Participation of cysteine 30 residue in the folding process of ovalbumin evaluated in a refolding experiment using cysteine mutants.

To understand the role of cysteine (SH) residues in the folding of hen ovalbumin (OVA), SH-mutated OVAs, in which each SH residue was replaced by alanine (C11A, C30A, C367A, and C382A), were prepared. SDS-PAGE analysis under non-reducing conditions showed that the C11A and C30A mutants produced a disulfide (SS) isomer in addition to a protein with a native SS bond (Cys73-Cys120). The susceptibility to elastase digestion suggested that the Cys73 residue in the SS isomer participates as a counterpart of the SS bond. Upon refolding of the SH-mutated OVAs under the denatured and SS-reduced states, only C30A failed to refold into an intact form. This indicated that the Cys30 residue plays an important role in correct refolding. To confirm this, each of the four SH-mutated OVAs, in which the original SS-forming sites (C11/73/120A, C30/73/120A, C73/120/367A, and C73/120/382A) were deleted, was constructed and expressed. The C11/73/120A and C30/73/120A mutants formed no SS form, in contrast to C73/120A as a control. Thus, we concluded that Cys30 participates in the correct folding of OVA, and that its SS bond (Cys11-Cys30) is transiently generated during the early folding stage to avoid misfolding, and then the native SS form of OVA is regenerated through SH-SS exchanges.

Characterization of a yam class IV chitinase produced by recombinant Pichia pastoris X-33.

A yam (Dioscorea opposita Thunb) class IV chitinase, whose genomic DNA was cloned by Mitsunaga et al. (2004), was produced by the recombinant Pichia pastoris X-33 in high yields such as 66 mg/L of culture medium. The chitinase was purified by column chromatography after Endoglycosidase H treatment and then characterized. It showed properties similar to the original chitinase E purified from the yam tuber reported by Arakane et al. (2000). This Pichia-produced chitinase also showed strong lytic activity against Fusarium oxysporum and Phytophthora nicotianae, wide pH and thermal stability, optimum activity at higher temperature such as 70 °C, and high substrate affinity, indicating that one can use this Pichia-produced yam chitinase as a bio-control agent.

A thiophene-containing compound as a matrix for matrix-assisted laser desorption/ ionization mass spectrometry and the electrical conductivity of matrix crystals.

The electrical conductivity of the matrix crystal might be a new factor to enhance matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sensitivity. In MALDI-MS, several compounds are used as a standard matrix. Utilization of such compounds is based on an a posteriori approach, but there is no theoretical guidance for selecting a matrix. In an attempt to further understand performance in MALDI-MS, we utilized peptide detection for random screening of a chemical library (12,383 compounds) for compounds with matrix functions in MALDI-MS. A lot of thiophene compounds were found to be a matrix, in which 2-[5-(2,4-dichlorobenzoyl)-2-thienyl] acetic acid (DCBTA) provided an important clue to measure the electrical conductivity of the matrix crystal, because the structure of DCBTA is analogous to conductive polymers and organic solar cells. Most of the crystals of standard matrices, such as alpha-cyano-4-hydroxycinnamic acid (CHCA), 3,5-dimethoxy-4-hydroxycinnamic acid [sinapinic acid, (SA)], and DCBTA showed electrical conductivity, whereas the conductivity of crystal was not observed in 2,5-dihydroxybenzoic acid (2,5-DHB). On the other hand, super-DHB using 2-hydroxy-5-methoxybenzoic acid [5-methoxysalicylic acid, (MSA)] as an additive to 2,5-DHB, improved the electrical conductivity of the crystal, that followed the enhancement of peak intensity in MS spectrum. These observations might indicate that the electrical conductivity of matrix crystals is a key consideration in obtaining efficient MALDI performance.

Contribution of structural reversibility to the heat stability of the tropomyosin shrimp allergen.

Tropomyosins are common heat-stable crustacean allergens. However, their heat stability and their effects on antigenicity have not been clarified. We purified tropomyosin in this study from raw kuruma prawns (Marsupenaeus japonicus) without heat processing. SDS-PAGE of the purified protein showed a band at approximately 35 kDa that cross-reacted with IgE from the serum of a shrimp-allergic patient, identifying it as Pen j 1. The circular dichroism spectrum of native Pen j 1 revealed the common α-helical structure of tropomyosins which easily collapsed upon heating to 80 °C. However, there were no insoluble aggregates after heating, and the protein regained its native CD spectral pattern after cooling to 25 °C. There was no significant difference in total IgG production between mice sensitized with native and heated Pen j 1. These results suggest that heat-denatured Pen j 1 refolded upon cooling and maintained its antigenicity following the heat treatment.

The role of the disulfide bridge in the stability and structural integrity of ovalbumin evaluated by site-directed mutagenesis.

To provide a molecular explanation of the role of the disulfide (SS) bridge in the thermostability and structural integrity of ovalbumin (OVA), we prepared SS-mutated OVAs in which SS-forming residues were replaced by Ala or Ser (C73A, C73S, C120A, and C73/120A), and compared the conformation, thermostability, susceptibility to elastase, and formation of heat-stable OVA (S-OVA) with those of the wild-type. The circular dichroism (CD) and tryptophan fluorescence spectra revealed that the SS-mutated OVAs assumed a native-like conformation similar to the wild-type. The thermal denaturation temperature for the SS-mutated OVAs was significantly lower than that for the wild-type. C73S, C120A, and C73/120A mutants converted to S-OVA on alkaline treatment. Analyses for elastase digestion fragments showed that a non-native SS bridge was generated in all SS-mutated OVAs, but non-native SS-pairing did not contribute to thermostability. Hence, we concluded that the presence of the original SS bridge in OVA contributes to conformational stability but is not directly related to the conversion to S-OVA.

Thermostabilization of ovalbumin by alkaline treatment: Examination of the possible roles of D-serine residues.

It was revealed from the crystal structure analysis of S-ovalbumin (S-OVA) formed by alkaline treatment that Ser164, Ser236, and Ser320 take the D-amino acid residue configuration (Yamasaki et al., J Biol Chem 2003; 278:35524-35530). To address the implications of a D-configuration for these Ser residues in S-OVA formation, three mutant OVAs (S164A, S236A, and S320A) were generated to compare their thermostabilities before and after alkaline treatment. Following alkaline treatment, S236A showed a marked increase in melting temperature similar to the wild type (DeltaT(m), +9 degrees C) which corresponded to the formation of S-OVA, whereas the increment in T(m) for both S164A and S320A was only 4.5 degrees C. Furthermore, the T(m) value of the double mutant S164/320A remained unchanged after alkaline treatment, supporting the relevance of Ser164 and Ser320 for thermostabilization of OVA. As Arg142 was predicted to interact with D-Ser164 upon S-OVA formation, it was substituted to Ala to generate R142A. The resulting increment in T(m) of mutant R142A after alkaline treatment was 5.8 degrees C. The double mutant R142/S320A was therefore prepared to eliminate the participation of Ser320 in thermostabilization, and its T(m) value was compared before and after alkaline treatment. As expected, the increase in T(m) for the double mutant was only 1.2 degrees C. Taken together, the data suggest that D-configuration of Ser164 caused by alkaline treatment favors interaction with Arg142 through conformational changes of the side chain. These results strongly supported the participation of the configurational inversion of both Ser164 and Ser320 residues in the formation of S-OVA.

Importance of N-glycosylation positioning for secretion and folding of ovalbumin.

To investigate the role of the carbohydrate chain of hen egg ovalbumin (OVA), potential N-glycosylation site-deletion OVA mutants were expressed in yeast. The secretion level of the N292Q and N292/311Q mutants was greatly reduced compared with the wild-type OVA. Furthermore, secretion of the mutants without a carbohydrate chain on Asn-292 could hardly be detected in the culture medium, even if an additional N-glycosylation site was introduced to the OVA molecule. The reduction in secretion level seems to be due to incorrectly folded protein. Moreover, the secretion levels of the wild-type and N311Q mutant reduced in a similar extent as those of the mutants without a carbohydrate chain on Asn-292 in calnexin-disrupted yeast. These results indicate that the carbohydrate chain attached to Asn-292 of OVA has an important role for the secretion and folding in the cells.