation of long cable-like copolymers of both proteins. The C terminus of MyBP-H is
involved in the formation of these myosin cables, for mutants
of MyBP-H lacking the C terminus fail to form the cables when
78
R. Gilbert and others
co-expressed with myosin heavy chain (Seiler et al., 1996). No
function has been assigned to the N-terminal unique sequence
of MyBP-H. Because of its proposed organization on the thick
filament, this peptide could theoretically interact either with the
N terminus of MyBP-C, an adjacent cross-bridge or some other
components of the thick filament. It is unlikely that it could
interact with the thin filament because of its short length.
MyBP-H can inhibit the actin-activated skeletal muscle myosin
ATPase in vitro (Yamamoto, 1984). With its cDNA now
available, it will be feasible to test whether this unique
sequence of MyBP-H is responsible for this ATPase inhibition.
Mutations of the cardiac isoform of MyBP-C in man have
been shown to cause familial hypertrophic cardiomyopathy
(FHC). Most but not all of the known mutations of MyBP-C
are characterized by deletions of their C terminus or by an
insertion within the C-terminal IgC2 motif, which probably
interferes with binding to myosin and/or titin (Watkins et al.,
1995; Bonné et al., 1995; Niimura et al., 1998; Carrier et al.,
1997; Yu et al., 1998). It is not clear how expression of these
mutated proteins cause FHC. The results of the present and
earlier studies (Gilbert et al., 1996; Koshida et al., 1995)
suggest that the mutations of MyBP-C which have disrupted
C-termini would have a lower association with the myofibrils
and be found in the sarcoplasm or rapidly degraded. FHC could
result from an insufficient quantity of MyBP-C in the thick
filament (i.e. haplo-insufficiency) assuming there is no upregulation of the second, wild-type MyBP-C allele. Since many
of these FHC patients have a mild phenotype it is also
conceivable that up-regulation of MyBP-H could partially
substitute for MyBP-C insufficiency. Expression of these
mutants could also affect the regulation of contraction or the
formation of myofibrils. Support for the latter view comes from
the prior demonstration that mutants of skeletal-type MyBP-C,
lacking portions of the C terminus, perturb myofibrillogenesis
when expressed in cultured muscle (Gilbert et al., 1996).
The authors express their sincere appreciation for the many helpful
suggestions and criticisms of this research by T. Mikawa, F. Reinach
and R. Welikson. Thanks are also expressed to F. E. StockDale
(Stanford University, CA) for antibody F59. Ms L. Ong and Ms C.
Siewert made important technical contributions to this study for which
the authors are very appreciative. This work was supported by grants
AR32147 and HL45458 from the NIH and generous private
contributions by Dr David Cofrin. R. Gilbert was an American Heart
Association postdoctoral fellow during the course of this study.
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