Insights into myosin regulatory and essential light chains: a focus on their roles in cardiac and skeletal muscle function,development and disease

Journal of Muscle Research and Cell Motility - The activity of cardiac and skeletal muscles depends upon the ATP-coupled actin–myosin interactions to execute the power stroke and muscle...     

Rabbit immunoglobulin lambda chains: Amino acid sequences of variable- and constant-region peptides from normal and allotype-suppressed rabbits

The amino acid sequences of fragments from the variable and constant regions of rabbit IgG λ-chains from different sources were determined and compared. The peptides had considerable homology with known sequences of human and murine λ-chains and, thus, could be readily aligned.Sequences of corresponding peptides from different sources and preparations reveal differences in the variable as well as the constant region. The latter probably reflect the existence of different alleles and/or isotypes at the c-locus, each coding for a different constant half of the λ-chain.Comparison of the rabbit λ-light-chain sequences with those of human and mouse λ-chains reveals a surprising degree of homology.The amino acid sequences of the peptides reported cover approximately one half of the total sequence of the light chains.     

Amino acid sequence of the constant region of immunoglobulin light chains from Rana catesbeiana

Little is known about the detailed structure of immunoglobulins in non-mammalian vertebrates. We have determined the amino acid sequence of the constant region of immunoglobulin light chains from the bullfrog, Rana catesbeiana. There appears to be one major type of light chain in this species. However, at position 153, about half of the chains have lysine, the remainder having arginine. Variation at this same or at an adjacent position is responsible for allotypic or isotypic variation in human kappa and lambda chains. At three positions (118, 119, and 181), residues that occur in all known kappa and lambda chains in other species are replaced in the Rana catesbeiana sequence. One of these unusual substitutions--the replacement of proline by cysteine at position 119--allows the formation of an extra intrachain disulfide bond within the constant domain, between positions 119 and 214. This bond appears to replace the usual disulfide bridge between heavy and light chains so that, in the immunoglobulins of this species, the light chains are not covalently bonded to heavy chains. The sequence of the Rana catesbeiana constant region is compared to sequences of a variety of mammalian light chain constant regions. We consider the implications of these comparisons for the timing of the divergence of kappa and lambda chains relative to the divergence of the lineages leading to amphibians and to mammals and birds.     

Systematic analysis of the regulatory and essential myosin light chain genes: genetic variants and mutations in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) can be caused by mutations in genes encoding for the ventricular myosin essential and regulatory light chains. In contrast to other HCM disease genes, only a few studies describing disease-associated mutations in the myosin light chain genes have been published. Therefore, we aimed to conduct a systematic screening for mutations in the ventricular myosin light chain genes in a group of clinically well-characterised HCM patients. Further, we assessed whether the detected mutations are associated with malignant or benign phenotype in the respective families. We analysed 186 unrelated individuals with HCM for the human ventricular myosin regulatory (MYL2) and essential light chain genes (MYL3) using polymerase chain reaction, single strand conformation polymorphism analysis and automated sequencing. We found eight single nucleotide polymorphisms in exonic and adjacent intronic regions of MYL2 and MYL3. Two MYL2 missense mutations were identified in two Caucasian families while no mutation was found in MYL3. The mutation Glu22Lys was associated with moderate septal hypertrophy, a late onset of clinical manifestation, and benign disease course and prognosis. The mutation Arg58Gln showed also moderate septal hypertrophy, but, in contrast, it was associated with an early onset of clinical manifestation and premature sudden cardiac death. In conclusion, myosin light chain mutations are a very rare cause of HCM responsible for about 1% of cases. Mutations in MYL2 could be associated with both benign and malignant HCM phenotype.     

Clonorchis sinensis: molecular cloning and localization of myosin regulatory light chain

One cDNA clone was purified from an adult Clonorchis sinensis cDNA library, and its deduced polypeptide sequence was found to be homologous with myosin regulatory light chain (MRLC) of invertebrates and vertebrates. Two amino-acid residues, Thr and Ser, were conserved at the phosphorylation sites that regulate the function of MRLCs. Recombinant C. sinensis MRLC (rCsMRLC) protein was produced and purified from Escherichia coli, and mouse anti-CsMRLC immune sera recognized a protein of molecular weight 24 kDa from a soluble protein preparation of C. sinensis. The CsMRLC protein was immunohistochemically localized to the muscle fibers of the subtegumental muscle layer and to the muscles of oral and ventral suckers. However, the rCsMRLC protein proved to be less useful antigen for the serodiagnosis of human clonorchiasis.The nucleotide sequence reported herein was submitted to GenBank and assigned accession number AY519356.     

Proteomic analysis of physiological versus pathological cardiac remodeling in animal models expressing mutations in myosin essential light chains

In this study we aimed to provide an in-depth proteomic analysis of differentially expressed proteins in the hearts of transgenic mouse models of pathological and physiological cardiac hypertrophy using tandem mass tag labeling and liquid chromatography tandem mass spectrometry. The Δ43 mouse model, expressing the 43-amino-acid N-terminally truncated myosin essential light chain (ELC) served as a tool to study the mechanisms of physiological cardiac remodeling, while the pathological hypertrophy was investigated in A57G (Alanine 57 → Glycine) ELC mice. The results showed that 30 proteins were differentially expressed in Δ43 versus A57G hearts as determined by multiple pair comparisons of the mutant versus wild-type (WT) samples with P < 0.05. The A57G hearts showed differential expression of nine mitochondrial proteins involved in metabolic processes compared to four proteins for ?43 hearts when both mutants were compared to WT hearts. Comparisons between ?43 and A57G hearts showed an upregulation of three metabolically important mitochondrial proteins but downregulation of nine proteins in ?43 hearts. The physiological model of cardiac hypertrophy (?43) showed no changes in the levels of Ca²⁺-binding proteins relative to WT, while the pathologic model (A57G) showed the upregulation of three Ca²⁺-binding proteins, including sarcalumenin. Unique differences in chaperone and fatty acid metabolism proteins were also observed in Δ43 versus A57G hearts. The proteomics data support the results from functional studies performed previously on both animal models of cardiac hypertrophy and suggest that the A57G- and not ?43- mediated alterations in fatty acid metabolism and Ca²⁺ homeostasis may contribute to pathological cardiac remodeling in A57G hearts.     

Preparation of frog myosin. Isolation and characterization of the light chains

Summary Frog myosin can be prepared with a good yield by precipitation of a high ionic strength extract betweenI 0.20 and 0.05 or by ammonium sulphate fractionation of actomyosin in the presence of Mg-ATP.Two alkali light chains, LC₁ and LC₃, along with one DTNB light chain LC₂ have been isolated by chromatography on ion exchange cellulose after urea dissociation. A supplementary light chain LC₁d present in variable amounts from one preparation to the other corresponds to a proteolysis product of LC₁.Their stoichiometry, molecular weight, amino acid composition, isoelectric point and peptide map have been determined.Their general proportions and structural properties show many similarities with rabbit skeletal muscle light chains.Amino acid compositions and peptide maps confirm that the additional band LC₁d comes from a proteolytic degradation affecting the N-terminal part of LC₁.B. Focant is Chercheur qualifié of the Fonds National de la Recherche Scientifique of Belgium.     

Mechanism of phosphorylation of the regulatory light chain of myosin from tarantula striated muscle

Contraction is modulated in many striated muscles by Ca²⁺-calmodulin dependent phosphorylation of the myosin regulatory light chain (RLC) by myosin light chain kinase. We have investigated the biochemical mechanism of RLC phosphorylation in tarantula muscle to better understand the basis of myosin-linked regulation. In an earlier study it was concluded that the RLC occurred as two species, both of which could be phosphorylated, potentiating contraction. Here we present evidence that only a single species exists, and that this can be phosphorylated at one or two sites. In relaxed muscle we find evidence for a substantial level of basal phosphorylation at the first site. This is augmented on activation, followed by partial phosphorylation of the second site. We find in addition that Ca²⁺ has a dual effect on light chain phosphorylation, depending on its concentration. At low concentration (relaxing conditions) only basal phosphorylation is observed, while at higher concentrations (activating conditions) RLC phosphorylation is stimulated. At still higher Ca²⁺ concentrations we find partial inhibition of RLC phosphorylation, suggesting an additional mechanism by which the muscle cell can fine tune contractile activity by controlling the level of free Ca²⁺.     

Essential and regulatory light chains of Placopecten striated and catch muscle myosins

Summary ATPase activities of molluscan adductor muscle myosins show both muscle and species specific differences: ATPase activity of catch muscle myosin is lower than that of phasic muscle myosin; a 4–5-fold difference exists between the activities of phasic striated muscle myosins from the bay scallop (Argopecten irradians) and sea scallop (Placopecten magellanicus). To characterize the light chains of these myosins we determined the cDNA sequences of the essential light chains and the regulatory light chains from Placopecten striated and catch muscle. The nucleotide sequences of the essential light chains from Placopecten striated and catch muscle myosins are identical and show 94% identity and 98% homology to the Argopecten essential light chain indicating that the tissue and species specific differences in ATPase activities are not due to the essential light chain. We identified three regulatory light chain isoforms, one from striated and two from catch muscle. Sequence differences were restricted to nucleotides encoding some of the N-terminal 52 amino acids. The three recombinant Placopecten regulatory light chain isoforms and the Argopecten regulatory light chain were incorporated into hybrid myosins that contained the essential light chain and heavy chain from Placopecten striated, Placopecten catch, or Argopecten striated muscle. Measurement of the ATPase activities of these hybrids indicates clearly that it is the myosin heavy chain and not the regulatory light chains that are responsible for the muscle and species specific differences in enzymatic activities. Analysis of genomic DNA indicated that these regulatory light chain isoforms are products of a single regulatory light chain gene that is alternatively spliced in the 5 region only.     

Effect of electrical stimulation and exercise on the phosphorylation state of myosin light chains from fish skeletal muscle

This paper investigates the phosphorylation of fish muscle myosin following electrical stimulation and exercise. Purified myosin isolated from the fast myotomal muscle of rainbow trout (Salmo gairdneri) shows three light chains on SDS polyacrylamide gels of molecular weights 16,000 (LC1) 18,000 (LC2) and 24,000 (LC3). The 18,000 dalton light chain (LC2) is capable of being phosphorylated and dephosphorylated in vitro by respectively rabbit myosin light chain kinase and phosphatase.The native phosphorylation state of fast muscle myosin has been investigated in rainbow trout and dogfish (Scyliorhinus canicula) using a technique involving freeze-clamping in liquid nitrogen (–159°C) and extraction of a light chain fraction in 5 M guanidine-HCl/ethanol. The two forms of the LC2 light chain, can be resolved in 8 M urea polyacrylamide gels at pH 8.6.Fast muscle from anaesthetised trout contained 0.16 moles phosphorylated LC/mole LC2. Electrical stimulation of isolated muscle under various conditions (1–10 s isometric tetani, 5–500 Hz) did not result in a significant increase in the phosphorylated form of the LC2 light chain. Qualitatively similar results were obtained for isolated fast muscles from dogfish (Scyliorhinus canicula). In five trout subject to strenuous exercise, two fish showed a slight increase in myosin phosphorylation (0.32 moles P LC/mole LC2) and three, no significant change (0.20 moles P LC/mole LC2).The lack of correlation between the phosphorylation state of LC2 light chain and electrical stimulation indicates that, unlike rabbit and frog skeletal muscle myosin, phosphorylation is not an integral part of the excitation-contraction cycle in fish muscle.     

Molecular cloning and sequencing of myosin light chains in human megakaryoblastic leukemia cells.

Myosin light chain genes of hematopoietic cells have yet to be characterized. We cloned the full-length cDNAs of 20 kDa regulatory myosin light chain (MLC-2) and 17 kDa essential myosin light chain (MLC-3) from Meg-01, a human megakaryoblastic leukemia cell line. Both MLC-2 and MLC-3 gene are transcribed ubiquitously in various hematopoietic cells. The MLC-2 open reading frame of 516 nucleotides encoding a protein of 172 residues was detected in cloned cDNA of 967 nucleotides. The Ca2+-binding domain and five phosphorylation sites were highly conserved. The deduced amino acid sequence has a 99.4% and 100% homology with that of human fetus brain and human lymphocyte, respectively. The MLC-3 open reading frame of 453 nucleotides encoding a protein of 151 residues was detected in cloned cDNA of 742 nucleotide. The MLC-3 protein is 99.3% identical to that of human fibroblasts. These results suggest that hematopoietic myosin light chain proteins are similar to those of other nonmuscle cells and smooth muscle, thus differing from skeletal and cardiac muscles.     

Expression of myosin heavy and light chains changes during pregnancy in the rat uterus

We investigated the in vivo expression of myosin heavy chains (MHC) and myosin light chains (MLC) in the rat uterus during pregnancy and post parturn (p. p.). According to their antigenic reactivity, we observed two smooth-muscle-specific MHC (SM-MHC) of 204 kDa and 200 kDa (SM1 and SM2 respectively) and one non-muscle-specific MHC band of 198 kDa (NM-MHC). Adult virgin female rats expressed SM1 and NM-MHC (65/35) but no SM2. During the first pregnancy NM-MHC expression decreased in favour of the SM1 form, the SM1/NM-MHC ratio being 82/12 after 20 days of pregnancy. SM2 is newly expressed in the first p. p. state, the SM1/SM2/NM-MHC ratio being 58/28/14 between 3 and 33 days p. p. During the second pregnancy, starting 34 days p. p., both SM2 and NM-MHC expression decreased, the SM1/SM2/NM-MHC ratio being 85/0/15 after 20 days of pregnancy. Two protein bands of approximately 154 kDa and 145 kDa (sodium dodecylsulphate electrophoresis), designated SMP1 and SMP2 respectively, with the same immunoreactivity as the SM-MHC were observed in vivo in the pregnant but not in the non-pregnant uterus. In addition, a Ca²⁺-independent protease, which uses MHC as substrate, is expressed in the pregnant but not in the non-pregnant rat uterus. Two isoforms of the 17-kDa MLC (LC_17a and LC_17b) exist in the rat uterus. Expression of the LC_17a isoform increased during the first pregnancy from 46% in virgin rats to 65% in uteri of rats 20 days pregnant. During the first p. p. state LC_17a expression decreased to 42% at 19 days p. p. then remained constant up to 33 days p. p. Again, during the second pregnancy LC_17a expression increased to the same extent as observed in the first pregnancy. Phosphorylation of the 20-kDa MLC forms of skinned uterus fibres derived from 6-days- and 20-days-pregnant rats (first and second pregnancy) increased upon Ca²⁺ activation but the phosphorylation levels were the same in both groups. As extrapolated from the force/velocity relation, the maximal shortening velocity (V _max) of chemically skinned uteri from 6-days pregnant-rats was 0.104 muscle length (ML) s^–1 and increased up to 0.201 ML s^–1 in uteri from 20-days-pregnant animals (first pregnancy). V _max remained high 2 days p. p. (0.2 ML s^–1) and decreased to 0.09 ML s^–1 19 days p. p. During the second pregnancy V _max was 0.1 ML for 6-days-pregnant uteri and increased up to 0.21 ML s^–1 after 20 days of pregnancy. Our results suggest a correlation between LC_17a expression and V _max rather than between MHC and V _max.     

Comparison of the body wall myosin heavy chain sequences from Onchocerca volvulus and Brugia malayi.

The complete coding sequence of Onchocerca volvulus myosin heavy chain has been determined from a series of overlapping cDNAs. The protein sequences from the 2 filarids, one responsible for subcutaneous filariasis, the other for lymphatic filariasis, show 92% identity, and are 1957 amino acids long. Each protein sequence is also equally related, with 75% identity, to MHC-B, the protein encoded by the unc-54 gene of the free-living nematode C.elegans. Such analysis is useful in phylogenetic studies among nematodes, as well as in structure-function relationships among myosin isolates.     

A comparison of the N-terminal amino acid sequences of early and late pools of anti-DNP and anti-DNP-p-aminobenzoylglutamate antibody light chains.

The N-terminal amino acid sequences of early and late pools of anti-DNP and anti-DNP-p-aminobenzoylglutamate (DNP-ABG) antibody light chains were quantitatively determined and compared. The amino acid composition at each locus of the N-terminal 20 amino acid residues of each light chain preparation was determined using automatic sequencing techniques coupled with high-pressure liquid chromatography and mass spectrometry. The sequence data obtained for the light chains corresponding to antibodies isolated early in the immune response (3-4 weeks) were essentially the same as those for light chains from antibodies isolated late in the response (12-14 weeks). In addition, it was observed that the sequence data obtained for the anti-DNP antibody light chain preparation were almost identical to those found for two anti-DNP-ABG antibody light "hain preparations. The sequence data obtained in the present study were compared with those obtained for normal rabbit light chains and with the composite sequence data published for other rabbit anti-hapten light chains.     

Electron microscopy of cardiac myosin: Its shape and properties as determined by the regulatory light chain

Summary Structural properties of dog cardiac myosin and the influence of the regulatory light chain (LC2) on the shape of myosin heads were investigated by electron microscopy. LC2 was reversibly removed using a neutral protease from myopathic hamsters (Margossian,J. Biol. Chem. 260 (1985) 13747–54). The distribution of myosin head length centred around 17 nm with the mean length being 18.9 nm. Statistical analysis suggested that myosin heads became more globular upon removal of LC2. No extensive aggregation of myosin could be detected after LC2 was dissociated, either by sedimentation velocity or by gels run under non-denaturing conditions. The centre-to-centre distance between heads remained constant at about 21 nm, regardless of the presence or absence of LC2. The distribution of length of the globular region reveals two peaks at 7.5 and 9.5 nm, suggesting an extended and a shorter configuration of this region. The decrease in mass at the head/tail junction upon LC2 removal suggests that it is the binding site for the regulatory light chains. A bend at 57 nm from the head/tail joint was sometimes noticed, corresponding to the myosin hinge region. In high resolution micrographs individual particles revealed invaginations along the contours of the head, possibly delineating the boundaries of structural domains within the head. The conformation of arrowheads in actin decorated with either subfragment 1 (S1) or heavy meromyosin (HMM) was investigated in the presence and absence of LC2.     

Expression of myosin heavy and light chains and phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the European hamster during hibernation and in summer

Summary We investigated the expression of myosin subunits (myosin heavy chains) as well as light chains and thein vivo phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the adult male European hamster (Cricetus cricetus L.). Two myosin heavy chain isoenzymes could be detected under native and denaturing electrophoretic conditions having high (-myosin heavy chain) and low (-myosin heavy chain) enzymatic activity. Enzymatic activity of- and-myosin heavy chain revealed a different temperature dependency. When temperature increased ATPase activity of the-myosin heavy chain isoenzyme increased relatively more than ATPase activity of the-myosin heavy chain isoenzyme. Summer animals expressed predominantly the-myosin heavy chain (79% of total myosin) while during hibernation the-myosin heavy chain expression increased to 53% of total myosin. Winter-active hamsters kept at 22°C and 12 h day/night rhythm showed the same myosin heavy chain isoenzyme pattern as summer-active animals. Two myosin light chain forms were expressed in the ventricle of all animal groups. thein vivo phosphorylation level of the phosphorylatable myosin light chain decreased from 45% in summer-active hamster to 23% during hibernation.