Light Meromyosin Region Research Articles

Human Skeletal myopathy myosin mutations disrupt myosin head sequestration.

Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle, and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyse the effects of common MYH7 and MYH2 mutations in the light meromyosin region of myosin (LMM). Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in-silico modelling showed that myosin coiled-coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients, and Mant-ATP chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with X-ray diffraction measurements to estimate myosin head order we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofibre mechanics experiments to investigate contractile function showed myofibre contractility was not affected. These findings indicate that the structural remodelling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.

Moderate Protein Oxidation Improves Bovine Myofibril Digestibility by Releasing Peptides in the S2 Region of Myosin: A Peptidomics Perspective.

This study aimed to investigate the influence of protein oxidation on the digestive properties of beef myofibrillar protein (MP). MP was treated with a hydroxyl radical-generating system containing various concentrations of H2O2. The increased content in a free sulfhydryl group and surface hydrophobicity indicated that oxidation treatment with 1 mM H2O2 induced unfolding of MP. Reducing and nonreducing SDS-PAGE results suggested that 10 mM H2O2 oxidation treatment resulted in aggregation of MP; meanwhile, the disulfide bond was the major covalent bond involved in aggregation. Peptidomics showed that peptides in the digestion products of MP were mainly derived from myosin tail. Moderate oxidation (1 mM H2O2) facilitated the release of peptide in the rod portion (S2) of myosin, whereas excessive oxidation (10 mM H2O2) inhibited peptide release in the light meromyosin region. This work presents insightful information for the crucial impact of oxidation on meat protein digestibility from the peptidomics perspective.

Characteristic stable structure of the myosin rod in dark muscle of yellowtail kingfish (Seriola aureovittata)

SummaryThe dark muscle of yellowtail kingfish is regularly consumed as fresh sashimi alongside with the ordinary muscle. Thus, in this study, three types of ATPase activities, chymotryptic digestion patterns as well as thermal unfolding of various myosin domains were determined to reveal the denaturation characterisation of myosin in dark muscle and compare it with normal muscle. The thermal inactivation rate of dark muscle myosin Ca2+‐ATPase at 30°C (7.3 × 10−4, sec−1) was significantly (P < 0.05) greater than that of ordinary muscle (5.9 × 10−4, sec−1), while dark muscle myosin became more stable than ordinary muscle one once bound with F‐actin. On the other hand, the rod region especially light meromyosin region of the dark muscle myosin was much more stable than ordinary myosin. These results characterised that myosin in the dark muscle of yellowtail kingfish is constituted with less stable head and more stable rod regions than those in the ordinary muscle.

Narrowing Beta-myosin hot spots in its association with hypertrophic cardiomyopathy

Abstract Introduction MYH7 encodes to β-cardiac myosin heavy chain, a large and pleomorphic protein, conformed by different and well-functionally characterized domains. This gene has an enormous contribution to hypertrophic cardiomyopathy (HCM) but also to other cardiomyopathies such as dilated, non-compaction and restrictive cardiomyopathy (and overlapping phenotypes). There are specific and adapted recommendations for classifying variants in this gene regarding its association with HCM; one of the aspects was the location of variants in the head domain, encompassing 181–937 residues as moderate ACMG criteria (Kelli et al., 2018). Objectives The purpose of this analysis was to determine if more specific regions of MYH7are enriched in HCM cases. This analysis could help to better characterise those regions where identifying a novel missense variant could reinforce their pathogenicity. Methods We included in the analysis 26,929 consecutives unrelated probands in which MYH7 was sequenced by NGS, referred from different countries and centres. We performed an enrichment analysis comparing the prevalence of rare missense variants (pre-established MAF cut-off 0.004%) in cases with the diagnosis of HCM versus their frequency in non-cardiomyopathy cases (internal controls, OR_int, comprising aortic diseases, channelopathies and dyslipidaemias) and in gnomAD population (external controls, OR_ext). Comparison between groups was performed using the Student's t-test; p value <0.05 was considered to indicate statistical significance. The rationale of this grouping was to avoid skewed analysis, given the high degree of overlapping cardiomyopathies observed in this gene. Results HCM was the diagnosis in 10,064 cases. 8,975 probands with a non-cardiomyopathy phenotype were used as internal controls; 7,890 cases with other MYH7-related phenotypes (restrictive, dilated, non-compaction and skeletal myopathies) were not included in the analysis. Rare missense variants in MYH7 were enriched in HCM cases (OR_int = 11.15; OR_ext =12.44) and in the pre-established hot spot head domain (OR_int = 16.43; OR_ext = 24.53). However, the regions with the highest odds ratios in both external and internal comparison were the central converter domain (residues 711–755, OR_int = 44.39; OR_ext = 50.27) and actin-binding region (residues 647–664, OR_int =18.77; OR_ext = 69.32). Conversely, the tail region (both S2 subfragment and light meromyosin region, LMM), had the lowest OR in this analysis. Conclusions Previously reported hotspot (head) is significantly enriched in HCM cases, but some specific regions within this domain, such as central converter and acting-binding region, could have a relatively higher contribution. These results reinforce the current approach, but on the other hand, show an asymmetric contribution within the head domain. This fact may have an impact on the reassessment of the variant classification in this gene towards a more precise approach. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Health in Code

Characteristic thermal denaturation profile of myosin in the longitudinal retractor muscle of sea cucumber (Stichoupus japonicas)

This study elucidated thermal denaturation profile of myosin in sea cucumber longitudinal muscle. Sea cucumber myosin structure was different from fish at its head/tail junction which could not be cleaved by EDTA. However, sea cucumber myosin in salt-dissolved form could be cleaved into heavy meromyosin (HMM) and light meromyosin (LMM) segments. Although sea cucumbers lived in cold water, its myosin stability was comparable to tropical tilapia, more stable than rainbow trout (a cold water fish). Upon heating, the sea cucumber myosin lost its salt-solubility rapidly, even before losing its ATPase activity. The quick loss of salt-solubility suggested a quick denaturation at light meromyosin region as revealed by chymotryptic digestion. These results suggested that sea cucumber myosin is consisted of very stable head region and unstable tail region, which is important for choosing proper heating conditions for sea cucumber processing.

Novel compound heterozygous mutations of the myosin heavy chain gene in patients with hypertrophic cardiomyopathy

background: Hypertrophic cardiomyopathy (HCM) is a multifactorial disorder, with mutations implicated in 14 sarcomeric and cytoskeletal genes, leading to genotypic and phenotypic heterogeneity, and a challenging genetic and clinical diagnosis. The genetic characteristics of HCM have been studied for more than two decades in various ethnic and racial groups, and many novel genetic variations have been reported. The myosin heavy chain gene is the most heavily implicated gene in HCM, with >200 reported mutations, the majority of which have been found in the head-rod junction. The rod portion of MYH7, coded by exons 29 to 40 and belonging to the light meromyosin (LMM) region, has not been characterized to the same extent as the head domain with respect to single-nucleotide polymorphisms (SNPs)/mutations. OBJECTIVE: To screen the conserved LMM region, constituting exons 27 to 39 (13 exons), to identify any pathogenic SNPs/variations in this region in a population of Indian patients. Methods: Molecular screening was performed by polymerase chain reaction-based single-strand conformation polymorphism analysis in 100 control individuals and 100 HCM patients. The variations were confirmed by sequencing. Insilico analysis was performed to analyze the effect of the respective variations. Results : Screening of exons 27 to 39 revealed three novel missense variations and one novel synonymous variation in exon 34. Interestingly, patients with these variations also exhibited compound heterozygosity, indicating exon 34 to be the ‘hotspot’ exon of the LMM region. Conclusion: The results of the present study emphasize the importance of the LMM (rod) region of the MYH7 gene and suggest that variations in the conserved region are likely to be more pathogenic, making screening of the entire gene for HCM diagnosis mandatory.

A Hypertrophic Cardiomyopathy-associated MYBPC3 Mutation Common in Populations of South Asian Descent Causes Contractile Dysfunction

Hypertrophic cardiomyopathy (HCM) results from mutations in genes encoding sarcomeric proteins, most often MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). A recently discovered HCM-associated 25-base pair deletion in MYBPC3 is inherited in millions worldwide. Although this mutation causes changes in the C10 domain of cMyBP-C (cMyBP-C(C10mut)), which binds to the light meromyosin (LMM) region of the myosin heavy chain, the underlying molecular mechanism causing HCM is unknown. In this study, adenoviral expression of cMyBP-C(C10mut) in cultured adult rat cardiomyocytes was used to investigate protein localization and evaluate contractile function and Ca(2+) transients, compared with wild-type cMyBP-C expression (cMyBP-C(WT)) and controls. Forty-eight hours after infection, 44% of cMyBP-C(WT) and 36% of cMyBP-C(C10mut) protein levels were determined in total lysates, confirming equal expression. Immunofluorescence experiments showed little or no localization of cMyBP-C(C10mut) to the C-zone, whereas cMyBP-C(WT) mostly showed C-zone staining, suggesting that cMyBP-C(C10mut) could not properly integrate in the C-zone of the sarcomere. Subcellular fractionation confirmed that most cMyBP-C(C10mut) resided in the soluble fraction, with reduced presence in the myofilament fraction. Also, cMyBP-C(C10mut) displayed significantly reduced fractional shortening, sarcomere shortening, and relaxation velocities, apparently caused by defects in sarcomere function, because Ca(2+) transients were unaffected. Co-sedimentation and protein cross-linking assays confirmed that C10(mut) causes the loss of C10 domain interaction with myosin LMM. Protein homology modeling studies showed significant structural perturbation in cMyBP-C(C10mut), providing a potential structural basis for the alteration in its mode of interaction with myosin LMM. Therefore, expression of cMyBP-C(C10mut) protein is sufficient to cause contractile dysfunction in vitro.

Cardiac Myosin Epitopes for Monitoring Progression of Rheumatic Fever

To the Editors: Rheumatic fever (RF) and rheumatic heart disease (RHD) have become rare in the developed world,1 but remain major causes of childhood diseases in developing countries and in some indigenous communities.2 In populations endemic for RF/RHD, additional factors other than those currently included in the Revised Jones Criteria would increase diagnostic sensitivity.3 Moreover, identification of robust markers for disease activity could provide better monitoring and delivery of health care for patients. It has been established that immune responses against group A streptococci (GAS) and heart tissue correlate with antibody and T-cell responses against cardiac myosin. Recently, in samples obtained from rheumatic carditis patients from India, Hawaii, and mainland United States, it was found that IgG antibodies targeted cardiac myosin epitopes in the S2 subfragment containing amino acid residues 842 to 992 and 1164 to 1272.4 It has been suggested that, regardless of the GAS M serotype involved in infection, S2 reactivity is similar among populations with rheumatic carditis. In the current exploratory study, we determined IgG reactivity in Australian serum samples to 32 S2 (rod region) and 50 LMM (light meromyosin) overlapping peptides from cardiac myosin using an enzyme-linked immunosorbent assay.4 Sera samples were obtained from 12 RF/RHD patients from the Townsville Hospital and the Royal Darwin Hospital (6 patients with acute rheumatic fever (ARF) with confirmed carditis and 6 patients with ARF/RHD on penicillin prophylaxis for more than a year) and 10 healthy controls. We also assessed IgG reactivity to the entire length of GAS M5 protein using 25 overlapping peptides (N-terminal, n = 10; B region, n = 8; and C region, n = 7). Reactivity to each peptide was determined in serum samples of each individual, and the cumulative optical density values were categorized into the different groups for comparison by either unpaired 2-tailed t tests (Fig. 1A, C) or ANOVA with Newman-Keuls multiple comparison post hoc test (Fig. 1B) on transformed data.FIGURE 1.: Immunoreactivity of Australian serum samples with peptides from the S2 and LMM regions of human cardiac myosin. A, ARF/RHD patients had higher reactivity to peptides from the S2 region of cardiac myosin compared with healthy controls (**P < 0.01). B, After prophylaxis for >12 months, patient reactivity to S2 1 and 2 (842–880) was comparable to healthy controls (*P < 0.05). C, Higher reactivity to the LMM region of cardiac myosin was also observed in ARF/RHD patients compared with healthy controls (**P < 0.01). Shown are mean optical density values + standard error of the mean.Significant differences between the patient and control group were observed in their antibody reactivity to myosin peptides. These observations are further proof of our previous suggestion that, regardless of the GAS M serotype involved in infection, S2 reactivity may be similar among populations with ARF.4 Significantly higher mean combined IgG reactivity to S2 (Fig. 1A) peptides was observed in patients. More importantly, within the patient group, we also found that IgG reactivity to S2 1 and 2 (842–880) was significantly higher in patients with ARF and confirmed carditis than in patients who have had prophylaxis for 12 months or longer (Fig. 1B). This observation warrants further assessment of antibody kinetics in patients with ARF undergoing treatment. Significantly higher mean combined IgG reactivity to LMM peptides was also observed in patients (Fig. 1C). IgG reactivity to GAS M5 N-terminal and B-repeat region peptides were minimal in patient and control groups, indicating that these subjects were most probably not exposed to GAS M5 (data not shown; GAS M5 is not a common isolate in circulation in Australia). However, the significantly high mean combined IgG reactivity observed in both patients and controls to GAS M protein C region peptides (data not shown), which have demonstrated high homology across the different GAS M types,1 indicated that both groups had been exposed to GAS. The assessment of antibody kinetics in patients with ARF undergoing treatment to determine whether antibody reactivity to S2 could be used as a marker of disease activity and the effectiveness of treatment and prophylaxis regimens warrants further investigation. Davina E. Gorton, PhD Brenda L. Govan, PhD Natkunam Ketheesan, MD, PhD Microbiology and ImmunologySchool of Veterinary and Biomedical Sciences James Cook University Townsville, Queensland, Australia Alan A. Sive, MD, FRACP Robert E. Norton, MD, FRCPA Paediatrics/Pathology Townsville Hospital Townsville, Queensland, Australia Bart J. Currie, FRACP, DTM&H Rebecca J. Towers, PhD Tropical and Emerging Infectious Diseases DivisionMenzies School of Health Research Darwin, Northern Territory, Australia Adita I. Mascaro-Blanco, BSc Madeleine W. Cunningham, PhD Department of Microbiology and ImmunologyUniversity of Oklahoma Health Sciences Center Oklahoma City, OK

Molecular cloning and mRNA expression analysis of myosin heavy chain (MyHC) from fast skeletal muscle of grass carp, Ctenopharyngodon idella

The myosin heavy chain (MyHC) is one of the major structural and contracting proteins of muscle. We have isolated the cDNA clone encoding MyHC of the grass carp, Ctenopharyngodon idella. The sequence comprises 5 934 bp, including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues. The deduced amino acid sequence showed 69% homology to rabbit fast skeletal MyHC and 73%–76% homology to the MyHCs from the mandarin fish, walleye pollack, white croaker, chum salmon, and carp. The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80% homology to the corresponding regions of other fish MyHCs. The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR. The MyHC gene showed the highest expression in the muscles compared with the kidney, spleen and intestine. Developmentally, there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage. The highest expression was detected in hatching larva. Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

Myosin filament depolymerizes in a low ionic strength solution containing l-histidine

Myosin, one of the major myofibrillar proteins, forms a filamentous polymer and is insoluble in physiological and low ionic strength solutions. We have shown that myosin is soluble in a low ionic strength solution containing L-histidine. In this study, to clarify the role of L-histidine in the solubilization of myosin, we investigated effects of L-histidine on the filament formation and the morphology of myosin at a low ionic strength. In the presence of L-histidine, myosin formed a filamentous polymer in a physiological ionic strength solution and dispersed in a low ionic strength solution. Transmission electron microscopy showed that light meromyosin (LMM), the rod region of myosin, in a low ionic strength solution containing L-histidine was longer than that in a high ionic strength solution without L-histidine. L-histidine causes the elongation of LMM region of myosin contributing to the weakening of the myosin filament and the dissociation of myosin in a low ionic strength solution.

G.P.7.10 Investigation of the patho-biology of MYH7 myopathy mutations

The β-cardiac myosin (β-MyHC) protein is a molecular motor fundamental to both the contractile and structural properties of the muscle sarcomere. Mutations in the gene encoding β-MyHC (MYH7) cause multiple disease phenotypes: early-onset distal myopathy (MPD1), myosin storage myopathy (MSM), hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Mutations causing HCM and DCM are spread across almost the entire gene, while those causing MPD1 and MSM are confined to exons encoding the C-terminal light-meromyosin (LMM) region of β-MyHC. How mutations located in the same region of MYH7 cause such a wide phenotypic range is as yet unknown. To investigate structural and functional effects of different β-MyHC mutants, full-length MYH7 and rod/LMM domain regions of interest were cloned for expression in mammalian cells or for recombinant protein expression. Fusion to enhanced green fluorescent protein (EGFP) allowed visualisation of the location of wildtype (WT) or mutant β-cardiac myosin in C2C12 myoblast and myotube cultures and in COS7 cells. Both WT and mutant proteins were able to arrange into ordered, striated structures in differentiated C2C12 myotubes. Analysis in COS7 cells however, suggested mutant β-MyHC showed reduced ability to form higher order structures compared to the WT protein. Secondary-structure analysis of recombinant expressed myosin tail fragments by circular dichroism (CD) revealed that both WT and mutant proteins are almost entirely α-helical. Mutant myosin tails however, tend to show a slight reduction in α-helical content. This reduction is amplified when temperature is increased to replicate physiological conditions. CD melt curve analysis indicated mutant β-MyHC proteins have decreased thermostability. Overall, the effects of the MYH7 LMM mutations on measured properties are less marked than might have been expected from missense mutations to proline or deletion or insertion of an amino-acid in a coiled coil.

Mapping of O‐linked β‐N‐acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle

O-linked beta-N-acetylglucosamine (O-GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O-GlcNAc modified. Moreover, it was demonstrated that O-GlcNAc moieties involved in contractile protein interactions could modulate Ca(2+) activation parameters of contraction. In order to better understand how O-GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O-GlcNAc modification in purified contractile protein homogenates. Using an MS-based method that relies on mild beta-elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O-GlcNAc site in the subdomain four of actin and four O-GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O-GlcNAc sites might modulate the actin-tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein-protein interactions but could also modulate the contractile properties of skeletal muscle.

Localization of the binding site of the C-terminal domain of cardiac myosin-binding protein-C on the myosin rod

cMyBP-C [cardiac (MyBP-C) myosin-binding protein-C)] is a sarcomeric protein involved both in thick filament structure and in the regulation of contractility. It is composed of eight IgI-like and three fibronectin-3-like domains (termed C0-C10). Mutations in the gene encoding cMyBP-C are a principal cause of HCM (hypertrophic cardiomyopathy). cMyBP-C binds to the LMM (light meromyosin) portion of the myosin rod via its C-terminal domain, C10. We investigated this interaction in detail to determine whether HCM mutations in beta myosin heavy chain located within the LMM portion alter the binding of cMyBP-C, and to define the precise region of LMM that binds C10 to aid in developing models of the arrangement of MyBP-C on the thick filament. In co-sedimentation experiments recombinant C10 bound full-length LMM with a K(d) of 3.52 microM and at a stoichiometry of 1.14 C10 per LMM. C10 was also shown to bind with similar affinity to LMM containing either the HCM mutations A1379T or S1776G, suggesting that these HCM mutations do not perturb C10 binding. Using a range of N-terminally truncated LMM fragments, the cMyBP-C-binding site on LMM was shown to lie between residues 1554 and 1581. Since it had been reported previously that acidic residues on myosin mediate the C10 interaction, three clusters of acidic amino acids (Glu1554/Glu1555, Glu1571/Glu1573 and Glu1578/Asp1580/Glu1581/Glu1582) were mutated in full-length LMM and the proteins tested for C10 binding. No effect of these mutations on C10 binding was however detected. We interpret our results with respect to the localization of the proposed trimeric collar on the thick filament.

T-cell mimicry of streptococcal M protein and cardiac myosin epitopes by T-cell clones from rheumatic heart disease

Mimicry between streptococcal M protein and cardiac myosin is important in the pathogenesis of rheumatic heart disease. M protein specific human T-cell clones derived from rheumatic carditis were crossreactive with human cardiac myosin, and laminin, a valve protein. Among the 11 CD4+ and CD8+ crossreactive T-cell clones, at least six different reactivity patterns were distinguished, suggesting different degrees of crossreactivity and a very diverse T-cell repertoire. The latter was confirmed by a heterogeneous Vβ-gene and CDR3 usage. The HLA-restriction and Th1 cytokine production in response to rM6 protein was preserved when the T-cell clones were stimulated by human cardiac myosin or other α-helical proteins, such as tropomyosin and laminin. The crossreactive human T-cell clones proliferated to B2 and B3A, dominant peptide epitopes in the B-repeat region of streptococcal M protein. In human cardiac myosin, epitopes were demonstrated in the S2 and light meromyosin (LMM) regions. The study of human T-cell clones from rheumatic heart disease revealed potential sites of T-cell mimicry between streptococcal M protein and human cardiac myosin and represents some of the most well-defined T-cell mimicry in human autoimmune disease.

Dimerization Specificity of Adult and Neonatal Chicken Skeletal Muscle Myosin Heavy Chain Rods

The dimerization specificity of the recombinantly expressed and purified rod domain of adult and neonatal chicken myosin heavy chain was analyzed using metal chelation chromatography. Our results indicate that full-length adult and neonatal rods preferentially formed homodimers when renatured from an equimolar mixture of the two isoforms denatured in guanidine hydrochloride. The contribution made toward the dimerization specificity by subdomains of the rod has been addressed by making a chimeric protein consisting of the subfragment 2 (S2) region of the adult isoform and the light meromyosin region of the neonatal isoform. The proportion of heterodimers formed in exchange experiments between the chimera and the neonatal and adult rods rose with increase in the sequence homology between the two exchanging proteins. This suggests that multiple regions of the rod domain of chicken MyHC including S2 can contribute toward dimerization specificity.

T cell mimicry and epitope specificity of cross-reactive T cell clones from rheumatic heart disease.

Mimicry between streptococcal M protein and cardiac myosin is important in the pathogenesis of rheumatic heart disease. M protein-specific human T cell clones derived from rheumatic carditis were cross-reactive with human cardiac myosin, and laminin, a valve protein. Among the 11 CD4(+) and CD8(+) cross-reactive T cell clones, at least 6 different reactivity patterns were distinguished, suggesting different degrees of cross-reactivity and a very diverse T cell repertoire. The latter was confirmed by a heterogeneous Vbeta gene and CDR3 usage. HLA restriction and Th1 cytokine production in response to rM6 protein were preserved when the T cell clones were stimulated by human cardiac myosin or other alpha-helical proteins, such as tropomyosin and laminin. The cross-reactive human T cell clones proliferated to B2 and B3A, dominant peptide epitopes in the B repeat region of streptococcal M protein. In human cardiac myosin, epitopes were demonstrated in the S2 and light meromyosin regions. In our study, T cell mimicry was defined as recognition of structurally related Ags involved in disease and recognized by the same T cell. Mimicry in our study was related to alpha-helical coiled coil proteins which have a repetitive seven-aa residue periodicity that maintains alpha-helical structure and thus creates a high number of degenerate possibilities for recognition by T cells. The study of human T cell clones from rheumatic heart disease revealed potential sites of T cell mimicry between streptococcal M protein and human cardiac myosin and represents some of the most well-defined T cell mimicry in human autoimmune disease.

Evidence for periodicity in the amino-acid sequence of myosin.

We have characterized CNBr digests of rabbit myosin heavy chain, total rod, light meromyosin and mouse heavy chain by combined isoelectric focusing and dodecylsulfate gel electrophoresis. Conditions for digest were chosen so as to have less than complete cleavage at most or all of the methionine residues. Examination of the gels in the dodecylsulfate dimension shows a remarkable periodicity in the molecular weights of fragments produced in all these digests. Nearly all lie in a monotonic sequence with an interval of approximately 3800 between them. One interpretation of this finding is that there is a high degree of periodicity in the position of methionines of, at the very least, the light meromyosin region. The impression of periodicity is further re-inforced by a regular distribution of fragments in the isoelectric focusing dimension of the gels as well as in the dodecylsulfate dimension. The heterogeneity in the isoelectric focusing dimension could, unlike that in the dodecylsulfate dimension, be artefactual (e.g. as a consequence of de-amidation and or cyanylation) or as a result of protein heterogeneity due to gene duplication. However, it is also possible that it too reveals a periodicity, this time in the distribution of charged residues in the amino-acid sequence.

Heat-induced structural changes and aggregation of walleye pollack myosin in the light meromyosin region

: Heat-induced structural changes and aggregation properties of walleye pollack myosin, light meromyosin (LMM) and heavy meromyosin (HMM) were investigated. According to the circular dichroism (CD) measurement, the α-helix content of the pollack myosin and LMM were estimated to be 72% and 90% at 5°C but decreased to 22% and 21% by increasing the temperature to 60°C with two transitions at 35°C and 50°C, respectively. In contrast, that of HMM decreased gradually from 37% to 33% by increasing the temperature from 5°C to 40°C, and decreased steeply to 20% above 50°C. These results indicate that the decrease in the α-helix content in the myosin molecule upon heating was attributable mainly to the decrease in the α-helix content in the LMM region. In contrast, 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence and light scattering intensity of both myosin and HMM were remarkably increased above 25°C and 35°C, respectively, while those of LMM showed only a slight change even above 60°C. Although LMM alone formed no aggregates detectable by the light scattering measurement, it formed coprecipitates with myosin but not with HMM upon heating at 40°C for 10 min. These facts suggest that LMM bind to the LMM region of the myosin. Further, it was found that myosin gel formed in a test tube by the same heating conditions was significantly weakened by coexistence of LMM. These results suggest that the association of the LMM region of myosin molecules is essential for the heat-induced gelation of myosin.

Mutations of the light meromyosin domain of the beta-myosin heavy chain rod in hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) is caused by mutations in 9 sarcomeric protein genes. The most commonly affected is beta-myosin heavy chain (MYH7), where missense mutations cluster in the head and neck regions and directly affect motor function. Comparable mutations have not been described in the light meromyosin (LMM) region of the myosin rod, nor would these be expected to directly affect motor function. We studied 82 probands with HCM in whom no mutations had been found in MYH7 exons encoding the head and neck regions of myosin nor in the other frequently implicated disease genes. Primers were designed to amplify exons 24 to 40 of MYH7. These amplimers were subjected to temperature modulated heteroduplex analysis by denaturing high-performance liquid chromatography. An Ala1379Thr missense mutation in exon 30 segregated with disease in three families and was not present in 200 normal chromosomes. The mutation occurred on two haplotypes, indicating that it was not a polymorphism linked with another disease-causing mutation. The position of this residue within the LMM region of myosin suggests that it may be important for thick filament assembly or for accessory protein binding. A further missense mutation in exon 37, Ser1776Gly, segregated with disease in a single family and was absent from 400 population-matched control chromosomes. Because the Ser1776 residue occupies a core position in the myosin rod at which the substitution of glycine is extremely energetically unfavorable, it is likely to disrupt the coiled-coil structure. We conclude that mutation of the LMM can cause HCM and that such mutations may act through novel mechanisms of disease pathogenesis involving myosin filament assembly or interaction with thick filament binding proteins.

Anti-Human Cardiac Myosin Autoantibodies in Kawasaki Syndrome

Kawasaki syndrome (KS) is the major cause of acquired heart disease in children. Although acute myocarditis is observed in most patients with KS, its pathogenesis is unknown. Because antimyosin autoantibodies are present in autoimmune myocarditis and rheumatic carditis, the purpose of the current study was to determine whether anticardiac myosin Abs might be present during the acute stage of KS. Sera from KS patients as well as age-matched febrile controls and normal adults were compared for reactivity with human cardiac myosin in ELISAs and Western blot assays. A total of 5 of 13 KS sera, as compared with 5 of 8 acute rheumatic fever sera, contained Ab titers to human cardiac myosin that were significantly higher than those found in control sera. Both cardiac and skeletal myosins were recognized in the ELISA by KS sera, although stronger reactivity was observed to human cardiac myosin. Only IgM antimyosin Abs from KS sera were significantly elevated relative to control sera. KS sera containing antimyosin Abs recognized synthetic peptides from the light meromyosin region of the human cardiac myosin molecule and had a different pattern of reactivity than acute rheumatic fever sera, further supporting the association of antimyosin Ab with KS. These Abs may contribute to the pathogenesis of acute myocarditis found in patients with KS.