Primary structure of myosin from the striated adductor muscle of the Atlantic scallop, Pecten maximus, and expression of the Regulatory Domain

We have determined the complete cDNA and deduced amino acid sequences of the heavy chain, regulatory light chain and essential light chain which constitute the molecular structure of myosin from the striated adductor muscle of the scallop, Pecten maximus. The deduced amino acid sequences of P. maximus regulatory light chain, essential light chain and heavy chain comprise 156, 156 and 1940 amino acids, respectively. These myosin peptide sequences, obtained from the most common of the eastern Atlantic scallops, are compared with those from three other molluscan myosins: the striated adductor muscles of Argopecten irradians and Placopecten magellanicus, and myosin from the siphon retractor muscle of the squid, Loligo pealei. The Pecten heavy chain sequence resembles those of the other two scallop sequences to a much greater extent as compared with the squid sequence, amino acid identities being 97.5% (A. irradians), 95.6% (P. magellanicus) and 73.6% (L. pealei), respectively. Myosin heavy chain residues that are known to be important for regulation are conserved in Pecten maximus. Using these Pecten sequences, we have overexpressed the regulatory light chain, and a combination of essential light chain and myosin heavy chain fragment, separately, in E. coli BL21 (DE3) prior to recombination, thereby producing Pecten regulatory domains without recourse to proteolytic digestion. The expressed regulatory domain was shown to undergo a calcium-dependent increase (7%) in intrinsic tryptophan fluorescence with a mid-point at a pCa of 6.6.     

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.     

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.     

Sequence variations in the surface loop near the nucleotide binding site modulate the ATP turnover rates of molluscan myosins

Summary The muscle and species-specific differences in enzymatic activity between Placopecten and Argopecten striated and catch muscle myosins are attributable to the myosin heavy chain. To identify sequences that may modulate these differences, we cloned and sequenced the cDNA encoding the myosin heavy chains of Placopecten striated and catch muscle. Deduced protein sequences indicate two similar isoforms in catch and striated myosins (97% identical); variations arise by differential RNA splicing of five alternative exons from a single myosin heavy chain gene. The first encodes the phosphate-binding loop; the second, part of the ATP binding site; the third, part of the actin binding site; the fourth, the hinge in the rod; and the fifth, a tailpiece found only in the catch muscle myosin heavy chain. Both Placopecten myosin heavy chains are 96% identical to Argopecten myosin heavy chain isoforms. Because subfragment-1 ATPase activities reflect the differences observed in the parent myosins, the motor domain is responsible for the variations in ATPase activities. In addition, data show that differences are due to V_max and not actin affinity. The sequences of all four myosin heavy chain motor domains diverge only in the flexible surface loop near the nucleotide binding pocket. Thus, the different ATPase activities of four molluscan muscle myosins are likely due to myosin heavy chain sequence variations within the flexible surface loop that forms part of the ATP binding pocket of the motor domain.     

Tuning smooth muscle contraction by molecular motors

As in striated muscle, smooth muscle cells (SMC) contract by Ca²⁺ activated cyclic interaction between actin and type II myosin. However, smooth muscle maintains tone at basal activating Ca²⁺ and low energetic cost during sustained activation. This review analyzes the regulation of phasic and tonic contraction of SMC on the molecular level. Type II myosin is the molecular motor also of smooth muscle contraction. Six myosin heavy chain (MHC) isoenzymes (four smooth muscle, two nonmuscle) and five myosin light chain (MLC) isoforms (two 17 kDa, two 20 kDa, one 23 kDa) are expressed in SMC. These myosin subunits could be generated by alternative splicing or by differential gene expression. Thus different myosin isoenzymes are generated which may be modified posttranslationally by phosphorylation, affecting the contractile state of the SMC. Furthermore, they may be part of distinct contractile systems which are targeted by different second messenger cascades and are recruited differentially during activation, electromechanical, and pharmacomechanical coupling. Low energy consumption, shortening velocity, and MLC20 phosphorylation at low Ca²⁺ activation levels during tone maintenance ("latch") could be explained by a switch from smooth muscle myosin to nonmuscle myosin activation upon prolonged activation.Abbreviations MHC Myosin heavy chains - MLC Myosin light chains - MLCK Myosin light chain kinase - MLCP MLC20 phosphatase - NM Nonmuscle - nt Nucleotide - SM Smooth muscle - SMC Smooth muscle cells     

Cloning of the cDNA encoding human nonmuscle myosin heavy chain-B and analysis of human tissues with isoform-specific antibodies

Summary Previously, we reported the sequence of cDNA clones encoding amino acids 63 through 723 of the human nonmuscle myosin heavy chain-B isoform. In this paper, we present the derived sequence of the remaining 1303 amino acids along with 5 and 3 untranslated sequences. We made use of the differences between the derived nonmuscle myosin heavy chain-A and-B amino acid sequences to raise isoform-specific antibodies. Immunoblot analysis reveals a differential expression of both myosin heavy chain isoforms in a variety of human adult and foetal tissues and cells. When extracts of human adult aorta were subjected to gel electrophoresis, two distinct Coomassie Blue-stained bands and a fused band were seen migrating at approximately 200 kDa. These bands can be detected with four different specific antibodies recognizing the two different smooth muscle myosin heavy chain isoforms (204 kDa and 200 kDa) and the two different nonmuscle myosin heavy chain isoforms (A and B). Using immunohistochemistry, we confirmed the presence of the four different isoforms in adult and foetal aortas.Present address: Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.     

Myosin-based cortical tension in Dictyostelium resolved into heavy and light chain-regulated components

Summary Cortical tension in most nonmuscle cells is due largely to force production by conventional myosin (myosin II) assembled into the cytoskeleton. Cytoskeletal contraction in smooth muscle and nonmuscle cells is influenced by the degree of myosin filament assembly, and by activation of myosin motor function via regulatory light chain phosphorylation. Recombinant Dictyostelium discoideum cell lines have been generated bearing altered myosin heavy chains, resulting in either constitutive motor function or constitutive assembly into the cytoskeleton. Analysis of these cells allowed stiffening responses to agonists, measured on single cells, to be resolved into an regulatory light chain-mediated component reflecting activation of motor function, and a myosin heavy chain phosphorylation-regulated component reflecting assembly of filaments into the cytoskeleton. These two components can account for all of the cortical stiffening response seen during tested in vivo contractile events.     

Antisense suppression of skeletal muscle myosin light chain-1 biosynthesis impairs myofibrillogenesis in cultured myotubes

Summary Although the alkali or essential light chains of skeletal muscle myosin are not required for actin-activated myosin ATPase activity, these myosin subunits are necessary for force transmission with in vitro actin motility assays and are believed to stabilize the -helical neck region of myosin subfragment-1. To probe the functions of the essential light chains during myofibril assembly, we used recombinant DNA procedures to deplete this light chain in cultured muscle. Retroviral expression vectors were constructed which encoded the exon-1 sequence of the myosin light chain-1 gene in antisense orientation. These vectors were applied to myogenic cells from embryonic chick and quail pectoralis muscle. Colonies expressing antisense RNA were selected in growth medium containing the neomycin analogue G-418, plus 5-bromo-2-deoxyuridine (BrdU) and triggered to differentiate by removal of the latter. Expression of antisense myosin light chain-1 mRNA impaired muscle development. In the antisense cultures there were more mononucleated cells, fewer and smaller myotubes which had poorly developed myofibrils and high levels of diffusely staining myosin heavy chain, not apparent in control myotubes. Protein synthesis in the myotube cultures was analyzed by ³⁵S-methionine labelling and two-dimensional gel electrophoresis. Except for a suppression of 80% of myosin light chain-1_f synthesis, the overall pattern of protein synthesis was not altered significantly. These studies suggest that retardation of myosin light chain-1_f accumulation inhibits or delays myofibrillogenesis.     

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.     

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.     

Complete sequence of human cardiac α-myosin heavy chain gene and amino acid comparison to other myosins based on structural and functional differences

We have obtained the 5820 nucleotide sequence encoding all 1939 amino acids of the human cardiac α-myosin heavy chain (α-MHC

1 The sequence has been deposited in the DDBJ base. (Accession no. D00943)

), as established by dideoxy sequencing of cloned cDNA, genomic DNA and polymerase chain reaction (PCR) amplification products. This sequence represents overlapping fragments of the entire coding sequence. Amino acid sequence comparison of the human cardiac α-MHC with the published human cardiac β-MHC have demonstrated that there are, at least, 7 isoform-specific divergent regions, including functionally important binding protein-related sites such as ATP, actin and myosin light chain. It has been reported that in the rat, there are 8 isoform-specific divergent regions. The 7th divergent area (residue area 1633-1657, which is thought to mediate thick filament formation) in the light meromyosin region in the rat is not apparent in the human. The amino acid compositions of cardiac α- and β-MHCs in the human and the rat, and human embryonic skeletal muscle and chicken gizzard smooth muscles were compared. Amino acid sequences in cardiac α- and β-MHCs in the human and the rat are well conserved. In the head portion, the amino acid composition divergence of human cardiac α-MHC is ranked between rat cardiac α-MHC and human cardiac β- or rat cardiac β-MHC; human skeletal muscle MHC is the most divergent of the myosin isoform examined. These data predict that human cardiac α-MHC may have undergone evolutionary changes toward obtaining the biochemical and physiological properties of cardiac β-MHC.     

Specificity of blebbistatin, an inhibitor of myosin II

Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. We have examined the specificity and potency of the drug by assaying its effects on the actin-activated MgATPase assay of diverse members of the myosin superfamily. Blebbistatin potently inhibits several striated muscle myosins as well as vertebrate nonmuscle myosin IIA and IIB with IC₅₀ values ranging from 0.5 to 5 μM. Interestingly, smooth muscle which is highly homologous to vertebrate nonmuscle myosin is only poorly inhibited (IC₅₀=80 μM). The drug potently inhibits Dictyostelium myosin II, but poorly inhibits Acanthamoeba myosin II. Blebbistatin did not inhibit representative myosin superfamily members from classes I, V, and X. This revised version was published online in July 2006 with corrections to the Cover Date.     

Complete sequence of human cardiac alpha-myosin heavy chain gene and amino acid comparison to other myosins based on structural and functional differences.

We have obtained the 5820 nucleotide sequence encoding all 1939 amino acids of the human cardiac alpha-myosin heavy chain (alpha-MHC), as established by dideoxy sequencing of cloned cDNA, genomic DNA and polymerase chain reaction (PCR) amplification products. This sequence represents overlapping fragments of the entire coding sequence. Amino acid sequence comparison of the human cardiac alpha-MHC with the published human cardiac beta-MHC have demonstrated that there are, at least, 7 isoform-specific divergent regions, including functionally important binding protein-related sites such as ATP, actin and myosin light chain. It has been reported that in the rat, there are 8 isoform-specific divergent regions. The 7th divergent area (residue area 1633-1657, which is thought to mediate thick filament formation) in the light meromyosin region in the rat is not apparent in the human. The amino acid compositions of cardiac alpha- and beta-MHCs in the human and the rat, and human embryonic skeletal muscle and chicken gizzard smooth muscles were compared. Amino acid sequences in cardiac alpha- and beta-MHCs in the human and the rat are well conserved. In the head portion, the amino acid composition divergence of human cardiac alpha-MHC is ranked between rat cardiac alpha-MHC and human cardiac beta- or rat cardiac beta-MHC; human skeletal muscle MHC is the most divergent of the myosin isoform examined. These data predict that human cardiac alpha-MHC may have undergone evolutionary changes toward obtaining the biochemical and physiological properties of cardiac beta-MHC.     

Partial purification of two myosin heavy chain kinases fromDictyostelium discoideum

Summary Myosin heavy chain kinase activity was identified in the high speed supernate of lysedDictyostelium amoebae and was precipated by 30–50% ammonium sulphate. In low ionic strength buffer, the activity bound tightly to a Cibacron Blue Sepharose column and eluted as a single peak with 1.0m NaCl. Gel filtration chromatography resolved the kinase into two activities, each of which phosphorylated the tail portion of purifiedDictyostelium myosin. One of these activities phosphorylated both serine and threonine residues of the heavy chain, while the other activity only phosphorylated threonine residues. Peptide mapping studies indicated thatin vivo andin vitro phosphorylation sites were identical. The heavy chain kinases required Mg²⁺ for activity but were unaffected by Ca²⁺ or calmodulin. The heavy chain kinases did not phosphorylateDictyostelium light chain, and also did not phosphorylate myosins from striated, smooth, or other nonmuscle sources.     

Nonmuscle myosin IIA with a GFP fused to the N-terminus of the regulatory light chain is regulated normally

Nonmuscle myosin II plays a crucial role in a variety of cellular processes (e.g., polarity formation, cell motility, and cytokinesis). It is composed of two heavy chains, two regulatory light chains and two essential light chains. The ATPase activity of the myosin II motor domain is regulated through phosphorylation of the regulatory light chain (RLC) by myosin light chain kinase. To study myosin function and localization in cellular processes, GFP-fused RLCs are widely used; however, the exact kinetic properties of myosins with bound GFP-RLC are poorly described. More importantly, it has not been shown that a regulatory light chain fused at its N-terminus with GFP can maintain the normal phosphorylation-dependent regulation of nonmuscle myosin or serve as a substrate for myosin light chain kinase. We coexpressed N-terminal GFP-RLC with a heavy meromyosin (HMM)-like fragment of nonmuscle myosin IIA and essential light chain to characterize the phosphorylation dynamics and in vitro kinetic properties of the resulting HMM. Myosin light chain kinase phosphorylates the GFP-RLC bound to HMM IIA with the same V_max as it does the wild type RLC bound to HMM IIA, but the K_m is about two fold higher for the GFP fusion protein, meaning that it is a somewhat poorer substrate. The steady-state actin-activated MgATPase activity of the GFP-RLC HMM is very low in the absence of phosphorylation demonstrating that the GFP moiety does not prevent formation of the off state. The actin-activated MgATPase activity of phosphorylated GFP-RLC-HMM and is about half that of wild type phosphorylated HMM. The ability of phosphorylated GFP-RLC-HMM to move actin filaments in the actin gliding assay is also slightly compromised. These data indicate that despite some kinetic differences the N-terminal GFP fusion to the regulatory light chain is a reasonable model system for studying myosin function in vivo.     

Partial sequence of connectin-like 1200K-protein in obliquely striated muscle of a polychaete (Annelida): Evidence for structural diversity from vertebrate and invertebrate connectins

Vertebrate striated muscle contains the giant elastic protein connectin that maintains the position of the A-band at the center of the sarcomere during repeated muscular contraction and relaxation. Connectin-like molecules may perform conserved functions in vertebrate and invertebrate striated and oblique muscles, although less is known about the structure of invertebrate connectins at present. The protein that maintains such a structure is present not only in vertebrate striated muscle, but also in invertebrate striated and oblique muscle. In the present study, we analyzed the partial primary structure of a 1200K-protein, which is a connectin-like protein that is expressed in Neanthes sp. body wall muscle that is in turn composed of oblique muscle. Antibody screening of a cDNA library of Neanthes sp. body wall muscle identified two different clones. Both clones coded for a sequence predominantly comprised of the four amino acids proline (P), glutamate (E), valine (V) and lysine (K). One clone included a PEVK-like repeat sequence flanked by an Ig domain, while the other clone comprised a distinct 14 amino acid repeat rich in PEVK residues, flanked by a non-repetitive unique sequence. The PEVK region is found in vertebrate connectin and is thought to generate elasticity and be responsible for passive tension of the muscle. The antibodies produced against a portion of each clone both reacted with bands corresponding to 1200 kDa present in Neanthes sp. body wall muscle. Therefore, our results demonstrate that this 1200K-protein is a connectin-like elastic protein and includes specific PEVK-like fragment. We suggest that this 1200K-protein plays a major role in maintaining the structure of oblique muscle in invertebrates.     

The amino acid sequence of the light chain of Acanthamoeba myosin IC

The amino acid sequence of the light chain of Acanthamoebamyosin IC deduced from the cDNA sequence comprises 149 aminoacids with a calculated molecular weight of 16739. All but the 3N-terminal residues were also determined by amino acid sequencingof the purified protein, which also showed the N-terminus to beblocked. Phylogenetic analysis shows Acanthamoeba myosin IC lightchain to be more similar to the calmodulin subfamily of EF-handcalcium-modulated proteins than to the myosin II essential lightchain or regulatory light chain subfamilies. In pairwisecomparisons, the myosin IC light chain sequence is most similarto sequences of calmodulins (50% identical) and a squidcalcium-binding protein (43% identical); the sequence is37% identical to the calcium-binding essential light chainof Physarum myosin II and 30% identical to the essentiallight chain of Acanthamoeba myosin II, and the essential lightchain and regulatory light chain of Dictyostelium myosin II. Thesequence predicts four helix-loop-helix domains with possiblecalcium-binding sites in domains I and III, suggesting thatcalcium may affect the activity of this unconventional myosin.This is the first report of the sequence of an unconventionalmyosin light chain other than calmodulin