Effect on catalysis by replacement of catalytic residue from hen egg white lysozyme to Venerupis philippinarum lysozyme.
ABSTRACT: Asn46Asp/Asp52Ser or Asn46Glu/Asp52Ser hen egg white lysozyme (HEL) mutant was designed by introducing the substituted catalytic residue Asp46 or Glu46, respectively, based on Venerupis philippinarum (Vp) lysozyme structure as a representative of invertebrate-type (i-type) lyzozyme. These mutations restored the bell-shaped pH-dependency of the enzyme activity from the sigmoidal pH-dependency observed for the Asp52Ser mutant. Furthermore both lysozyme mutants possessed retaining mechanisms like Vp lysozyme and HEL. The Asn46Glu/Asp52Ser mutant, which has a shorter distance between two catalytic residues, formed a glycosyl adduct in the reaction with the N-acetylglucosamine oligomer. Furthermore, we found the accelerated turnover through its glycosyl adduct formation and decomposition. The turnover rate estimated from the glycosyl formation and decomposition rates was only 20% of the observed hydrolysis rate of the substrate. Based on these results, we discussed the catalytic mechanism of lysozymes.
Project description:Association rate constants for antigen/antibody associations have been computed by Brownian Dynamics simulations of D. L. Ermak and J. A. McCammon, J. Chem. Phys. 69:1352-1360, 1978. The model of monoclonal antibody (mAb) D44.1 is based on crystallographic data (B. C. Braden et al., J. Mol. Biol. 243:767-781, 1994). Electrostatic forces that steer the antigen to the antibody-combining site are computed by solving the linearized Poisson-Boltzmann equation. D44. 1-HEL complex displays very similar association motifs to a related anti-lysozyme antibody, HyHEL-5-HEL system. The computed association rate constants are comparable in the two systems, although the experimental affinity constants differ by three orders of magnitude (D. Tello et al., Biochem. Soc. Trans. 21:943-946, 1993; K. A. Hibbits et al., Biochemistry. 33:3584-3590, 1994). Simulations suggest that the origin of the differences in the affinity come from dissociation rate constants. We have also carried out simulation experiments on a number of mutant antibody fragment-HEL associations to address the role of electrostatics and, to a limited extent, the orientational aspects of association.
Project description:The LytC lysozyme of Streptococcus pneumoniae forms part of the autolytic system of this important pathogen. This enzyme is composed of a C-terminal CM (catalytic module), belonging to the GH25 family of glycosyl hydrolases, and an N-terminal CBM (choline-binding module), made of eleven homologous repeats, that specifically recognizes the choline residues that are present in pneumococcal teichoic and lipoteichoic acids. This arrangement inverts the general assembly pattern of the major pneumococcal autolysin, LytA, and the lytic enzymes encoded by pneumococcal bacteriophages that place the CBM (made of six repeats) at the C-terminus. In the present paper, a three-dimensional model of LytC built by homology modelling of each module and consistent with spectroscopic and hydrodynamic studies is shown. In addition, the putative catalytic-pair residues are identified. Despite the inversion in the modular arrangement, LytC and the bacteriophage-encoded Cpl-1 lysozyme most probably adopt a similar global fold. However, the distinct choline-binding ability and their substrate-binding surfaces may reflect a divergent evolution directed by the different roles played by them in the host (LytC) or in the bacteriophage (Cpl-1). The tight binding of LytC to the pneumococcal envelope, mediated by the acquisition of additional choline-binding repeats, could facilitate the regulation of the potentially suicidal activity of this autolysin. In contrast, a looser attachment of Cpl-1 to the cell wall and the establishment of more favourable interactions between its highly negatively charged catalytic surface and the positively charged chains of pneumococcal murein could enhance the lytic activity of the parasite-encoded enzyme and therefore liberation of the phage progeny.
Project description:During treatment with protein therapeutics, such as monoclonal antibodies, the development of anti-drug antibodies is a serious side-effect of modern pharmacology. Anti-drug antibodies are produced as the number and exposure to therapeutic proteins increase. In this context, less immunogenic responses could diminish these noxious effects. Biophysical characterization of antigens, that is size, chemical composition, physical form, and degrability, are known to influence the outcome of immune responses. Here, using chemical modification, we have prepared oligomers of hen egg lysozyme (HEL), 3- to 5-mer, as a typical antigen in immunology and evaluated the efficacy as a tolerogen in HEL-specific antibody responses. Our results clearly demonstrated that pre-exposed the HEL-oligomers into mice effectively suppressed HEL-specific IgG responses regardless of the cross-linking mode. Therefore, the oligomerization is a method to induce tolerogenicity of proteins and may emerge as a promising strategy to control the production of undesirable anti-protein drug antibodies.
Project description:In contrast to hen egg-white lysozyme, which retains the beta-configuration of the substrate in the product, T4 lysozyme (T4L) is an inverting glycosidase. The substitution Thr-26 --> His, however, converts T4L from an inverting to a retaining enzyme. It is shown here that the Thr-26 --> His mutant is also a transglycosidase. Indeed, the transglycosylation reaction can be more effective than hydrolysis. In contrast, wild-type T4L has no detectable transglycosidase activity. The results support the prior hypothesis that catalysis by the Thr-26 --> His mutant proceeds via a covalent intermediate. Further mutations (Glu-11 --> His, Asp-20 --> Cys) of the T26H mutant lysozyme indicate that the catalytic mechanism of this mutant requires Glu-11 as a general acid but Asp-20 is not essential. The results help provide an overall rationalization for the activity of glycosidases, in which a highly conserved acid group (Glu-11 in T4L, Glu-35 in hen egg-white lysozyme) on the beta-side of the substrate acts as a proton donor, whereas alterations in the placement and chemical identity of residues on the alpha-side of the substrate can lead to catalysis with or without retention of the configuration, to transglycosidase activity, or to the formation of a stable enzyme-substrate adduct.
Project description:Single molecule bioelectronic circuits provide an opportunity to study chemical kinetics and kinetic variability with bond-by-bond resolution. To demonstrate this approach, we examined the catalytic activity of T4 lysozyme processing peptidoglycan substrates. Monitoring a single lysozyme molecule through changes in a circuit's conductance helped elucidate unexplored and previously invisible aspects of lysozyme's catalytic mechanism and demonstrated lysozyme to be a processive enzyme governed by 9 independent time constants. The variation of each time constant with pH or substrate crosslinking provided different insights into catalytic activity and dynamic disorder. Overall, ten lysozyme variants were synthesized and tested in single molecule circuits to dissect the transduction of chemical activity into electronic signals. Measurements show that a single amino acid with the appropriate properties is sufficient for good signal generation, proving that the single molecule circuit technique can be easily extended to other proteins.
Project description:We have previously produced a transgenic mouse line for hen egg lysozyme (HEL), an experimental model for analyzing tolerance to self-antigens at the peptide level. We have now characterized transgenic mice with HEL blood levels below 2 ng/ml, where significant T cell proliferative responses to HEL and its immunodominant peptide were observed. This HEL-low transgenic model was chosen because it mimics physiological conditions in which autoreactive T lymphocytes, recognizing self-components expressed at very low levels, persist without inducing a break in tolerance. Furthermore, in H-2d mice, HEL-specific T lymphocytes are triggered by a single immunodominant region, allowing us to compare the HEL-specific T cell V beta repertoires of transgenic and nontransgenic animals against a single peptide presented as self or foreign, respectively. We found that a V beta 8.2-D beta 1-J beta 1.5 rearrangement is found in response to HEL in all nontransgenic mice, whereas this V beta-restricted response is absent in HEL-low transgenic animals. At the nucleotide level, this rearrangement results from the trimming of the genomic segments during VDJ or DJ joining, without N additions, suggesting that the dominant rearrangement is selected early during fetal or neonatal life, before the expression of terminal deoxynucleotidyl transferase. In HEL-low transgenic mice, no dominant rearrangements are found as alternatives to the one observed in normal mice. Instead, each transgenic animal uses a different set of V beta-J beta combinations in its response to the immunodominant HEL peptide. In nontransgenic mice, besides the dominant V beta 8.2-D beta 1-J beta 1.5 combination, minor V beta repertoires were found which differed in each animal and were distinct from the rearrangements used by individual transgenic mice. These findings suggest that the T cell response to an immunodominant peptide involves a "public" V beta repertoire found in all animals and a "private" one which is specific to each individual.
Project description:BACKGROUND:Lysozyme purified from duck eggs (DEL) has long been used as a model antigen as a counterpoint to the enzyme purified from hen eggs (HEL). However, unlike the single C-type variant found in hen eggs, duck eggs contain multiple isoforms: I, II and III. We recently reported the structures of isoforms I and III from Pekin duck (Anas platyrhynchos) and unequivocally determined the sequences of all three isoforms by mass spectrometry. Here we present the crystal structure of isoform II (DEL-II). RESULTS:Lysozyme isoform II was purified from isoforms I and III using ion-exchange and gel-filtration chromatography, then crystallized. X-ray diffraction data were collected to 1.15 Å resolution and the structure of DEL-II was solved by molecular replacement using the structure of DEL-I as the search model. It contains two molecules in the crystallographic asymmetric unit: both molecules display a canonical C-type lysozyme fold and electron density consistent with the expected sequence. The most significant difference between the two molecules concerns different conformations of a surface loop containing one of the expected amino acid differences between the isoforms. CONCLUSIONS:The structure of DEL-II supports the primary sequence as elucidated by a combination of amino acid sequencing, DNA sequencing and mass spectrometry, with strong electron density confirming it to be an S37G G71R variant of DEL I, and differing from hen egg lysozyme at a total of 21 amino acid positions.
Project description:In APCs, presentation by MHC II molecules of the chemically dominant peptide from the protein hen egg white lysozyme (HEL) generates different conformational isomers of the peptide-MHC II complexes (pMHC). Type B pMHCs are formed in early endosomes from exogenous peptides in the absence of H2-DM, whereas in contrast, type A pMHC complexes are formed from HEL protein in late vesicles after editing by H2-DM. Thus, H2-DM edits off the more unstable pMHC complexes, which are not presented from HEL. In this study, we show that type B pMHC complexes were presented from HEL protein only after stimulation of dendritic cells (DC) with TLR ligands or type I IFN. Type I IFN contributed to most TLR ligand-induced type B pMHC generation, as presentation decreased in DC lacking the receptor for type I IFNs (IFNAR1(-/-)). In contrast, presentation of type A pMHC from HEL and from peptide was minimally affected by TLR ligands. The relative effectiveness of CD8?(+) DC or CD8?(-) DC in presenting type B pMHC complexes varied depending on the TLR ligand used. The mechanisms of generation of type B pMHC from HEL protein with TLR stimulation did not involve H2-DM or release of peptides. DC from H2-DM-deficient mice in the presence of TLR ligands presented type B pMHC. Such DC showed a slight enhancement of HEL catabolism, but peptide release was not evident. Thus, TLR ligands and type I IFN alter the pathways of presentation by MHC II molecules of DC such that type B pMHCs are generated from protein Ag.
Project description:It has been well established that in the oxidative folding of hen egg white lysozyme (HEL), which has four SS linkages in the native state (N), three des intermediates, i.e., des[76-94], des[64-80], and des [6-127], are populated at 20 °C and N is dominantly formed by the oxidation of des[64-80] and des[6-127]. To elucidate the temperature effects, the oxidative folding pathways of HEL were reinvestigated at 5-45 °C in the presence of 2 M urea at pH 8.0 by using a selenoxide reagent, DHSox. When reduced HEL was reacted with 1-4 equivalents of DHSox, 1S, 2S, 3S, and 4S intermediate ensembles with 1-4 SS linkages, respectively, were produced within 1 min. After the oxidation, 3S was slowly converted to the des intermediates with formation of the native structures through SS rearrangement. At 5 °C, des[76-94] was populated in the largest amount, but the oxidation to N was slower than that of des[64-80] and des[6-127]. At 35 °C, on the other hand, des[64-80] and des[6-127] were no longer stable, and only des[76-94] was populated. The results suggested that the major folding pathways of HEL can be switched from one to the other by temperature control.
Project description:4-O-?-Di-N-acetylchitobiosyl moranoline (2) and 4-O-?-tri-N-acetylchitotriosyl moranoline (3) were produced by lysozyme-mediated transglycosylation from the substrates tetra-N-acetylchitotetraose, (GlcNAc)4, and moranoline, and the binding modes of 2 and 3 to hen egg white lysozyme (HEWL) was examined by inhibition kinetics, isothermal titration calorimetry (ITC), and x-ray crystallography. Compounds 2 and 3 specifically bound to HEWL, acting as competitive inhibitors with Ki values of 2.01 × 10(-5) and 1.84 × 10(-6) m, respectively. From ITC analysis, the binding of 3 was found to be driven by favorable enthalpy change (?Hr°), which is similar to those obtained for 2 and (GlcNAc)4. However, the entropy loss (-T?Sr°) for the binding of 3 was smaller than those of 2 and (GlcNAc)4. Thus the binding of 3 was found to be more favorable than those of the others. Judging from the Kd value of 3 (760 nm), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. X-ray crystal structure of HEWL in a complex with 3 showed that compound 3 binds to subsites -4 to -1 and the moranoline moiety adopts an undistorted (4)C1 chair conformation almost overlapping with the -1 sugar covalently bound to Asp-52 of HEWL (Vocadlo, Davies, G. J., Laine, R., and Withers, S. G. (2001) Nature 412, 835-838). From these results, we concluded that compound 3 serves as a transition-state analogue for lysozyme providing additional evidence supporting the covalent glycosyl-enzyme intermediate in the catalytic reaction.