Life-Long Wheel Running Attenuates Age-Related Fiber Loss in the Plantaris Muscle of Mice: a Pilot Study.

The purpose of this study was to investigate whether long-term wheel running would attenuate age-related loss of muscle fiber. Male ICR mice were divided into young (Y, n=12, aged 3 months), old-sedentary (OS, n=5, aged 24 months), and old-exercise (OE, n=6, aged 24 months) groups. The OE group started spontaneous wheel running at 3 months and continued until 24 months of age. Soleus and plantaris muscles were fixed in 4% paraformaldehyde buffer. The fixed muscle was digested in a 50% NaOH solution to isolate single fiber and then fiber number was quantified. The masses of the soleus and plantaris muscles were significantly lower at 24 months than at 3 months of age, and this age-related difference was attenuated by wheel running (P<0.05). Soleus muscle fiber number did not differ among the groups. In the plantaris muscle, the fiber number in the OS group (1 288±92 fibers) was significantly lower than in the Y group (1 874±93 fibers), and this decrease was attenuated in the OE group (1 591±80 fibers) (P<0.05). These results suggest that age-related fiber loss occurs only in the fast-twitch fiber-rich muscle of mice, and that life-long wheel running exercise can prevent this fiber loss.

Social Detachment Influenced Muscle Mass and Strength during the COVID-19 Pandemic in Japanese Community-Dwelling Older Women.

Social detachment due to coronavirus disease (COVID-19) has caused a decline in physical activity, leading to sarcopenia and frailty in older adults. This study aimed to compare muscle mass, strength, and function values in older women before and after the first wave of the COVID-19 pandemic (April-May 2020). Furthermore, changes in muscle measures across women who experienced different levels of impact on their social participation due to the COVID-19 pandemic were examined. Muscle mass (total, trunk, and appendicular muscle), grip strength, oral motor skills, social interactions (social network and participation), and social support were assessed in 46 Japanese community-dwelling older women (mean, 77.5 y; range 66-93 y) before and after the first wave of the COVID-19 pandemic. Trunk muscle mass significantly decreased after the first wave of the pandemic. When comparing changed values between the enhanced/maintained and reduced group during the pandemic, significant group difference was observed in trunk muscular mass, grip strength, and oral motor skills. Intriguingly, those who enhanced social participation had a positive change of grip strength values, showing that social participation might influence muscle function during the COVID-19 pandemic.

Molecular orientation in the stacked dimer form of 5"AMP in aqueous solution. A study by the NMR-desert method.

Molecular orientation of 5"-AMP in its stacked dimeric form in neutral aqueous solutions has been investigated at room temperature through the NMR-DESERT method proposed earlier by Akasaka et al. (J. Magn. Resonance, (1975) 18, 328-343). The effect of deuterium substitution of H8 of the adenine ring on the relaxation rate of H2 has become appreciable with increasing concentration of 5"-AMP, which should be attributed to the intermolecular H2-H8 interaction between adjacent adenine rings in the stacked dimer of 5"-AMP. The reciprocal sixth-power-averaged distance between H8 and H2 of the adjacent adenine rings in the stacked dimeric form obtained from the differential relaxation rate for H2 has been found to be almost constant (3.6 +/- 0.2 A S.D.) in the whole concentration range studied (0.1--1.0 M). The result has presented a direct proof of the existence of trans-stacking with a relatively large proportion (more than 60%) in the stacked dimeric form of 5"-AMP.

A new electrophoretic variant of hemoglobin (Ogi) in which a leucine residue is replaced by an arginine residue at position 34 of the alpha-chain.

Hemoglobin Ogi, in which an arginine is substituted for a leucine residue at position 34 of the alpha-chain, was detected in a Japanese family. Although slightly increased oxygen affinity is associated with this amino acid substitution in the alpha 1 beta 1 contact, it is without obvious deleterious effect on the hematological parameters of the individuals heterozygous for this variant.

Unfolding rates of globular proteins determined by kinetics of proteolysis.

A convenient method for the determination of unfolding rates of small globular proteins under physiological conditions was developed using digestion with proteases. The apparent first-order rate constants for digestion of lysozyme with thermolysin and with Pronase at pH 8 and 50 degrees C were shown to be saturated with increases of concentrations of these proteases. The maximum rate constants extrapolated were identical in digestions with two different proteases, and were found to be equal to the unfolding rate constant of lysozyme. Similarly, the unfolding rate constant of RNase A at pH 8 and 50 degrees C, and those of lysozyme, RNase A and beta-lactoglobulin at pH 8 and 40 degrees C, were determined by the digestion method. Thus, it was shown that digestion by proteases proceeds mainly via the unfolded state of proteins.

Analysis of the relationship between enzyme activity and its internal motion using nuclear magnetic resonance: 15N relaxation studies of wild-type and mutant lysozyme.

A mutant lysozyme where R14 and H15 are deleted together has higher activity and a similar binding ability to an inhibitor, trimer of N-acetylglucosamine ((NAG)3), compared with wild-type lysozyme. Since this has been attributed to intrinsic protein dynamic properties, we investigated the relationship between the activity and the internal motions of proteins. Backbone dynamics of the free and the complex forms with the (NAG)3 have been studied by measurement of the 15N T1 and T2 relaxation rates and NOE determinations at 600 MHz. Analysis of the data using the model-free formalism showed that the generalized order parameters (S2) were almost the same in wild-type and mutant lysozyme in unbound state, indicating that the mutation had little effect on the global internal motions. On the other hand, in the presence of (NAG)3, although some signals located around the active site were broadened or decreased in intensity because of strong perturbation by (NAG)3, there were several residues that showed increased or decreased backbone S2 in the complexed lysozymes. A comparison of the internal motions of the wild-type and mutant complexes showed a number of distinct dynamic differences between them. In particular, many residues located at or near active-site regions (turn 1, strand 2, turn 2 and long loop), displayed greater backbone dynamics reflecting the order parameter in mutant complex relative to mutant free. Furthermore, the Rex values at the loop C-D region, which was considered to be important for enzymatic activity, significantly increased. From these results, it was suggested that variations in the dynamics of these regions may play an important role in the enzyme activity.

Formation of alpha-helix 88-98 is essential in the establishment of higher-order structure from reduced lysozyme.

Lys96 and Lys97 in lysozyme are located at the C terminus of alpha-helix 88-98. The positive charges of these residues are supposed to stabilize the helical structure, and these residues are conserved as the basic amino acids among c-type lysozymes. The renaturation rate of reduced mutant lysozyme, where both Lys96 and Lys97 were mutated together to Ala, was slower than that of native lysozyme at pH 8.0 and 37 degrees C by SH-SS interchange reactions. In order to investigate the reason, the peptide fragment 36-105 (where we can obtain information of the interaction between helix 88-98 and Trp62 and Trp63 residues) was prepared. CD spectra were compared between peptide fragment 36-105 and its acetylated form, where the positive charges of Lys96 and Lys97 were eliminated, and it was elucidated that the displacement of positive charges at the C terminus of the helix caused the shift of the advantageous structure of the fragment from alpha-helix to coil. Moreover, we obtained evidence that there was interaction of the helix with Trp62 and/or Trp63, which maintained a thermodynamically stable higher-order structure. Therefore, these results suggest that the formation of alpha-helical structure in 88-98 is a significant factor in the establishment of native structure from reduced lysozyme.

A single amino acid substitution in a self protein is sufficient to trigger autoantibody response.

We determined if a single amino acid substitution in a self protein causes autoantibody responses. Mouse lysozyme (ML) was used as a model self protein, and a mutant ML (F57L ML) was prepared by replacing 57Phe of ML to Leu, an approach which resulted in introducing into ML the immunogenic sequence of peptide 50-61 of hen egg lysozyme (HEL) restricted to I-A(k) MHC class II molecule. We found that F57L ML but not native ML primed HEL specific T cells and triggered ML specific autoantibody responses in B10.A and C3H mice (I-A(k), I-E(k)). Peptide regions, ML 14-69 and ML 98-130, were major epitopes of autoantibodies in both strains of mice. These findings indicate that a single amino acid substitution in self proteins can cause an autoantibody response when the mutated region is presented by MHC class II molecules and recognized by T cells.

Analysis of the internal motion of free and ligand-bound human lysozyme by use of 15N NMR relaxation measurement: a comparison with those of hen lysozyme.

Human lysozyme has a structure similar to that of hen lysozyme and differs in amino acid sequence by 51 out of 129 residues with one insertion at the position between 47 and 48 in hen lysozyme. The backbone dynamics of free or (NAG)3-bound human lysozyme has been determined by measurements of 15N nuclear relaxation. The relaxation data were analyzed using the Lipari-Szabo formalism and were compared with those of hen lysozyme, which was already reported (Mine S et al.. 1999, J Mol Biol 286:1547-1565). In this paper, it was found that the backbone dynamics of free human and hen lysozymes showed very similar behavior except for some residues, indicating that the difference in amino acid sequence did not affect the behavior of entire backbone dynamics, but the folded pattern was the major determinant of the internal motion of lysozymes. On the other hand, it was also found that the number of residues in (NAG)3-bound human and hen lysozymes showed an increase or decrease in the order parameters at or near active sites on the binding of (NAG)3, indicating the increase in picosecond to nanosecond. These results suggested that the immobilization of residues upon binding (NAG)3 resulted in an entropy penalty and that this penalty was compensated by mobilizing other residues. However, compared with the internal motions between both ligand-bound human and hen lysozymes, differences in dynamic behavior between them were found at substrate binding sites, reflecting a subtle difference in the substrate-binding mode or efficiency of activity between them.

Stabilization of hen egg white lysozyme by a cavity-filling mutation.

Stabilization of a protein using cavity-filling strategy has hardly been successful because of unfavorable van der Waals contacts. We succeeded in stabilizing lysozymes by cavity-filling mutations. The mutations were checked by a simple energy minimization in advance. It was shown clearly that the sum of free energy change caused by the hydrophobicity and the cavity size was correlated very well with protein stability. We also considered the aromatic-aromatic interaction. It is reconfirmed that the cavity-filling mutation in a hydrophobic core is a very useful method to stabilize a protein when the mutation candidate is selected carefully.

Construction of a yeast expression system with positive selection for gene insertion in the absence of a specific phenotype.

A positive-selection system of cloned inserts in Escherichia coli has been devised using a streptomycin-resistant (Smr) gene and streptomycin-dependent (Smd) E. coli. A vector, pHA394, based on the yeast expression vector pAM82 has the Smr gene, which inactivates streptomycin (Sm) and invalidates the streptomycin dependence, resulting in a very low transformation efficiency. Replacement of the Smr gene by the recombinant vector allows high-frequency transformation. This system was applied to the lysozyme gene. After the yeast secretion signal was fused to the lysozyme gene using an intermediate vector, pHA474, the Smr gene of pHA394 was replaced by the fusion gene, followed by transformation of Smd E. coli. Analysis of the transformants showed that the plasmid gene contained 100% of the lysozyme gene.

High-level expression of uniformly 15N-labeled hen lysozyme in Pichia pastoris and identification of the site in hen lysozyme where phosphate ion binds using NMR measurements.

The non-enzymatic deamidation of Asn to Asp is known to occur in proteins and peptides and is accelerated by phosphate buffer [Tyler-Cross, R. and Schirch, V. (1991) J. Biol. Chem. 25, 22549-22556]. We attempted to identify the site in lysozyme where a phosphate ion binds by means of 1H-15N HSQC measurements of 15N-labeled lysozyme, which was successfully obtained using Pichia pastoris. As a result, we found that the phosphate ion was preferentially bound to Asn-103 in hen lysozyme. The method presented here may be useful for identifying the binding site of a protein with low molecular weight substances.

Reduced immunogenicity of monomethoxypolyethylene glycol-modified lysozyme for activation of T cells.

Chemical modification of proteins with monomethoxypolyethylene glycol (mPEG) will reduce the immunogenicity of proteins. In the present study, we evaluated the effect of mPEG modification on the capacity of hen egg-white lysozyme (HEL) to stimulate T cells. Lymph node cells (LNCs) from mice immunized with HEL or with mPEG-HEL conjugate were cultured with these antigens, then we measured the proliferation and IL-2 production. mPEG-modification lowered the T cell activating capacity of HEL, both in vitro and in vivo. Neither toxicity, nor antigen non-specific immunosuppressive capacity was observed with mPEG-HEL and unconjugated mPEG. Suppressor cells were unlikely to be generated in the mPEG-HEL-primed LNCs. We next examined the behavior of mPEG-HEL during antigen processing. The capacity of HEL and mPEG-HEL to be incorporated by live cells was much the same. However, the susceptibility to various proteases, including endosomal/lysosomal enzymes, was significantly decreased by mPEG modification. The increased resistance of mPEG-HEL to proteolytic degradation implied that the conjugate was poorly presented to T cells. This may be an important factor related to the low immunogenicity of mPEG modified proteins.

Engineering of lysozyme.

As the most extensively investigated model protein, the protein engineering of lysozyme is described. By utilizing modifications made possible by chemical or gene engineering methods, we can get a better understanding of protein behaviour and we can also improve their properties. The results of the protein engineering of lysozyme are described, which give some ideas for a better understanding of the physiological function of proteins, their stabilization, and how to engineer a novel protein.

Thermodynamic and kinetic stabilities of hen-egg lysozyme and its chemically modified derivatives: analysis of the transition state of the protein unfolding.

For the stabilization of a protein against irreversible denaturation caused by rapid reaction of the unfolded form of the protein (kinetic stabilization), the free energy change of activation for unfolding should be increased. First, we demonstrated that this strategy was effective to stabilize a protein against protease digestion. For kinetic stabilization, it is important to stabilize a protein at a site where the local structures are largely unfolded in the transition state for unfolding. We developed a method to find such sites by comparison of the thermodynamic stabilities and the unfolding rate constants between unmodified and modified proteins. Application of this method to analyze the transition state of hen-egg lysozyme using some chemically modified derivatives is also described. Moreover, it was confirmed that the protease digestion method is superior to the relaxation method for estimation of the unfolding rate constant. Namely, the protease digestion method may be useful in analyzing the transition state of protein unfolding.

Specific carbodiimide-binding mechanism for the selective modification of the aspartic acid-101 residue of lysozyme in the carbodiimide-amine reaction.

A mechanism for the selective modification of Asp-101 in hen egg-white lysozyme with an amine nucleophile catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was investigated using ethanolamine as a nucleophile at pH 5.0 and room temperature. In the presence of N-acetyl-D-glucosamine (NAG) and its oligomers [(NAG)n, n = 2 and 3] under the conditions with which about 90% of lysozyme was calculated to form complexes, the formation of Asp-101 modified lysozyme decreased markedly but to different degrees, that is (NAG)3 was the most and NAG the least effective. When the lysozyme derivative, in which Trp-62 in the active site cleft was oxidized to oxindolealanine (Ox-62 lysozyme), was used in place of native lysozyme, the formation of Asp-101 modified derivative decreased to about half, which was similar to the decrease in the presence of (NAG)2. In the presence of 0.5 M NaCl, on the other hand, the formation of Asp-101 modified lysozyme was considerably enhanced. From these observations, it is concluded that EDC binds to the active site cleft of lysozyme to specifically activate Asp-101. The affinity of EDC to the active site of lysozyme is partly due to the hydrophobic interaction of EDC with the Trp-62 residue at sub-site B of lysozyme. EDC is an activating reagent for carboxyl groups unlike most active site-directed reagents which produce final products directly. Therefore, the active site-directed nature of EDC was very useful because it made it possible to selectively introduce various amines as needed at a particular carboxyl group of lysozyme.

Analysis of the transition state in the unfolding of hen lysozyme by introduction of Gly-Pro and Pro-Gly sequences at the same site.

We developed a sensitive method for analyzing the conformation of the transition state in the unfolding of hen lysozyme. The activation free energy changes of mutant lysozymes with Gly-Pro and Pro-Gly sequences at the same sites (Gly47Pro47', Pro47Gly47', Gly101Pro102, Pro101Gly102, Gly117Pro118, Pro117Gly118, Gly121Pro122, and Pro121Gly122 lysozymes) were obtained for the unfolding in aqueous solution at pH 5.5 and 35 degrees C. Since we had shown that the difference of energies of the unfolded state in lysozymes having an introduced Gly-Pro or Pro-Gly sequence at the same site was much smaller than the difference of energies of the folded states [Motoshima, H., Ueda, T., Hashimoto, Y., Tsutsumi, M., and Imoto, T. (1995) J. Biochem. 118, 1138-1144], we could estimate the difference of energies of the folded and the transition states unequivocally. We defined the phi-value as the ratio of the difference in the free energy change in the transition state to that in the free energy change in the folded state between lysozymes with Gly-Pro and Pro-Gly sequences at the same site. The phi-values gave information on how much the mutated sites retained the folded structure in the transition state. These values were 0.45 around position 47, which is located in the beta-sheet structure, 0.12 at position 101-102, which is located in the loop at the upper part of the active site, 0.17 at position 117-118, which is located in the beta-turn and 0.64 at position 121-122, which is located in the 3(10)-helix. Therefore, in the transition state in the unfolding of lysozyme, it was found that the 3(10)-helical region had a similar structure to the intact region, while both the beta-turn and the loop at the upper part of the active site were considerably unfolded. The beta-sheet structure was also moderately disrupted in the transition state.

Binding of calcium to lysozyme and its derivatives.

Bindings of calcium to lysozyme and its derivatives were studied by UV difference spectroscopy at various pH's. The binding constant was ca. 40 m-1 at around neutral pH. The binding caused proton release from lysozyme and did not inhibit the binding of tri-N-acetylglucosamine to lysozyme. In the presence of 0.2 M Ca2+, lysozyme showed 26% of the activity of the free enzyme toward hexa-N-acetylglucosamine but the cleavage pattern was similar to that of the free enzyme. Thus, calcium was predicted to bind near the catalytic carboxyls to cause inhibition of lysozyme activity. It was found from the results of protease digestion that calcium binding shifted the native-denatured transition in lysozyme toward the native state.

An improved method for preparing lysozyme with chemically 13C-enriched methionine residues using 2-aminothiophenol as a reagent of thiolysis.

Jones et al. have reported that the epsilon-carbons of methionine residues in myoglobin can be enriched with stable isotope (13C) in two steps, i.e., methylation of methionine residues with 13CH3I in the protein and thiolysis using dithiothreitol [Jones, W.C., Rothgeb, T.M., and Gurd, F.R.N. (1976) J. Biol. Chem. 251,7452-7460]. Using their method, we failed to prepare active lysozyme in which the epsilon-carbons of methionine residues are enriched with 13C, because many side reactions took place under the thiolysis condition (pH 10.5, 37 degrees C). When we employed 2-aminothiophenol as a reagent for thiolysis, the reduction proceeded under a weakly acidic condition to afford fully active lysozyme, in which the epsilon-carbons of two methionine residues were enriched with 13C, in a 30% yield. Analysis of the 13C-edited NOESY spectra of 13C-enriched methionine lysozyme in the absence and presence of a substrate analogue indicated the occurrence of conformational change around Met 105 in lysozyme.

Construction of a screening system for selecting lysozyme mutants unable to form a stable structure from random mutants.

To collect folding information, we screened and analyzed the recombinant hen lysozyme mutants which were not secreted from yeast. As model mutants, Leu8Arg, Ala10Gly, and Met12Arg were prepared by site-directed mutagenesis and analyzed as to whether they were secreted from yeast or not. Consequently, Ala10Gly was found to be secreted from yeast, but Leu8Arg and Met12Arg were not. Next, these mutants were expressed in Escherichia coli and refolded in vitro. As a result, Ala10Gly folded as the wild-type did. Leu8Arg efficiently refolded in renaturation buffer containing glycerol. Met12Arg did not refold even in the presence of glycerol. These results show that the Ala10Gly mutation does not affect folding or stability, that Leu8Arg is too unstable to be secreted from yeast, and that Met12Arg may be very unstable or the mutation affects the folding pathway. We screened the mutants that were not secreted by yeast from a randomly mutated lysozyme library, and obtained Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln. These two mutants were expressed in E. coli and then refolded in the presence of urea or glycerol. These mutants were refolded only in the presence of glycerol. Each single mutant of Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln was independently prepared and folded in vitro. The results showed that Leu25Arg and Leu56Gln were the dominant mutations, respectively, which cause destabilization. These results show that the mutant lysozymes which were not secreted from yeast may be unstable or have a defect in the folding pathway. Thus, we established a screening system for selecting mutants which are unable to form a stable structure from random mutants.