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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.

A two-phase sequential dynamic change in the secondary structure of hen egg lysozyme (Lys) adsorbed on solid substrates was observed. The first phase involved fast conversion of α-helix to random/turns (within the first minute or at very low coverage or high substrate wettability) with no perceptible change in β-sheet content. The second phase (1–1200 min), however, involved a relatively slow conversion from α-helix to β-sheet without a noticeable change in random/turns. An important finding of this work is that the concentration of lysozyme in the adsorbed state has a substantial effect on the fractional content of secondary structures. Attenuated total reflection Fourier transform infrared (ATR/FTIR) spectroscopy, along with a newly-developed optimization algorithm for predicting the content of secondary structure motifs, was used to correlate the secondary structure and the amount of adsorbed lysozyme with the surface wettability of six different flat nanoporous substrates. Although three independent variables, surface wettability, solution concentration and time for adsorption, were used to follow the fractional structural changes of lysozyme, the results were all normalized onto a single plot with the amount adsorbed as the universal independent variable. Consequently, lateral interactions among proteins likely drive the transition process. Direct intermolecular force adhesion measurements between lysozyme and different functionalized self-assembled alkanethiol monolayers confirm that hydrophobic surfaces interact strongly with proteins. The lysozyme-unfolding pathway during early adsorption appears to be similar to that predicted by published molecular modeling results. Proteins 2004. © 2004 Wiley-Liss, Inc.

We previously found that autoantibodies against mouse lysozyme (ML) were strongly induced in normal BALB/c mice when immunized with mutant ML that has triple mutations rendering the dominant T-cell epitope of hen egg lysozyme (HEL), HEL 107-116. As T cells specific for HEL 107-116 were primed in these mice, the anti-ML immunoglobulin G (IgG) responses would be the result of collaborations between autoreactive B cells specific for ML and T cells specific for HEL 107-116. Serum IgG responses against ML were dominantly focused on the ML 14-69 region, indicating that B cells responding to the epitope escape tolerance. In the present study, we prepared several monoclonal antibodies (mAbs) specific for ML 14-69 and examined their antigen specificities in detail, to characterize the nature of the remaining B-cell repertoire specific for ML. mAbs specific for ML 14-69 interacted weakly with soluble, native ML, but the interactions were strengthened by denaturation of ML. The apparent affinity constants between these mAbs and ML showed an increase, ranging from six- to 80-fold, by denaturation of ML. Therefore, these mAbs were more specific for the denatured determinant than for the determinant in the native structure. These results indicate that a substantial number of autoreactive B cells, specific for the unfolded conformation of ML, escape tolerance and are dominantly involved in the autoantibody response to ML. Our finding provides important information to understand the naturally occurring autoreactive B-cell repertoire in normal mice.

In the present study, we investigated whether or not the amyloid-β protein (Aβ) peptide itself spontaneously generates free radicals using electron spin resonance (ESR) spectroscopy while also monitoring the aggregational state of Aβ and Aβ-induced cytotoxicity. The present results demonstrated a four-line spectrum in the presence of both Aβ40 and Aβ42 with Ntert-butyl-α-phenylnitrone (PBN), but not in the presence of PBN alone in phosphate-buffered saline (PBS). The fact that the four-line spectrum obtained for the Aβ/PBN in PBS was completely abolished in the presence of the iron-chelating agent Desferal demonstrated the observed four-line spectrum to be iron-dependent. The present study also revealed that either Aβ40 or Aβ42 with PBN in phosphate buffer (PB) did not produce any definite four-line spectrum. Both a thioflavine-T (Th-T) fluorometric assay and circular dichroism (CD) spectroscopy showed the amyloid fibril formation of Aβ in PBS to be much higher than that of Aβ in PB. Moreover, Aβ-induced cytotoxicity assays showed Aβ incubated in PBS to be more cytotoxic than that incubated in PB. These results thus suggest that Aβ-associated free radical generation is strongly influenced by the aggregational state of the peptides.

It is important to identify characteristics that confer on proteins the potential to induce allergenic sensitization and allergenic disease. Protein allergens carry T-cell epitopes that are capable of inducing a type 2 T helper (Th2) cell response. There is limited information regarding factors that govern the allergenicity of proteins. We previously reported that a decrease in the conformational stability of hen-egg lysozyme (HEL) enhanced its capacity to activate HEL-specific T cells owing to the increased susceptibility to intracellular antigen processing. To determine whether the conformational stability of HEL makes for a critical contribution to allergenic sensitization in vivo, we immunized BALB/c mice with HEL derivatives of different conformational stability, but which retained a similar three-dimensional structure. The magnitude of in vivo T-cell responses, evaluated by ex vivo proliferative responses of lymph node T cells from mice primed with various HEL derivatives, was inversely correlated with conformational stability, as was interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production by splenic T cells in response to HEL. Immunization of the least stable derivative led to a potent IL-4 response and to immunoglobulin E (IgE) antibody production. We propose that the intrinsic allergenicity of proteins can be determined by the degree of conformational stability.

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.

Conjugation of proteins with polyethylene glycol (PEG) has been reported to make the proteins tolerogenic. Native proteins are also potentially tolerogenic when given without adjuvants. We compared immunotolerogenic activities between PEG-conjugated and native hen egg-white lysozyme (HEL). BALB/c mice were given consecutive weekly intraperitoneal administrations of PEG-conjugated HEL, unmodified HEL or phosphate-buffered saline (PBS), for 3 weeks, then challenged with HEL in Freund's complete adjuvant. The pretreatment with PEG-HEL tolerized both T-cell and humoral responses, while native HEL tolerized only the T-cell response. Immunoglobulin G1 (IgG1) antibody was already elevated in HEL-pretreated mice prior to challenge immunization, followed by suppressed IgG2a and IgG2b, but spared IgG1 production after the antigen challenge, whereas PEG-HEL-pretreated mice produced no antibody in all IgG subclasses prior and subsequent to the antigen-challenge. Production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by lymphoid cells in response to HEL in vitro was markedly suppressed in both the antigen-pretreated groups, while suppression of IL-4 production was evident only in PEG-HEL-, not in HEL-pretreated animals. Involvement of suppressor cells in these tolerance states was found to be unlikely, and the immunological property of PEG-HEL differed from deaggregated HEL that was similar to the original HEL. These results suggest a unique immunotolerogenic activity of PEG-conjugated proteins to suppress both T-helper type-1 (Th1)- and Th2-type responses, the result being extensive inhibition of all IgG subclass responses, while tolerance induction by unconjugated soluble proteins tends to be targeted on Th1-, but spares Th2-type responses.

On irradiation, diepoxy[15]annulenone 1 and monobromo-diepoxy[15]annulenone 2 undergo an intriguing rearrangement, whereby all of the ether and carbonyl oxygens of 1 and 2 can travel freely and change their positions in the rings to give 15 position isomers in all.

Proteins are usually in an equilibrium between the folded and the unfolded state. Therefore, for the stabilization of proteins against reversible denaturation, the free energy change for the unfolding should be increased by stabilizing the folded state by lowering the energy level of the folded state or by destabilizing the unfolded state by raising the energy level of the unfolded state. On the other hand, various processes can be coupled with the unfolded state of proteins. For example, protease digestion of proteins at physiological temperature may be one of such processes. The process would lead to an irreversible denaturation. For the stabilization of proteins against the irreversible denaturation coupled with the unfolded state, a kinetic stabilization is important, that is, the activation free energy for the unfolding should be increased. Methods for the kinetic stabilization were discussed. Finally, the irreversible chemical deterioration of proteins was considered.

I have pursued research on lysozymes for 42 years. During that time, I made Several new findings, some of them by chance. My enjoyment of the following areas is reviewed: the story of tryptophan; protease digestion mechanisms; peptide mapping with RP-HPLC; gene engineering; renaturation of protein; catalytic residues; fluctuation and function; stabilization; folding; antigenecity; tolerance; and various lysozymes.

Das vollstandige Manuskript dieser Zuschrift erscheint in: Angew. Chem. Suppl. 1983, 480. DOI: 10.1002/ange.198304800

Das vollstandige Manuskript dieser Zuschrift erscheint in: Angew. Chem. Suppl. 1983, 243. DOI: 10.1002/ange.198302430

Das vollstandige Manuskript dieser Zuschrift erscheint in Angew. Chem. Suppl. 1983, 471. DOI: 10.1002/ange.198304710

The complete manuscript of this communication appears in: Angew. Chem. Suppl. 1983, 480. DOI:10.1002/anie.198304800

The complete manuscript of this communication appears in: Angew. Chem. Suppl. 1983, 243. DOI:10.1002/anie.198302430

The complete manuscript of this communication appears in: Angew. Chem. Suppl. 1983, 471. DOI:10.1002/anie.198304710