Epitope analysis of Japanese cedar pollen allergen Cry j2 with a human IgM class monoclonal antibody 404-117.

Japanese cedar pollen allergen Cry j2 is a causal allergen of seasonal pollinosis in Japan. To analyze B cell epitopes of Cry j2, we established two human-mouse hybridomas secreting IgM class human monoclonal antibodies to Cry j2. A pin-peptide enzyme-linked immunosorbent assay with synthesized icosa peptides showed that 404-117 monoclonal antibody bound to peptides #11-13 with cry j2 amino acid sequence of 101F-L140. Detailed analysis with octa peptides and alanine substituted peptides indicated that an amino acid sequence of 118FKVD121 was an essential for antibody binding. When K119 (Asn) was substituted with alanine, 404-117 monoclonal antibody did not bind to the alanine substituted peptide. We concluded that the 118FKVD121 sequence might have a very important role in early recognition by Cry j2-specific B cells, which could act as antigen presenting cells.

Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger.

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater kcat/Km values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.

A novel metabolic pathway for glucose production mediated by α-glucosidase-catalyzed conversion of 1,5-anhydrofructose.

α-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form α-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for α-glucosidases. α-Glucosidase-catalyzed hydration produced α-glucose from AF, which plateaued with time. Combined reaction with α-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger α-glucosidase (31AG), which is stable in organic solvent, produced ethyl α-glucoside from AF in 80% ethanol. The findings indicate that α-glucosidases catalyze trans-addition. This is the first report of α-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.

A historical perspective for the catalytic reaction mechanism of glycosidase; so as to bring about breakthrough in confusing situation.

Various catalytic reaction models have been proposed as the reaction mechanisms of glycosidases, but a reasonable and unitary model capable of interpreting both "inverting" and "retaining" glycosidase reactions remains to be established. As for the models proposed to date, the nucleophilic displacement mechanism and the oxocarbenium ion intermediate mechanism are widely known, but recently the former is widely accepted, and so the general tendency of world opinion appears to favor it. This reaction model, however, is considered to comprise some inconsistencies that cannot be neglected from the viewpoint of reactivity in organic chemistry. While the nucleophilic displacement mechanism is often applied to reactions of glycosidases, it appears unlikely that such reactions actually occur. This review argues that the oxocarbenium ion intermediate reaction mechanism is more rational than the nucleophilic displacement reaction mechanism, as the action mode of glycosidases and related enzymes.

Catalytic reaction mechanism based on alpha-secondary deuterium isotope effects in hydrolysis of trehalose by European honeybee trehalase.

Trehalase, an anomer-inverting glycosidase, hydrolyzes only alpha,alpha-trehalose in natural substrates to release equimolecular beta-glucose and alpha-glucose. Since the hydrolytic reaction is reversible, alpha,alpha-[1,1'-(2)H]trehalose is capable of synthesis from [1-(2)H]glucose through the reverse reaction of trehalase. alpha-Secondary deuterium kinetic isotope effects (alpha-SDKIEs) for the hydrolysis of synthesized alpha,alpha-[1,1'-(2)H]trehalose by honeybee trehalase were measured to examine the catalytic reaction mechanism. Relatively high k(H)/k(D) value of 1.53 for alpha-SDKIEs was observed. The data imply that the catalytic reaction of the trehalase occurs by the oxocarbenium ion intermediate mechanism. In addition, the hydrolytic reaction of glycosidase is discussed from the viewpoint of chemical reactivity for the hydrolysis of acetal in organic chemistry. As to the hydrolytic reaction mechanism of glycosidases, oxocarbenium ion intermediate and nucleophilic displacement mechanisms have been widely recognized, but it is pointed out for the first time that the former mechanism is rational and valid and generally the latter mechanism is unlikely to occur in the hydrolytic reaction of glycosidases.

Function-unknown glycoside hydrolase family 31 proteins, mRNAs of which were expressed in rice ripening and germinating stages, are alpha-glucosidase and alpha-xylosidase.

In rice (Oryza sativa L., var Nipponbare) seeds, there were three mRNAs encoding for function-unknown hydrolase family 31 homologous proteins (ONGX-H1, ONGX-H3 and ONGX-H4): ONGX-H1 mRNA was expressed in ripening stage and mRNAs of ONGX-H3 and ONGX-H4 were found in both the ripening and germinating stages [Nakai et al., (2007) Biochimie 89, 49-62]. This article describes that the recombinant proteins of ONGX-H1 (rONGXG-H1), ONGX-H3 (rONGXG-H3) and ONG-H4 (rONGXG-H4) were overproduced in Pichia pastoris as fusion protein with the alpha-factor signal peptide of Saccharomyces cerevisiae. Purified rONGXG-H1 and rONGXG-H3 efficiently hydrolysed malto-oligosaccharides, kojibiose, nigerose and soluble starch, indicating that ONGX-H1 and ONGX-H3 are alpha-glucosidases. Their substrate specificities were similar to that of ONG2, a main alpha-glucosidase in the dry and germinating seeds. The rONGXG-H1 and rONGX-H3 demonstrated the lower ability to adsorb to and degradation of starch granules than ONG2 did, suggesting that three alpha-glucosidases, different in action to starch granules, were expressed in ripening stage. Additionally, purified rONGXG-H4 showed the high activity towards alpha-xylosides, in particular, xyloglucan oligosaccharides. The enzyme hardly hydrolysed alpha-glucosidic linkage, so that ONGX-H4 was an alpha-xylosidase. Alpha-xylosidase encoded in rice genome was found for the first time.

Molecular cloning of cDNA for trehalase from the European honeybee, Apis mellifera L., and its heterologous expression in Pichia pastoris.

cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5' untranslated region (UTR) of 869 bp, and the 3' UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The Mr of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast Pichia pastoris as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase.

Molecular cloning of cDNAs and genes for three alpha-glucosidases from European honeybees, Apis mellifera L., and heterologous production of recombinant enzymes in Pichia pastoris.

cDNAs encoding three alpha-glucosidases (HBGases I, II, and III) from European honeybees, Apis mellifera, were cloned and sequenced, two of which were expressed in Pichia pastoris. The cDNAs for HBGases I, II, and III were 1,986, 1,910, and 1,915 bp in length, and included ORFs of 1,767, 1,743, and 1,704 bp encoding polypeptides comprised of 588, 580, and 567 amino acid residues, respectively. The deduced proteins of HBGases I, II, and III contained 18, 14, and 8 putative N-linked glycosylation sites, respectively, but at least 2 sites in HBGase II were unmodified by N-linked oligosaccharide. In spite of remarkable differences in the substrate specificities of the three HBGases, high homologies (38-44% identity) were found in the deduced amino acid sequences. In addition, three genomic DNAs, of 13,325, 2,759, and 27,643 bp, encoding HBGases I, II, and III, respectively, were isolated from honeybees, and the sequences were analyzed. The gene of HBGase I was found to be composed of 8 exons and 7 introns. The gene of HBGase II was not divided by intron. The gene of HBGase III was confirmed to be made up of 9 exons and 8 introns, and to be located in the region upstream the gene of HBGase I.

Multiple forms of alpha-glucosidase in rice seeds (Oryza sativa L., var Nipponbare).

Two isoforms of alpha-glucosidases (ONG2-I and ONG2-II) were purified from dry rice seeds (Oryza sativa L., var Nipponbare). Both ONG2-I and ONG2-II were the gene products of ONG2 mRNA expressed in ripening seeds. Each enzyme consisted of two components of 6kDa-peptide and 88kDa-peptide encoded by this order in ONG2 cDNA (ong2), and generated by post-translational proteolysis. The 88kDa-peptide of ONG2-II had 10 additional N-terminal amino acids compared with the 88kDa-peptide of ONG2-I. The peptides between 6kDa and 88kDa components (26 amino acids for ONG2-I and 16 for ONG2-II) were removed by post-translational proteolysis. Proteolysis induced changes in adsorption and degradation of insoluble starch granules. We also obtained three alpha-glucosidase cDNAs (ong1, ong3, and ong4) from ripening seeds. The ONG1, ONG2, and ONG4 genes were situated in distinct locus of rice genome. The transcripts encoding ONG2 and ONG3 were generated by alternative splicing. Members of alpha-glucosidase multigene family are differentially expressed during ripening and germinating stages in rice.

Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA.

alpha-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.

Structural elements to convert Escherichia coli alpha-xylosidase (YicI) into alpha-glucosidase.

Escherichia coli YicI, a member of glycoside hydrolase family (GH) 31, is an alpha-xylosidase, although its amino-acid sequence displays approximately 30% identity with alpha-glucosidases. By comparing the amino-acid sequence of GH 31 enzymes and through structural comparison of the (beta/alpha)(8) barrels of GH 27 and GH 31 enzymes, the amino acids Phe277, Cys307, Phe308, Trp345, Lys414, and beta-->alpha loop 1 of (beta/alpha)(8) barrel of YicI have been identified as elements that might be important for YicI substrate specificity. In attempt to convert YicI into an alpha-glucosidase these elements have been targeted by site-directed mutagenesis. Two mutated YicI, short loop1-enzyme and C307I/F308D, showed higher alpha-glucosidase activity than wild-type YicI. C307I/F308D, which lost alpha-xylosidase activity, was converted into alpha-glucosidase.

Glucoamylase originating from Schwanniomyces occidentalis is a typical alpha-glucosidase.

A starch-hydrolyzing enzyme from Schwanniomyces occidentalis has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of S. occidentalis. The enzyme showed high catalytic efficiency (k(cat)/K(m)) for maltooligosaccharides, compared with that for soluble starch. The product anomer was alpha-glucose, differing from glucoamylase as a beta-glucose producing enzyme. These findings are striking characteristics of alpha-glucosidase. The DNA encoding the enzyme was cloned and sequenced. The primary structure deduced from the nucleotide sequence was highly similar to mold, plant, and mammalian alpha-glucosidases of alpha-glucosidase family II and other glucoside hydrolase family 31 enzymes, and the two regions involved in the catalytic reaction of alpha-glucosidases were conserved. These were no similarities to the so-called glucoamylases. It was concluded that the enzyme and also S. occidentalis glucoamylase, had been already reported, were typical alpha-glucosidases, and not glucoamylase.

Aglycon specificity profiling of alpha-glucosidases using synthetic probes.

We designed and synthesized hydrogen bond based probes 1-8 with the exception of known glycosidase inhibition mechanisms, and aglycon specificity of 11 different sources of alpha-glucosidases were investigated using their probes. Probe 4 (2,6-anhydro-1-deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-D-glycero-D-ido-heptitol) showed a potent inhibition of S. cerevisiae alpha-glucosidase among all alpha-glucosidases. Probe 4 was found to be a competitive inhibitor for S. cerevisiae alpha-glucosidase with Ki 0.13 mM.

Localization of alpha-glucosidases I, II, and III in organs of European honeybees, Apis mellifera L., and the origin of alpha-glucosidase in honey.

Three kinds of alpha-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an alpha-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey alpha-glucosidase, were almost the same as those of alpha-glucosidase III. Specific antisera against the alpha-glucosidases were prepared to examine the localization of alpha-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that alpha-glucosidase I was present in ventriculus, and alpha-glucosidase II, in ventriculus and haemolymph. alpha-Glucosidase III, which became apparent to be honey alpha-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted alpha-glucosidase III.

Overexpression and characterization of two unknown proteins, YicI and YihQ, originated from Escherichia coli.

The proteins encoded in the yicI and yihQ gene of Escherichia coli have similarities in the amino acid sequences to glycoside hydrolase family 31 enzymes, but they have not been detected as the active enzymes. The functions of the two proteins have been first clarified in this study. Recombinant YicI and YihQ produced in E. coli were purified and characterized. YicI has the activity of alpha-xylosidase. YicI existing as a hexamer shows optimal pH at 7.0 and is stable in the pH range of 4.7-10.1 with incubation for 24h at 4 degrees C and also is stable up to 47 degrees C with incubation for 15 min. The enzyme shows higher activity against alpha-xylosyl fluoride, isoprimeverose (6-O-alpha-xylopyranosyl-glucopyranose), and alpha-xyloside in xyloglucan oligosaccharides. The alpha-xylosidase catalyzes the transfer of alpha-xylosyl residue from alpha-xyloside to xylose, glucose, mannose, fructose, maltose, isomaltose, nigerose, kojibiose, sucrose, and trehalose. YihQ exhibits the hydrolysis activity against alpha-glucosyl fluoride, and so is an alpha-glucosidase, although the natural substrates, such as alpha-glucobioses, are scarcely hydrolyzed. alpha-Glucosidase has been found for the first time in E. coli.

Purification and characterization of Acremonium implicatum alpha-glucosidase having regioselectivity for alpha-1,3-glucosidic linkage.

alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 51,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(0)/K(m) value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)-O-alpha-maltosyl-maltose were novel saccharides.

Two potent competitive inhibitors discriminating alpha-glucosidase family I from family II.

The inhibition kinetics for isoacarbose (a pseudotetrasaccharide, IsoAca) and acarviosine-glucose (pseudotrisaccharide, AcvGlc), both of which are derivatives of acarbose, were investigated with various types of alpha-glucosidases obtained from microorganisms, plants, and insects. IsoAca and AcvGlc, competitive inhibitors, allowed classification of alpha-glucosidases into two groups. Enzymes of the first group were strongly inhibited by AcvGlc and weakly by IsoAca, in which the K(i) values of AcvGlc (0.35-3.0 microM) were 21- to 440-fold smaller than those of IsoAca. However, the second group of enzymes showed similar K(i) values, ranging from 1.6 to 8.0 microM for both compounds. This classification for alpha-glucosidases is in total agreement with that based on the similarity of their amino acid sequences (family I and family II). This indicated that the alpha-glucosidase families I and II could be clearly distinguished based on their inhibition kinetic data for IsoAca and AcvGlc. The two groups of alpha-glucosidases seemed to recognize distinctively the extra reducing-terminal glucose unit in IsoAca.

Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus.

Tyrosinase (monophenol, L-DOPA:oxygen oxidoreductase) was isolated from the ink of the squid, Illex argentinus. Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit. The tyrosinase activity of ST94 was enhanced by proteolysis with trypsin to form a protein, termed ST94t, with a molecular mass of 127.6 kDa. The amino acid sequence of the subunit was deduced from N-terminal amino acid sequencing and cDNA cloning, indicating that the subunit of ST94 is synthesized as a premature protein with 625 amino acid residues and an 18-residue signal sequence region is eliminated to form the mature subunit comprised of 607 amino acid residues with a deduced molecular mass of 68,993 Da. ST94 was revealed to contain two putative copper-binding sites per a subunit, that showed sequence similarities with those of hemocyanins from mollusks, tyrosinases from microorganisms and vertebrates and the hypothetical tyrosinase-related protein of Caenorhabditis elegans. The squid tyrosinase was shown to catalyze the oxidation of monophenols as well as o-diphenols and to exhibit temperature-dependency of o-diphenolase activity like a psychrophilic enzyme.

Purification, characterization, and sequence analysis of two alpha-amylase isoforms from azuki bean, Vigna angularis, showing different affinity towards beta-cyclodextrin sepharose.

Two alpha-amylase isoforms designated VAAmy1 and VAAmy2 were purified from cotyledons of germinating seedlings of azuki bean (Vigna angularis). VAAmy1 apparently had lower affinity towards a beta-cyclodextrin Sepharose column than VAAmy2. Molecular weights of VAAmy1 and VAAmy2 were estimated to be 47,000 and 44,000, respectively. However, no considerable difference was found between them in effects of pH, temperature, CaCl2, and EDTA, as well as the kinetic parameters for amylose (average degree of polymerization 17): kcat, 71.8 and 55.5 s(-1), Km, 0.113 and 0.097 mg/ml; for blocked 4-nitrophenyl alpha-D-maltoheptaoside: kcat, 62.4 and 85.3 s(-1), Km, 0.22 and 0.37 mM, respectively. Primary structures of the two enzymes were analyzed by N-terminal sequencing, cDNA cloning, and MALDI-TOF mass spectrometry, implying that the two enzymes have the same peptide. The results indicated that the low affinity of VAAmy1 towards beta-cyclodextrin Sepharose was due to some modification on/near carbohydrate binding site in the limited sequence regions, resulting in higher molecular weight.

Identification of essential ionizable groups and evaluation of subsite affinities in the active site of beta-D-glucosidase F1 from a Streptomyces sp.

Partial amino acid sequences, the essential ionizable groups directly involved in catalytic reaction, and the subsite structure of beta-D-glucosidase purified from a Streptomyces sp. were investigated in order to analyze the reaction mechanism. On the basis of the partial amino acid sequences, the enzyme seemed to belong to the family 1 of beta-glucosidase in the classification of glycosyl hydrolases by Henrissat (1991). Dependence of the V and Km values on pH, when the substrate concentration was sufficiently lower than Km, gave the values of 4.1 and 7.2 for the ionization constants, pKe1 and pKe2 of essential ionizable groups 1 and 2 of the free enzyme, respectively. When the dielectric constant of the reaction mixture was decreased in the presence of 10% methanol, the pKe1 and pKe2, values shifted to higher, to +0.60 and +0.35 pH unit, respectively. The findings supported the notion that the essential ionizable groups of the enzyme were a carboxylate group (-COO-, the group 1) and a carboxyl group (-COOH, the group 2). The subsite affinities Ai's in the active site were evaluated on the basis of the rate parameters of laminarioligosaccharides. Subsites 1 and 2 having positive Ai values (A1 was 1.10 kcal/mol and A2 was 4.98 kcal/mol) were considered to probably facilitate the binding of the substrate to the active site. However, the subsites 3 and 4 showed negative Ai values (A3 was -0.21 kcal/mol and A4 was -2.8 kcal/mol).