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Cardiovascular Research 2003 60(2):388-396; doi:10.1016/j.cardiores.2003.07.001
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Copyright © 2003, European Society of Cardiology

Biomolecular interactions between human recombinant β-MyHC and cMyBP-Cs implicated in familial hypertrophic cardiomyopathy

Jeanne Flavignya,1, Philippe Robertb,1, Jean-Claude Camelinb, Ketty Schwartza, Lucie Carrier*,a and Isabelle Berrebi-Bertrandb

aINSERM U582, Institut de Myologie, Bâtiment Babinski, CHU Pitié-Salpêtrière, 47 Bld de l'Hôpital, 75651 Paris Cedex 13, France
bLaboratoires GlaxoSmithKline, Saint-Grégoire, France

*Corresponding author. Tel.: +33-1-42-16-57-15; fax: +33-1-42-16-57-00. Email address: l.carrier{at}myologie.chups.jussieu.fr

Received 13 April 2003; revised 8 July 2003; accepted 28 July 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Cardiac myosin-binding protein C (cMyBP-C) is a component of sarcomere that contains at least three putative myosin-binding sites. Mutations in its gene are implicated in familial hypertrophic cardiomyopathy (FHC) and most of them are predicted to produce C-terminal truncated cMyBP-Cs. The aim of the present study was to analyze whether cMyBP-C truncated mutants resulting from FHC mutations interact in vitro with human β-MyHC. Methods: Recombinant proteins were produced using the baculovirus/insect cell system, and wild type and three truncated cMyBP-Cs were purified using metal affinity chromatography. The interaction between recombinant proteins was analyzed in real time using biosensor technology on immobilized anti-β-MyHC antibodies. Results: Biomolecular interaction with β-MyHC was detected for both wild type cMyBP-C and a truncated mutant lacking half of the C-terminal C10 domain. In contrast, no interaction with β-MyHC was found for two truncated cMyBP-Cs lacking at least the C5–C9 region. Conclusions: Biosensor technology allows in vitro analysis of the interaction between human β-MyHC and cMyBP-C mutants resulting from FHC mutations. The data show that the interaction depends on the size of the truncation. This suggests that, in the context of FHC, impairment of suitable interaction between β-MyHC and some of the truncated cMyBP-Cs may promote degradation of the truncated proteins and therefore contribute to the development of the disease.

KEYWORDS Cardiomyopathy; Contractile apparatus; Hypertrophy; Biosensors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiac MyBP-C (cMyBP-C) is a component of sarcomere, which is arrayed transversely in the cross-bridge bearing C-zone of the A-band and which plays an important structural and functional role during cardiac contraction in health and disease (for reviews, see Refs. [1,2]). The structure of cMyBP-C features eight IgC2-like and three FN3 domains as well as a MyBP-C motif between C1 and C2 domains. Cardiac MyBP-C is at least a myosin-interacting partner, which is able to bind the myosin heavy chain (MyHC) through at least three different sites that allow the compaction of myosin filaments [3]. The interactions with the light meromyosin (LMM) part and the S2 fragment of MyHC were first described in the 1970s [4–6]. It is only in the 1990s that the MyBP-C domains involved in the interaction with MyHC were determined. The myosin LMM-binding site is located in the C-terminal domain (C10 domain) of both fast-skeletal and cardiac isoforms [7–9], and the myosin S2-binding site in the MyBP-C motif of cMyBP-C [10]. Moreover, we identified by hydrophobic cluster analysis a third putative myosin-binding site in the N-terminal domain (C0 domain) of human cMyBP-C [11]. For human cMyBP-C, only the interaction with S2 myosin through the MyBP-C motif was demonstrated by biochemistry analyses [10]. We further showed that the presence of one, two or three putative myosin-binding sites allowed incorporation of human cMyBP-Cs into the A-band of the sarcomere [11].

Mutations in the human cMyBP-C gene are associated with familial hypertrophic cardiomyopathy (FHC), which is a myocardial disease associated with septal asymmetric hypertrophy, myocardial disarray and increase of interstitial fibrosis [12]. FHC is genetically heterogeneous and involves mutations in at least 11 different genes encoding components of the cardiac sarcomere (for review, see Refs. [13–16]). Most of the families present mutations in the cMyBP-C and β-MyHC genes [17]. Moreover, most of the cMyBP-C gene mutations disrupt the reading frame and are expected to produce C-terminal truncated proteins lacking at least part of the C10 myosin-binding domain [18–20]. It has been shown that four different truncated proteins resulting from cMyBP-C gene mutations are instable both in myocardial tissue of patients [21–23] as well as ex vivo after transfection of fetal rat cardiomyocytes with human cMyBP-C cDNAs [11].

The aim of the present study was to analyze whether cMyBP-C truncated mutants resulting from FHC mutations interact in vitro with human β-MyHC. For this purpose, human recombinant full-length β-MyHC, wild type cMyBP-C and three truncated mutants resulting from FHC mutations and containing one, two or three myosin-binding sites were produced using the baculovirus/insect cell system. The interaction between the two partners (β-MyHC lysate and purified cMyBP-Cs) was analyzed in real time using biosensor technology with immobilized anti-β-MyHC antibodies. The results show biomolecular interaction between β-MyHC and either wild type or truncated cMyBP-C lacking half of the C10 domain after preincubation of the partners. In contrast, no interaction was detected between β-MyHC and either cMyBP-Cs lacking at least the C5–C10 domains. These data suggest that, in the context of FHC, impairment of suitable interaction between some C-terminal truncated cMyBP-Cs and β-MyHC could contribute to the development of FHC.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Antibodies
The rabbit anti-cardiac MyBP-C polyclonal antibody, raised against a recombinant cMyBP-C fragment (C0–C1 domains) produced in Escherichia coli, was generously provided by Dr. M. Gautel (London, Great Britain). The mouse anti-histidine monoclonal antibody was from Sigma-Aldrich. The monoclonal antibody against β-MyHC was from Erfa-Canada (Westmount, Canada) hybridoma 3–48. The secondary goat anti-mouse horseradish peroxydase and goat anti-rabbit horseradish peroxydase antibodies were purchased from DAKO.

2.2. Generation of recombinant baculoviruses encoding human β-MyHC and cMyBP-Cs
The cDNA encoding human β-MyHC was generously provided by Dr. A. J. Marian (Houston, USA). In order to produce different human truncated cMyBP-Cs with one, two or three myosin-binding sites, three FHC mutations, M0, M1 and M6, were selected (Fig. 1) [18,20]. The three mutations result in COOH-terminal truncated proteins: M0t contains the C0–C4 domains, M1t contains the C0–C1 domains, M6t contains the first 1219 normal cMyBP-C residues followed by respectively 2, 25 and 19 additional amino acid residues added by the mutations. The procedure used to construct M0 and M1 cMyBP-C cDNAs has been described previously [11]. The M6 mutated cDNA (insertion (+12 bp)/deletion (–4 bp) in exon 33) was generated by PCR mutagenesis in two steps from the wild type cMyBP-C cDNA. First, a 309 bp fragment at the 3' end of the wild type cDNA was excised using appropriate enzymes (HindIII in 5' and XhoI at the 3' end). The deletion of 4 bp (ACCT) was then introduced by PCR. The PCR modification was assessed by enzymatic digestion since the deletion abolishes an AvaII restriction site and sequencing according to Sanger's method using fluorescent dideoxynucleotides [24]. After gel purification of the deleted PCR fragment, the 12 bp insertion (i.e. duplication of 3675–3686 at the position 3691) was introduced by the same PCR approach. The mutated PCR fragment was finally reintroduced into the wild type cDNA using the HindIII and XhoI cloning sites. The production of M6t protein was assessed by Western blot after transfection of the M6 cDNA in COS cells (data not shown). Recombinant baculoviruses were generated in two phases using a transposition system developed by Life Technologies. We first cloned the five cDNAs encoding human β-MyHC, wild type and truncated cMyBP-Cs into two different pFastBac shuttle vectors containing a poly-histidine tag at the 5' position of the multiple cloning site. Following this, we generated recombinant bacmid DNAs containing a single copy of the sense-oriented recombinant β-MyHC and cMyBP-Cs cDNA fragments after transformation into E. coli DH10-Bac bacteria strain. The cDNA encoding the human full-length β-MyHC was subcloned in the pFastBac Hta vector between NruI, introduced by PCR, and HindIII cloning sites. The different cMyBP-C cDNAs were subcloned in pFastBac Htc shuttle vectors between EcoRI and XhoI cloning sites. Recombinant baculoviruses were extracted by alkaline lysis and analyzed by Southern blot according to the supplier's instructions (data not shown).


Figure 1
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  		Fig. 1 Schematic representation of the wild type cMyBP-C protein and three of its truncated forms found in human FHC. The wild type protein contains eight Ig-C2 like modules (empty boxes), the MyBP-C motif (empty rectangle) and three fibronectin type-III modules (grey boxes). Horizontal arrows above the diagram indicate the three myosin-binding sites. The M6t cMyBP-C lacks half of the C10 domain but contains 19 additional amino acid residues (black rectangle at the C-terminal end). The M0t cMyBP-C lacks the C5–C10 domains and contains two additional amino acid residues. The M1t cMyBP-C is only composed of the C0–C1 domains plus 25 additional amino acid residues and contains the third putative myosin-binding site.

 
2.3. Culture and infection of Sf9 insect cells
Spodoptera frugiperda (Sf9) insect cells were maintained at +27 °C in serum-free SF-900 II SFM medium supplemented with 5 UI/ml penicillin and 5 µg/ml streptomycin (Life technologies). Transfection of Sf9 insect cells was performed with CellFectinTM reagent to constitute the initial recombinant baculovirus stock. Virus amplification was achieved in a suspension culture mode until having 1 l of viral suspension with a titer up to 107 pfu/ml at a density of 7 x 105 cells/ml and using a multiplicity of infection (MOI) ranging from 0.1 to 0.01. This viral stock was then used to infect 1 x 109 Sf9 cells for the production of recombinant proteins in the 2 l spinner at a MOI of 5 for β-MyHC, wild type and M6t cMyBP-Cs and a MOI of 2 for M0t and M1t cMyBP-Cs at +27 °C for 3 days. Infected cells were then collected after infection and centrifuged for 10 min at 1000 x g.

2.4. Analysis of recombinant proteins
The Sf9 cell pellets were lysed overnight at 4 °C in 50 mM Tris HCl, pH 8.5, 10 mM β-mercapto-ethanol, 1% NP-40 and 0.5% anti-proteases cocktail used for purification of poly(Histidine)-tagged proteins containing 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), bestatin, pepstatin A, E-64 and phosphoramidon. The next day, the lysates were centrifuged (1 h, 4 °C, 20000 rpm). For cMyBP-Cs, the supernatant was collected. For β-MyHC, the pellet containing the insoluble protein of interest was resuspended in 20 mM Na2HPO4, pH 7.4, 0.5 M NaCl, 0.5% anti-protease cocktail, and kept one more night at 4 °C. The overnight suspension was centrifuged (1 h, 4 °C, 20000 rpm) and the supernatant containing soluble β-MyHC was collected. Electrophoresis analyses were performed on the supernatants. Briefly, 15 µl of each sample was diluted in a Laemmli buffer [25], heated 5 min at 95 °C and loaded on 7.5% or 10% SDS-PAGE gel. After running (1 h, 150 V), the gels were either stained by Coomassie blue (Novex) or transferred to polyvinylidene fluoride (PVDF) membrane (Biotrace PVDF, Pall-Gelman) in 10% methanol (1 h, 10 °C, 100 V). The details of saturation, antibodies hybridization and chemiluminescence revelation have been described previously [11]. The monoclonal anti-histidine and the polyclonal anti-cMyBP-C antibodies were diluted at 1:1000 and the Erfa anti-β-MyHC antibodies at 1:1000. Both the goat anti-mouse and anti-rabbit horseradish peroxidase secondary antibodies were diluted at 1:2000.

2.5. Purification of recombinant proteins
The purification steps were semi-automatically performed with an AKTATM Explorer for affinity chromatography (Amersham-Pharmacia). Briefly, prepacked Hi-Trap chelating HP columns (1 ml) were loaded with 0.1 M NiSO4 solution in order to immobilize Ni2+ ions for histidine residue capture. The solubilized lysates were injected after a 1:2 dilution in loading buffer A (20 mM NaHPO4, 0.5 M NaCl, pH 8.5, 10 mM imidazole). Hi-Trap columns were washed with five column volumes of buffer A. Bound proteins were eluted with a 0–100% linear gradient of elution buffer B (20 mM NaHPO4, 0.5 M NaCl, pH 8.5, 0.5 M imidazole) and collected as 1 ml fractions. Each peak was dialyzed against a volatile 50 mM ammoniac/formic acid buffer solution (pH 7.4) for 2 days in order to remove high salt concentration. After lyophylisation, proteins were resuspended in a minimal volume of HEPES-buffered saline (HBS-EP: 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, pH 7.4), the vehicle buffer used in the biosensor experiments. The level and the specificity of purification products were assessed after SDS-PAGE electrophoresis both by Coomassie coloration and Western blot.

2.6. Procedure of covalent immobilization of monoclonal anti-β-MyHC antibodies on CM5 chip
The schematic representation of the biosensor experiments is shown in Fig. 2. The aminosilane surface of the sensor chip CM5 (carboxymethyl dextran) was activated by injection of a 1/1 mixture of N-hydroxysuccimide and ethylenecarbodiimide at 10 µl/min in order to form reactive esters. After activation, 50 or 100 µg/ml of monoclonal anti-β-MyHC antibodies in 10 mM Na-acetate pH 4.5 was immobilized on flow cell 2. Injection of a solution of ethanolamine deactivates excess reactive esters. A continuous flow rate of HBS-EP (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, pH 7.4) is maintained during the experiments and between the various injections. An activated/deactivated flow cell 1 was used as a reference flow cell in order to control non-specific binding and bulk refractive index changes. Regeneration of the surface was achieved in 10 mM glycine, pH 1.5.


Figure 2
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  		Fig. 2 Schematic representation of the biosensor experiments. (A) Without preincubation of β-MyHC and cMyBP-C; (B) after preincubation of β-MyHC and cMyBP-C. In both cases, the sensor chip CM5 was first activated with Dextran, and then covered with anti-β-MyHC antibodies. Then, β-MyHC was captured by its antibodies alone (A) or together with cMyBP-C (B). The last step in (A) consisted of capture cMyBP-C on β-MyHC.

 
2.7. Direct molecular interaction analysis between recombinant human β-MyHC and cMyBP-Cs on the biosensor chip
Once covalently coupled to the CM5 sensor chip, the anti-β-MyHC antibodies were used to capture β-MyHC. Therefore, 30 or 80 µl of myosin (supernatant in HBS-EP) was injected at a flow rate of 10 µl/min on flow cells 1 and 2. When β-MyHC binds, the refractive index of the medium at the surface of the sensor is changed in direct proportion to the bound mass. This change was detected according to the principle of surface plasmon resonance (SPR) and was expressed in resonance units (RU). Regeneration was done using 10 mM glycine pH 1.5 at a flow rate of 10 µl/min. This allows washout of the unspecific binding. After the capture of β-MyHC on monoclonal antibodies previously bound to CM5 surface, 10–80 µl (10–80 µg/ml) of wild type cMyBP-C were injected in the flux at a flow rate of 3 µl/min.

2.8. Detection of the interaction between recombinant β-MyHC and cMyBP-Cs after preincubation of the partners
A mix containing two recombinant partners (20 µl myosin+20 µl cMyBP-C+60 µl HBS-EP) was preincubated for 20 min at room temperature (RT). The mix (30 or 80 µl) was injected at 10 µl/min on the immobilized anti-β-MyHC antibodies. As controls, the same experiments were performed after preincubation of 20 µl of each recombinant protein for 20 min at RT in 80 µl of HBS-EP. Regeneration was achieved using 10 µl NaCl 1 M/NaOH 50 mM for either myosin or cMyBP-Cs alone and 10 µl glycine, pH 3 at 10 µl/min when the proteins were incubated together.

2.9. Biacore analyses
Graphs were prepared with Biaevaluation 3.0 software. All calculations were performed after subtraction of the reference flow cell 1 where neither the antibody nor the myosin was coupled to the chip but was otherwise treated as the experimental flow cell 2.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Production of recombinant human β-MyHC and cMyBP-Cs in insect cells
Expression of recombinant human β-MyHC, wild type and truncated mutant cMyBP-Cs was analyzed at different post-infection times by SDS-PAGE electrophoresis. As shown in Fig. 3A, a maximal expression of both β-MyHC and cMyBP-C proteins was observed between 48 and 96 h after infection, which is characteristic of a prototypic overexpression scheme in the chosen system. The synthesis of recombinant proteins then decreased as the virus entered in its lytic phase. After successive rounds of amplification for reaching high titer recombinant baculovirus stocks, 109 Sf9 cells were infected at previously optimal determined multiplicities of infection (MOI=5 for wild type cMyBP-C, M6t cMyBP-C and β-MyHC; MOI=2 for M0t and M1t cMyBP-Cs) in order to produce large amounts of recombinant proteins. Three days after infection, cell lysates were analyzed by SDS-PAGE electrophoresis and Western blot. Fig. 3B shows the results before purification of recombinant cMyBP-Cs. The Coomassie gel shows for each protein a clear band at the expected size, which was confirmed by Western blot analysis with specific antibodies (144 kDa for wild type cMyBP-C, 138 kDa for M6t, 74 kDa for M0t, 30 kDa for M1t). To analyze the biomolecular interaction between the two partners with SPR technology, at least one of the partners should be purified [26]. Therefore, the human recombinant cMyBP-Cs was purified by immobilized metal affinity chromatography. Fig. 3C illustrates the results obtained after purification of each cMyBP-C and shows a high degree of purification. As for the recombinant human β-MyHC, Fig. 3D shows a major band at the expected size around 200 kDa in the Coomassie gel. This was confirmed by Western blot analysis with the anti-β-MyHC antibodies. In addition, a second band of about 150 kDa was also detected by the antibody, which probably corresponds to the HMM part of β-MyHC. This second band should not interfere with the biosensor analysis since the full-length β-MyHC amount is largely higher than the HMM one (Fig. 3D, Coomassie). The presence of this proteolytic fragment of β-MyHC is probably due to the absence of myosin-light chains which are known to stabilize the myosin molecule at the head/hinge region [27,28].


Figure 3
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  		Fig. 3 (A) Time course optimization of recombinant proteins synthesis in recombinant bacmid-infected Sf9 cells as analyzed by Western blot and quantified using Storm 860 phosphorimager and Image Quant software. (B) Coomassie staining and Western blot analysis of crude lysates of human recombinant wild type cMyBP-C and three truncated cMyBP-Cs. (C) Coomassie staining of purified cMyBP-Cs. (D) Coomassie staining and Western blot analysis of crude lysates of human recombinant β-MyHC. The molecular weights were calculated by using a protein standard mixture (New England Biolabs). Proteins were resolved on 7.5% and 10% acrylamide gels (10 µl of crude lysate or 100 ng of purified cMyBP-Cs).

 
3.2. Biomolecular interactions between recombinant human β-MyHC and cMyBP-Cs on the biosensor
To specifically capture β-MyHC, anti-β-MyHC antibodies were first covalently immobilized on the biosensor surface. Two antibody concentrations were tested, 100 and 50 µg/ml. They gave rise to +14091 and +7719 RU, respectively (Fig. 4A and B). This corresponds to an estimated quantity of 0.6–1.5 ng of immobilized antibodies. The amount of immobilized antibodies influences the binding response of β-MyHC on the antibodies as shown in Fig. 4C and D: the injection of β-MyHC gave a rise of +506.9 and +194.4 RU after immobilization of 100 and 50 µg/ml of antibodies on the surface, respectively (Fig. 4C and D). Since both responses were significant, the lowest concentration of 50 µg/ml was used in the following experiments. As a control for non-specific binding, we systematically and simultaneously used an empty activated/deactivated flow cell, which was treated in the same way. Importantly, this never gave any signal (data not shown). We also tested two other types of negative controls (empty baculovirus or β-MyHC alone in the absence of the antibodies), which finally could not be used because of technical problems during regeneration of the surface and the precipitation of β-MyHC in the absence of antibodies.


Figure 4
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  		Fig. 4 Covalent immobilization of 100 µg/ml (A) or 50 µg/ml (B) anti-β-MyHC antibodies on biosensor CM5 chip followed by regeneration of the surface with ethanolamine–HCl pH 8.5. (C) Sensorgram showing the binding of β-MyHC (30 µl of supernatant) on the flow cell where 100 µg/ml of monoclonal antibody was covalently coupled to the CM5 surface followed by the addition of 10 µl (10 µg/ml) of wild type cMyBP-C at a 3 µl/min flow rate. (D) Sensorgram showing the binding of β-MyHC (30 µl of supernatant) on the flow cell where 50 µg/ml of monoclonal antibody was covalently coupled to the CM5 surface followed by the addition of 30 µl (30 µg/ml) of wild type cMyBP-C at a 3 µl/min flow rate. Abbreviations used: FC1, flow cell 1; FC2, flow cell 2; NHS/EDC, N-hydroxysuccimide/ethylenecarbodiimide.

 
To detect interaction between partners, each purified cMyBP-C was directly injected to the coupled β-MyHC after regeneration of the surface. The wild type cMyBP-C (Fig. 4C and D) or the three truncated cMyBP-Cs (data not shown) did not interact with the coupled β-MyHC. The absence of direct interaction between partners may be due to a conformational change of immobilized β-MyHC, which would hide the cMyBP-C-binding sites. Such conformational changes have been already reported in other biosensor experiments [29].

To allow interaction between the two partners, a preincubation step was therefore performed before the capture of the putative complex to the immobilized anti-β-MyHC antibodies (see Methods). Fig. 5 presents a typical sensorgram obtained after preincubation of β-MyHC with each cMyBP-C. With wild type cMyBP-C, a SPR response of 314 RU was obtained, whereas myosin or wild type cMyBP-C alone exhibits a SPR response of 200.7 and 21.5 RU, respectively. This gives a significant specific rise of +91.8 RU (Fig. 5A). This enhancement of mass transport clearly indicates that during the preincubation step, wild type cMyBP-C and β-MyHC formed a complex in which myosin is still able to bind with the anti-β-MyHC precoupled to the biosensor. With M6t cMyBP-C, a significant specific rise of +85 RU was obtained (Fig. 5B). Again, this indicates that M6t cMyBP-C and β-MyHC formed a complex during the preincubation step, which is still able to bind with the anti-β-MyHC precoupled biosensor. One should note, however, that M6t cMyBP-C alone exhibited a significant SPR response of 133 RU when compared to wild type cMyBP-C alone (21.5 RU, Fig. 5A). This was not expected but could be due to the presence of the 19 additional residues resulting from the mutation (see Methods). With M0t cMyBP-C, a non-significant specific rise of –1.8 RU was obtained (Fig. 5C). This indicates that the putative M0t cMyBP-C/β-MyHC complex did not interact with the immobilized antibodies. With the M1t cMyBP-C, only a small specific rise of +2.4 RU was obtained (Fig. 5D). However, this is not significant when compared to the SPR response of 6.8 RU obtained with M1t cMyBP-C alone. Again, this indicates that the putative M1t cMyBP-C/β-MyHC complex did not interact with the immobilized antibodies. To test the hypothesis of an inappropriate ratio between the two partners, we performed different experiments with variable ratios of truncated cMyBP-Cs and β-MyHC lysate. Again, no signal was obtained with M0t or M1t cMyBP-Cs (data not shown).


Figure 5
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  		Fig. 5 (A) Sensorgrams showing binding of the wild type cMyBP-C/β-MyHC complex on the captured anti-β-MyHC antibodies after a prior 20 min preincubation of the two proteins. The respective binding of β-MyHC and wild-type cMyBP-C when injected alone on captured anti-β-MyHC antibodies is also shown. (B) Sensorgrams showing binding of the M6t cMyBP-C/β-MyHC complex on captured anti-β-MyHC antibodies after 20 min of preincubation of the two proteins and the respective binding of β-MyHC and M6t cMyBP-C when injected alone. (C) Sensorgrams showing binding of the M0t cMyBP-C/β-MyHC complex after 20 min of preincubation of the two proteins and respective binding of β-MyHC and M0t cMyBP-C when injected alone. (D) Sensorgrams showing binding of the M1t cMyBP-C/β-MyHC complex after 20 min of preincubation of the two proteins and the respective binding of β-MyHC and M1t cMyBP-C when injected alone. Either 30 or 80 µl of β-MyHC was injected at the same flow rate (3 µl/min) but at different kinetics. This results in identical β-MyHC concentrations in all cases.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Using the baculovirus/insect cell expression system, we produced recombinant human β-MyHC, wild type cMyBP-C and three C-terminal truncated cMyBP-Cs resulting from FHC mutations. Moreover, using the surface plasmon resonance technology, this study provides evidence that (i) β-MyHC and wild type cMyBP-C clearly interact together, (ii) β-MyHC also binds to truncated cMyBP-C lacking half of the C10 domain and (iii) no interaction was found between β-MyHC and truncated cMyBP-Cs lacking at least the C5–C10 domains.

To express human β-MyHC and the different cMyBP-Cs, we chose the baculovirus/insect cell expression system, which respects the eukaryotic environment including post-translational modifications and therefore takes into account protein folding and allows a large-scale protein production. With this system, we obtained for the first time both full-length and expected truncated human cMyBP-Cs resulting from FHC mutations. Similarly for human β-MyHC, we generated a significant yield of the full-length protein. To analyze the biomolecular interaction between the two partners, we used Biacore's technology. This is a highly sensitive, non-invasive method which relies on surface plasmon resonance and enables detection of biomolecular interaction in real time (in seconds) without requiring any labels and in the respect of native protein conformation. The potential weaknesses of this method namely false positive response, sensor chip surface regeneration concerns, needs of a large amount of purified and homogeneous samples of proteins were managed in our work using multiple appropriate controls as well as purified proteins and the use of specific antibodies for the capture. To detect protein interaction with the surface plasmon technology, at least one of the partners should be purified [26]. The anti-histidine tag purification technique was successful for cMyBP-Cs. Therefore, the crude lysate of β-MyHC was used for the interaction experiments with purified cMyBP-Cs and the β-MyHC was selectively captured on the sensor chip with anti-β-MyHC antibodies.

To allow interaction between the two partners, a preincubation step was performed before the capture of the putative cMyBP-C/β-MyHC complex to the immobilized anti-β-MyHC antibodies. In the conditions used during the preincubation step, β-MyHC is probably organized in correctly formed filaments through its LMM part as it was demonstrated before in a similar range of pH and ionic strength [3,5,30–33]. In that configuration, the wild type cMyBP-C and β-MyHC formed a complex that is captured on the antibodies. This indicates that interaction probably occurs between the LMM region of β-MyHC and the C10 domain of cMyBP-C [7–9]. The truncated M6t cMyBP-C, which lacks half of the C10 domain, also interacts with β-MyHC. This suggests that the integrity of the C10 domain is not necessary for the binding to myosin. This is supported by previous data obtained with C10 recombinant fast-skeletal MyBP-C mutants showing that three amino acids, located in positions 37, 73 and 74, are required in the interaction with recombinant LMM [34]. Out of them, only residue 37 is present in the truncated M6t cMyBP-C, suggesting that this residue may be sufficient to allow LMM-myosin binding. In addition, another myosin-binding domain could also be involved in the interaction, as has been suggested recently for the C7 domain of the skeletal isoform [35]. Moreover, it has been shown recently that cMyBP-C forms dimers between the C5–C7 domains of one molecule and C8–C10 domains of another [36]. This process could occur during the preincubation step and therefore stabilize the cMyBP-C/β-MyHC complex.

In contrast to what was found with wild type and M6t cMyBP-Cs, the putative complexes formed during preincubation between β-MyHC and truncated M0t or M1t cMyBP-C mutants could not be captured by the anti-β-MyHC antibodies. This was not expected since M0t contains at least the S2-myosin binding domain located in the MyBP-C motif [10] and M1t contains the third putative myosin-binding domain located in the C0 domain [11]. A potential explanation could be that the affinities of the N-terminal myosin-binding sites of cMyBP-C are lower than that of the C-terminal myosin-binding domain, which would prevent the interaction in this in vitro system. Another likely explanation could be the weakness of the complex formed during preincubation due to the absence of the C5–C10 domains in the truncated mutants, which prevents the dimerization of cMyBP-C molecules and therefore putative stabilization of the complex [36]. The impaired interaction that we have found between β-MyHC and some truncated cMyBP-C mutants may promote the degradation of truncated proteins and therefore contribute to the development of FHC. This is consistent with the fact that in humans, truncated cMyBP-Cs resulting from frameshift mutations were not detected in cardiac tissue of patients [21–23]. Moreover, our recent data obtained after infection of neonatal rat cardiomyocytes with recombinant adenoviruses showed that two human truncated cMyBP-Cs resulting from FHC frameshift mutations promote lysosome- and proteasome-dependent degradation, suggesting that these degradation pathways are involved in human FHC [37].

In conclusion, biosensor technology allows in vitro analysis of the interaction between human β-MyHC and cMyBP-C mutants resulting from FHC mutations. Our results show that some of the truncated cMyBP-C mutants cannot interact with β-MyHC in vitro. This suggests that, in the context of FHC, impairment of suitable interaction between β-MyHC and some of the truncated cMyBP-Cs may participate in the development of the disease.


   Acknowledgements
 
The authors thank Marie-Paule Laville and Laurence Tourtelier from GlaxoSmithKline Laboratories for their precious technical help. We are grateful to Dr. A.J. Marian (Houston, TX) for the gift of the full-length cDNA encoding human β-MyHC and to Dr. M. Gautel (London, Great Britain) for the gift of the anti-cardiac MyBP-C antibody. This work was supported by the Institut National de la Santé et de la Recherche Médicale, by the Association Française contre les Myopathies and by GlaxoSmithKline Laboratories.


   Notes
 
1 Contributed equally to this work. Back

Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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