Yuki Chijiiwa

Role of disulfide bonds in goose‐type lysozyme

The role of the two disulfide bonds (Cys4–Cys60 and Cys18–Cys29) in the activity and stability of goose‐type (G‐type) lysozyme was investigated using ostrich egg‐white lysozyme as a model. Each of the two disulfide bonds was deleted separately or simultaneously by substituting both Cys residues with either Ser or Ala. No remarkable differences in secondary structure or catalytic activity were observed between the wild‐type and mutant proteins. However, thermal and guanidine hydrochloride unfolding experiments revealed that the stabilities of mutants lacking one or both of the disulfide bonds were significantly decreased relative to those of the wild‐type. The destabilization energies of mutant proteins agreed well with those predicted from entropic effects in the denatured state. The effects of deleting each disulfide bond on protein stability were found to be approximately additive, indicating that the individual disulfide bonds contribute to the stability of G‐type lysozyme in an independent manner. Under reducing conditions, the thermal stability of the wild‐type was decreased to a level nearly equivalent to that of a Cys‐free mutant (C4S/C18S/C29S/C60S) in which all Cys residues were replaced by Ser. Moreover, the optimum temperature of the catalytic activity for the Cys‐free mutant was downshifted by about 20 °C as compared with that of the wild‐type. These results indicate that the formation of the two disulfide bonds is not essential for the correct folding into the catalytically active conformation, but is crucial for the structural stability of G‐type lysozyme.

Mutant Mouse Lysozyme Carrying a Minimal T Cell Epitope of Hen Egg Lysozyme Evokes High Autoantibody Response

Self proteins including foreign T cell epitope induce autoantibodies. We evaluated the relationship between the size of foreign Ag introduced into self protein and the magnitude of autoantibody production. Mouse lysozyme (ML) was used as a model self protein, and we prepared three different ML derivatives carrying T cell epitope of hen egg white lysozyme (HEL) 107–116, i.e., heterodimer of ML and HEL (ML-HEL), chimeric lysozyme that has residue 1–82 of ML and residue 83–130 of HEL in its sequence (chiMH), and mutant ML that has triple mutations rendering the most potent T cell epitope of HEL (sequence 107–116). Immunization of BALB/c mice with these three ML derivatives induced anti-ML autoantibody responses, whereas native ML induced no detectable response. In particular, mutML generated a 104 times higher autoantibody titer than did ML-HEL. Anti-HEL107–116 T cell-priming activities were almost similar among the ML derivatives. The heterodimerization of mutant ML and HEL led to significant reduction of the autoantibody response, whereas the mixture did not. These results show that size of the nonself region in modified self Ag has an important role in determining the magnitude of the autoantibody response, and that decrease in the foreign region in a modified self protein may cause high-titered autoantibody response.

The Amino Acid Sequence of Satyr Tragopan Lysozyme and Its Activity

The amino acid sequence of satyr tragopan lysozyme and its activity was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 103 (Asn to Ser), 106 (Ser to Asn), and 121 (His to Gln) comparing with Temmincks tragopan lysozyme and five amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), 101 (Asp to Gly) and 103 (Asn to Ser) with chicken lysozyme. The time course analysis using N-acetylglucosamine pentamer as a substrate showed a decrease of binding free energy change, 1.1 kcal/mol at subsite A and 0.2 kcal/mol at subsite B, between satyr tragopan and chicken lysozymes. This was assumed to be responsible for the amino acid substitutions at subsite A-B at position 101 (Asp to Gly), however another substitution at position 103 (Asn to Ser) considered not to affect the change of the substrate binding affinity by the observation of identical time course of satyr tragopan lysozyme with turkey and Temmincks tragopan lysozymes that carried the identical amino acids with chicken lysozyme at this position. These results indicate that the observed decrease of binding free energy change at subsites A-B of satyr tragopan lysozyme was responsible for the amino acid substitution at position 101 (Asp to Gly).

The Role of Arg114 at Subsites E and F in Reactions Catalyzed by Hen Egg-White Lysozyme

To understand better the role of subsites E and F in lysozyme-catalyzed reactions, mutant enzymes, in which Arg114, located on the right side of subsites E and F in hen egg-white lysozyme (HEL), was replaced with Lys, His, or Ala, were prepared. Replacement of Arg114 with His or Ala decreased hydrolytic activity toward an artificial substrate, glycol chitin, while replacement with Lys had little effect. Kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)5, revealed that the replacement for the Arg residue reduced the binding free energies of E-F sites and the rate constant of transglycosylation. The rate constant of transglycosylation for R114A was about half of that for the wild-type enzyme. 1H-NMR analysis of R114H and R114A indicated that the structural changes induced by the mutations were not restricted to the region surrounding Arg114, but rather extended to the aromatic side chains of Phe34 and Trp123, of which the signals are connected with each other through nuclear Overhauser effect (NOE) in the wild-type. We speculate that such a conformational change causes differences in substrate and acceptor binding at subsites E and F, lowering the efficiency of glycosyl transfer reaction of lysozyme.

Amino Acid Sequence of Egyptian Goose Egg-White Lysozyme and Effects of Amino Acid Substitution on the Enzymatic Activity

The amino acid sequence of Egyptian goose lysozyme (EGL) from egg-white and its enzymatic properties were analyzed. The established sequence had the highest similarity to wood duck lysozyme (WDL) with five amino acid substitutions, and had eighteen substitutions difference from hen egg-white lysozyme (HEL). Tyr34 and Gly37 were found at subsites E and F of the active site when compared with HEL. The experimental time-course characteristics of EGL against the N-acetylglucosamine pentamer substrate, (GlcNAc)5, revealed higher production of (GlcNAc)4 and lower production of (GlcNAc)2 when compared with HEL. The saccharide-binding ability of subsites A–C in EGL was also found to be weaker than in HEL. An analysis of the enzymatic reactions of five mutants in respect of positions 34, 37 and 71 in HEL indicated the time-course characteristics of EGL to be caused by the combination of three substitutions (F34Y, N37G and G71R) between HEL and EGL. A computer simulation of the EGL-catalyzed reaction suggested that the time-course characteristics of EGL resulted from the difference in the binding free energy for subsites A, B, E and F and the rate constant of transglycosylation between EGL and HEL.

The Mutational Effect of Ile58 at Subsite C in Hen Egg-White Lysozyme on Substrate Binding, Enzymatic Activity, and Protein Stability

Ile58 of hen egg-white lysozyme (HEL) is buried in the interior of the molecule and is considered to participate in sugar residue binding at subsite C through hydrophobic interaction. The contribution of Ile58 to lysozyme function and stability was investigated by replacement of Ile58 with less hydrophobic residues, Val (I58V) and Ala (I58A). Replacement of Ile58 with Ala decreased substrate binding ability to an N-acetylglucosamine trisaccharide, (GlcNAc)3, and a GlcNAc polymer, chitin, whereas replacement with Val had little effect. Similar results were obtained as to enzymatic activity toward both the bacterial cell substrate and glycol chitin. Kinetic analysis by substrate (GlcNAc)5 revealed that replacement of the Ile residue reduced the sugar residue affinity at subsite C and the rate constant of glycosidic bond cleavage. The rate constant of glycosidic cleavage for mutant I58A was about one-third of that for the wild-type. Guanidine hydrochloride unfolding experiments showed that mutants I58V and I58A were less stable than the wild-type, by 1.88 and 2.88 kcal/mol respectively. Moreover, the stability of the protein inserted at this position decreased linearly with decreasing hydrophobicity of the inserted residue. It appears that the hydrophobicity of Ile58 is an important factor in the efficient substrate binding, enzymatic reaction, and structural stability of HEL.

Application of the amino acid analysis for the detection of AGE-proteins of the Maillard reaction

Abstract Recent studies demonstrated N-(carboxymethyl) lysine (CML) and N-(carboxyethyl) lysine (CEL) in several tissues. To elucidate the mechanism of the formation of advanced glycation end product (AGE) structure during incubation of protein with glucose, we established the quantitative analysis system of CML and CEL by amino acid analyzer with cation exchange and post column ninhydrin detection system and applied for the detection of CML and CEL in AGE structure. We found the formation of CML from Amadori product by the reaction of Fe2+ with H2O2. Further, the formation of CEL in glucose-modified proteins was inhibited by aminoguanidine but enhanced by phosphate.