Solubility-enhanced gMYL6 fused with a hexa-lysine tag promotes the cytotoxicity of human NK cells.

Goat myosin light chain 6 (gMYL6) is a constituent of certain extracted immunization-induced goat anti-cancer bioactive peptides (ACBPs). However, little is known about its activity onto NK cells which are the basic cellular attackers in cancer immunotherapy for patients with malignancies. Because of the complicated extraction process and low yield of gMYL6 out of the goat ACBPs' mixture, the Nano-flow liquid chromatography and C-terminal polycationic tag expression strategy were used to identify and enrich the peptide to investigate its bioactivity against cancers/tumors. The solubility-enhanced gMYL6 fused with a hexa-lysine tag showed a capacity of promoting the NK cells' cytotoxicity, making it a novel promising heterogeneous peptide cytokine against cancers.

Purification, Characterization, and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkii.

Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.

Molecular cloning and functional analysis of MRLC2 in Tianfu, Boer, and Chengdu Ma goats.

To determine the molecular basis of heterosis in goats, fluorescence quantitative polymerase chain reaction (PCR) was performed to investigate myosin-regulatory light chain 2 (MRLC2) gene expression in the longissimus dorsi muscle tissues of the Tianfu goat and its parents, the Boer and Chengdu Ma goats. The goat MRLC2 gene was differentially expressed in the crossbreed, and the purebred mRNA were isolated and identified using fluorescence quantitative reverse transcription-PCR (RT-PCR). The complete coding sequence of MRLC2 was obtained using the cDNA method, and the full-length coding sequence consisted of 513 bp encoding 172 amino acids. The EF-hand superfamily domain of the MRLC2 protein is well conserved in caprine and other animals. The deduced amino acid sequence of MRLC2 shared significant identity with MRLC2 from other mammals. Phylogenetic tree analysis revealed that the MRLC2 protein was closely related to MRLC2 in other mammals. Several predicted miRNA target sites were found in the coding sequence of caprine MRLC2 mRNA. Analysis by RT-PCR showed that MRLC2 mRNA was present in the heart, stomach, liver, spleen, lung, small intestine, kidney, leg muscle, abdominal muscle, and longissimus dorsi muscles. In particular, the high expression of MRLC2 mRNA was detected in the longissimus dorsi, leg muscle, abdominal muscle, stomach, and heart, but low levels of expression were also observed in the liver, spleen, lung, small intestine, and kidney. The expression of the MRLC2 gene was upregulated in the longissimus dorsi muscle of Boer and Tianfu goats, and it was moderately upregulated in Chengdu Ma goats.

Mapping of O-linked beta-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.

IQ motif selectivity in human IQGAP1: binding of myosin essential light chain and S100B.

IQGAPs are cytoskeletal scaffolding proteins which link signalling pathways to the reorganisation of actin and microtubules. Human IQGAP1 has four IQ motifs each of which binds to calmodulin. The same region has been implicated in binding to two calmodulin-like proteins, the myosin essential light chain Mlc1sa and the calcium and zinc ion binding protein S100B. Using synthetic peptides corresponding to the four IQ motifs of human IQGAP1, we showed by native gel electrophoresis that only the first IQ motif interacts with Mlc1sa. This IQ motif, and also the fourth, interacts with the budding yeast myosin essential light chain Mlc1p. The first and second IQ motifs interact with S100B in the presence of calcium ions. This clearly establishes that S100B can interact with its targets through IQ motifs in addition to interacting via previously reported sequences. These results are discussed in terms of the function of IQGAP1 and IQ motif recognition.

A highly sensitive method for quantification of myosin light chain phosphorylation by capillary isoelectric focusing with laser-induced fluorescence detection.

Activation of myosin II by phosphorylation of the 20 kDa regulatory light chains (LC20) has been implicated in numerous contractile and motile events, e.g., smooth muscle contraction, cytokinesis, and cell migration. The ability to analyze LC20 phosphorylation in minute samples is critical to determine the importance of LC20 phosphorylation in diverse physiological processes. We have developed a method for the separation and quantification of unphosphorylated, monophosphorylated, and diphosphorylated LC20 with a detection limit of 1 pg (50 amol). LC20 is initially isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted to a polyvinlyidene difluoride (PVDF) membrane. The region of the membrane containing the LC20 band (identified by electrophoresis of purified LC20 in a neighboring lane) is cut out and fluorescently labeled with Alexa Fluor 488 C5 maleimide. The labeled LC20 is eluted from the membrane with detergent and subjected to capillary isoelectric focusing (CIEF) to separate unphosphorylated, mono-, and diphosphorylated LC20, which are detected and quantified by laser-induced fluorescence (LIF). A linear relationship between log(peak area) and log(LC20 amount) is observed over the range of 50 amol-150 fmol. Quantification of LC20 phosphorylation by CIEF with LIF detection was compared with three commonly used methods with much lower levels of sensitivity: urea/glycerol-PAGE with Western blotting, phosphorylation by [gamma-32P]ATP with Cerenkov counting, and phosphorylation by [gamma-32P]ATP followed by SDS-PAGE, autoradiography, and scanning densitometry. All four methods gave very similar quantitative results, the major difference being that the new method exhibits 3000-fold enhanced sensitivity. This method is therefore applicable to quantitative analysis of phosphorylation of minute quantities of LC20.

Crystallization, X-ray characterization and selenomethionine phasing of Mlc1p bound to IQ motifs from myosin V.

Mlc1p is a calmodulin-like protein from the budding yeast Saccharomyces cerevisiae, where it has been identified as a subunit of a class V myosin, Myo2p, and a binding partner of an IQGAP-like protein, Iqg1p. Through its interactions with these two proteins, Mlc1p plays a role in polarized growth and cytokinesis. Mlc1p has been crystallized in complexes with four different IQ target motifs from the neck region of Myo2p: IQ2, IQ3, IQ4 and IQ2-IQ3 (referred to as IQ2,3). Electron-density maps for two of the complexes (Mlc1p-IQ4 and Mlc1p-IQ2,3) were obtained from multiple anomalous dispersion (MAD) experiments based on selenomethionine derivatives. The other two structures (Mlc1p-IQ2 and Mlc1p-IQ3) were determined by molecular replacement using the partially refined structure of Mlc1p-IQ2,3 as a search model.

The protein phosphatase inhibitor cantharidin alters vascular endothelial cell permeability.

In this study, we characterized the effects of the protein phosphatases type 1 (PP 1) and type 2A (PP 2A) inhibitor cantharidin in endothelial cells. We identified catalytic subunits of PP 1alpha, PP 2Aalpha, and PP 2Abeta immunologically in bovine aortic endothelial cells. Moreover, we detected mRNAs coding for catalytic subunits of PP 1alpha, PP 1beta, and PP 2Aalpha by hybridization with specific DNA probes in total RNA from these cells. Okadaic acid and cantharidin inhibited the activities of catalytic subunits of PP 1 (okadaic acid, 0.01-1 microM; cantharidin, 1-100 microM) and PP 2A (okadaic acid, 0.1 nM to 1 microM; cantharidin, 0.1-100 microM) separated by column chromatography in a concentration-dependent manner. Moreover, cantharidin (1 microM to 1 mM) increased the phosphorylation state of endothelial proteins including the regulatory light chains of myosin without affecting cytosolic calcium concentrations. Cantharidin (5-100 microM) increased the permeability of cultured endothelial cells in a time- and concentration-dependent manner. We suggest that inhibition of PP 1 and PP 2A activities by cantharidin increases endothelial permeability by enhancing the phosphorylation state of endothelial regulatory proteins. Thus, cantharidin might be a useful tool to study the function of protein phosphatases in endothelial barrier function.

MLCK-A, an unconventional myosin light chain kinase from Dictyostelium, is activated by a cGMP-dependent pathway.

Dictyostelium myosin II is activated by phosphorylation of its regulatory light chain by myosin light chain kinase A (MLCK-A), an unconventional MLCK that is not regulated by Ca2+/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with Con A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A's activation mechanism. MLCK-A does not alter its cellular location upon treatment of cells with Con A, nor does it localize to the myosin-rich caps that form after treatment. However, MLCK-A activity rapidly increases 2- to 13-fold when Dictyostelium cells are exposed to Con A. This activation can occur in the absence of MLCK-A autophosphorylation. cGMP is a promising candidate for an intracellular messenger mediating Con A-triggered MLCK-A activation, as addition of cGMP to fresh Dictyostelium lysates increases MLCK-A activity 3- to 12-fold. The specific activity of MLCK-A in cGMP-treated lysates is 210-fold higher than that of recombinant MLCK-A, which is fully autophosphorylated, but lacks threonine-166 phosphorylation. Purified MLCK-A is not directly activated by cGMP, indicating that additional cellular factors, perhaps a kinase that phosphorylates threonine-166, are involved.

Characterization of pI(Cln) binding proteins: identification of p17 and assessment of the role of acidic domains in mediating protein-protein interactions.

pICln is a ubiquitous and abundant 27 kDa soluble protein that is localized primarily to the cytoplasm. The protein has been proposed to be a swelling-activated anion channel or a channel regulator. Recent studies, however, have cast significant doubt on these hypotheses, and the function of pI(Cln) therefore remains unknown. To further characterize the physiological role of pI(Cln), we have begun to identify the proteins that bind to it and the amino acid domains that mediate pICln protein-protein interactions. Using affinity assays and immunoprecipitation we have identified three proteins in C6 glioma cells with molecular masses of 17 kDa, 29 kDa and 72 kDa that bind selectively to pI(Cln). Microsequencing revealed that p17 is the non-muscle isoform of the alkali myosin light chain. pI(Cln) contains three acidic amino acid domains termed AD1, AD2 and AD3. Mutation of AD1 and/or AD2 had no effect on p17, p29 and p72 binding. However, binding of p72 was lost when four acidic amino acid residues were mutated in AD3, which is located at the carboxy terminus. A truncation peptide containing the last 29 amino acids of pI(Cln) was able to bind p72 normally. These results indicate that the carboxy terminus is necessary for p72-pI(Cln) interaction. Based on these and other findings, we propose that pI(Cln) is a protein responsible for regulating the structure and function of the cytoskeleton, and/or a protein involved in mediating interactions between components of intracellular signal transduction pathways.

Subunit interactions within an expressed regulatory domain of chicken skeletal myosin. Location of the NH2 terminus of the regulatory light chain by fluorescence resonance energy transfer.

The regulatory domain (RD), or neck region of the myosin head, consists of two classes of light chains that stabilize an alpha-helical segment of the heavy chain. RD from chicken skeletal muscle myosin was prepared in Escherichia coli by coexpression of a 9-kDa heavy chain fragment with the essential light chain. Recombinant regulatory light chain (RLC), wild type or mutant, was added separately to reconstitute the complex. The affinity of RD for divalent cations was determined by measuring the change in fluorescence of a pair of heavy chain tryptophans upon addition of calcium or magnesium. The complex bound divalent cations with high affinity, similar to the association constants determined for native myosin. The intrinsic fluorescence of the tryptophans could be used as a donor to measure the fluorescence resonance energy transfer distance to a single labeled cysteine engineered at position 2 on RLC. Dansylated Cys2 could also serve as a donor by preparing RLC with a second cysteine at position 79 which was labeled with an acceptor probe. These fluorescence resonance energy transfer distances (24-30 A), together with a previous measurement between Cys2 and Cys155 (Wolff-Long, V. L., Tao, T., and Lowey, S. (1995) J. Biol. Chem. 270, 31111-31118) suggest a location for the NH2 terminus of RLC that appears to preclude a direct interaction between the phosphorylatable serine and specific residues in the COOH-terminal domain.

Essential light chain exchange in smooth muscle myosin.

The exchange of 17-kDa essential light chain (LC17) in smooth muscle myosin was carried out by incubating myosin with a 10-fold molar excess of exogenously added LC17 over the corresponding endogenous light chain in the presence of trifluoperazine and 4.5 m ammonium chloride. Porcine aorta myosin contains two kinds of LC17 isoform, LC17nm and LC17gi, while chicken gizzard myosin contains only one kind of LC17 isoform. As LC17gi can be separated from LC17nm and gizzard LC17 by urea-gel electrophoresis, LC17nm in aorta myosin and LC17 in gizzard myosin were exchanged with LC17gi and LC17gi in aorta myosin was exchanged with LC17nm, and the degree of exchange was estimated by urea-gel electrophoresis. Under the optimal conditions (6 and 10 degrees C for aorta and gizzard myosin, respectively), nearly 90% of exchange, which is close to the theoretical value, was achieved for the former combinations, and a slightly lower exchange was obtained for the latter. The LC17-exchanged myosins contained stoichiometric amounts of the heavy and light chains and retained the original nature in the phosphorylation-dependent actin-activated ATPase activity, 6S-10S conformational transition, and filament assembly.

Chemo-mechanical energy transduction in relation to myosin isoform composition in skeletal muscle fibres of the rat.

1. ATP consumption and force development were determined in single skinned muscle fibres of the rat at 12 degrees C. Myofibrillar ATPase consumption was measured photometrically from NADH oxidation which was coupled to ATP hydrolysis. Myosin heavy chain (MHC) and light chain (MLC) isoforms were identified by gel electrophoresis. 2. Slow fibres (n = 14) containing MHCI and fast fibres (n = 18) containing MHCIIB were compared. Maximum shortening velocity was 1.02 +/- 0.63 and 3.05 +/- 0.23 lengths s-1, maximum power was 1.47 +/- 0.22 and 9.59 +/- 0.84 W l-1, and isometric ATPase activity was 0.034 +/- 0.003 and 0.25 +/- 0.01 mM s-1 in slow and in fast fibres, respectively. 3. In fast as well as in slow fibres ATP consumption during shortening increased above isometric ATP consumption. The increase was much greater in fast fibres than in slow fibres, but became similar when expressed relative to the isometric ATPase rate. 4. Efficiency was calculated from mechanical power and free energy change associated with ATP hydrolysis. Maximum efficiency was larger in slow than in fast fibres (0.38 +/- 0.04 versus 0.28 +/- 0.03) and was reached at a lower shortening velocity. 5. Within the group of fast fibres efficiency was lower in fibres which contained more MLC3f. We conclude that both MHC and essential MLC isoforms contribute to determine efficiency of chemo-mechanical transduction.

Length-dependent potentiation and myosin light chain phosphorylation in rat gastrocnemius muscle.

Changes in muscle length affect the degree of staircase potentiation in skeletal muscle, but the mechanism by which this occurs is unknown. In this study, we tested the hypothesis that length-dependent change in staircase is modulated by phosphorylation of the myosin regulatory light chains (RLC), since this is believed to be the main mechanism of potentiation. In situ isometric contractile responses of rat gastrocnemius muscle during 10 s of repetitive stimulation at 10 Hz were analyzed at optimal length (Lo), Lo - 10%, and Lo + 10%. The degree of enhancement of developed tension during 10 s of repetitive stimulation was observed to be length dependent, with increases of 118.5 +/- 7.8, 63.1 +/- 3.9, and 45.6 +/- 4.1% (means +/- SE) at Lo - 10%, Lo, and Lo + 10%, respectively. Staircase was accompanied by increases in the average rate of force development of 105.6 +/- 7.7, 55.6 +/- 4.1, and 37.2 +/- 4.4% for Lo - 10%, Lo, and Lo + 10%, respectively. RLC phosphorylation after 10 s of 10-Hz stimulation was higher than under resting conditions but not different among Lo - 10% (40 +/- 3.5%), Lo (35 +/- 3.5%), and Lo + 10% (41 +/- 3.5%). This study shows that there is a length dependence of staircase potentiation in mammalian skeletal muscle that may not be directly modulated by RLC phosphorylation. Interaction of RLC phosphorylation with length-dependent changes in Ca2+ release and intermyofilament spacing may explain these observations.

Inhibition of myogenesis in mouse C2 cells by double-stranded phosphorothioate oligodeoxynucleotides containing mef-1 sequence.

Phosphorothioate oligonucleotides containing the muscle creatinine kinase enhancer sequence (mef-1) and a mutant of the enhancer sequence (mmef-1) were tested for their ability to block muscle differentiation in mouse C2 cells in culture. Maximum inhibition of fusion of myoblasts was observed at 10 microM concentration of mef-1 oligomer. No appreciable inhibition of fusion with the mmef-1 oligomer at the same concentration was observed. Synthesis of myogenin, muscle creatinine kinase, and myosin heavy chain polypeptides were reduced in mef-1 oligomer-treated cells. In contrast, no significant reduction in the synthesis of these polypeptides in mmef-1-treated cells was detected. The overall protein synthesis was not affected. These results suggest that muscle differentiation may be disrupted by competition of the oligomer with the endogenous promoter for specific transcription factor(s).

Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers.

To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers.

The regulatory light chains of myosin modulate cross-bridge cycling in skeletal muscle.

We investigated the kinetics of Ca2+ activation of skeletal muscle contraction elicited by the photolysis of caged Ca2+. Previously we showed that partial extraction of the 18-kDa regulatory light chains (RLCs) of myosin decreased the rate of force development and was subsequently increased by approximately 20% following reconstitution with RLCs (Potter, J. D., Zhao, J. and Pan, B. S. (1992) FASEB J. 6, A1240). We extend here the RLC-extraction study to the complete removal of the RLCs. The complete removal of RLCs was achieved by a combination of 5,5'-dithiobis-(2-nitrobenzoic acid) and EDTA treatment followed by reduction of oxidized sulfydryl groups by dithiothreitol. Under these conditions the complete extraction of RLCs was accompanied by the extraction of endogenous troponin C, resulting in the loss of isometric tension. Steady state force was restored to 65-75% following troponin C reconstitution and increased to 75-85% as a result of RLC reincorporation into the fibers. The rates of force transients generated by UV-flash photolysis of 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N' -tetrakis[(oxycarbonyl)methyl]-1,2-ethanediamine) or nitrophenyl-EGTA, photoliberating bound Ca2+, decreased 2-fold after RLC extraction and troponin C reconstitution and then increased to the values of intact fibers after RLC reconstitution. These results support our earlier findings that the regulatory light chains of myosin play an important role in the kinetics of cross-bridge cycling.