Yohichi Hashimoto

Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves

Abstract Two l -arabino- d -galactan-containing glycoproteins having a potent inhibitory activity against eel anti-H agglutinin were isolated from the hot saline extracts of mature radish leaves and characterized to have a similar monosaccharide composition that consists of l -arabinose, d -galactose, l -fucose, 4-O-methyl- d -glucuronic acid, and d -glucuronic acid residues. The chemical structure features of the carbohydrate components were investigated by carboxyl group reduction, methylation, periodate oxidation, partial acid hydrolysis, and digestion with exo- and endo-glycosidases, which indicated a backbone chain of (1→3)-linked β- d -galactosyl residues, to which side chains consisting of α-(1→6)-linked d -galactosyl residues were attached. The α- l -arabinofuranosyl residues were attached as single nonreducing groups and as O-2- or O-3-linked residues to O-3 of the β- d -galactosyl residues of the side chains. Single α- l -fucopyranosyl end groups were linked to O-2 of the l -arabinofuranosyl residues, and the 4-O-methyl-β- d -glucopyranosyluronic acid end groups were linked to d -galactosyl residues. The O-α- l -fucopyranosyl-(1→2)-α- l -arabinofuranosyl end-groups were shown to be responsible for the serological, H-like activity of the l -arabino- d -galactan glycoproteins. Reductive alkaline degradation of the glycoconjugates showed that a large proportion of the polysaccharide chains is conjugated with the polypeptide backbone through a 3-O- d -galactosylserine linkage.

Activation of a limulus coagulation factor G by (1 → 3)-g⊝d-glucans

Abstract Various oligo- and poly-saccharides differing in sugar compositions and types of linkage were examined for their ability to activate a limulus coagulation factor G, the first protease in an alternative coagulation cascade of the horseshoe crab, Tachypleus tridentatus , whose amebocytes have originally been known to contain a lipopolysacchaide(LPS)-driven pathway leading to the formationof coagulin gel. Linear and branched (1 → 3)-β- d -glucans and mixed linkage (1 → 3),(1 → 4)-β- d -glucans were found to exhibit the ability to activate factor G at concentrations of 10 −8 –10 −10 g/mL as assayed by amidolytic activity of the clotting enzyme. Laminaran oligosaccharides, laminaran dextrins (number-average mol. wt. ≦ 5800), and other polysaccharides including carboxymethylcellulose, amylose, starch, d -fructans, α- l -arabinan, β- d -xylans, (1 → 3)-β- d -galactan, water-soluble chitin derivatives, chondroitin, chondroitin sulfates, hyaluronan, keratan sulfate, heparins, heparan sulfate, and LPSs were virtually inactive as activators even at a concentration of 10 −7 g/mL. The activating ability increased with increasing number-average mol. wt. (6800–216 000) of linear (1 → 3)-β- d -glucans. The apparent activating ability of gyrophoran, nigeran, and yeast α- d -mannan was largely abolished by digestion with a highly purified Arthrobacter luteus endo-(1 → 3)-β- d -glucanase, which provided supportive evidence for the activation to be ascribed to contaminating (1 → 3)-β- d -glucan(s). Possible participation of ordered structures of (1 → 3)-β- d -glucans in the activation of factor G is discussed.

Amino acid sequences of metalloendopeptidases specific for acyl-lysine bonds from Grifola frondosa and Pleurotus ostreatus fruiting bodies.

The complete amino acid sequences of two lysine-specific zinc metalloendopeptidases (EC 3.4.24), Grifola frondosa metalloendopeptidase (GFMEP) and Pleurotus ostreatus metalloendopeptidase (POMEP), from the fruiting bodies of these two edible mushrooms have been established based on the sequence information of the peptides generated from the reduced and alkylated GFMEP and POMEP by proteolytic digestions using GFMEP, trypsin, and other proteinases as well as by several chemical cleavages. From the sequences, it was found that GFMEP and POMEP were polypeptides composed of 167 and 168 amino acid residues, from which their molecular weights were calculated to be 18,040.5 and 17,921.3 in accord with the observed (M+H)+ values of 18,028 and 17,927, respectively, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Two disulfide bonds in GFMEP were found to link Cys5 to Cys75 and Cys77 to Cys97. An unusual post-translational modification of GFMEP was corroborated to be a partial attachment of a single mannose to Thr42. Comparison of the sequences revealed that overall identity between the enzymes was 61.3%. Although a highly homologous sequence was not found in sequence data bases except for a consensus zinc-binding sequence, HEXXH, both metalloendopeptidases somewhat resembled a family of metalloproteinases categorized as deuterolysin. These proteases together with GFMEP and POMEP do not have conserved third and/or fourth liganding amino acid residues seen in metzincin or thermolysin superfamily proteins and belong to a novel zinc metalloendopeptidase superfamily.

An L-arabino-D-galactan and an L-arabino-D-galactan-containing proteoglycan from radish (Raphanus sativus) seeds

Abstract An l -arabino- d -galactan and an l -arabino- d -galactan-containing proteoglycan were isolated from hot phosphate-buffered saline extracts of radish seeds by ethanol fractionation, ion-exchange chromatography, and gel filtration, and found homogeneous by ultracentrifuge analysis and high-voltage electrophoresis. The proteoglycan consisted of 86% of a polysacchraide component containing β- l -arabinose and d -galactose as major sugar constituents, together with small proportions of d -xylose, d -glucose, and uronic acids, and 9% of a hydroxyproline-containing protein. Methylation analysis, periodate oxidation, and enzymic degradations indicated a backbone chain of (1a3)-linked β- d galactosyl residues with side chains at O-6 of (1a6)-linked β- d -galactosyl residues and uronosyl groups. The α- l -arabinofuranosyl residues were located mainly in the outer regions as nonreducing groups, as well as O-2- or -5-linked inner chain residues, and O-2,5- or -3,5-linked branching residues. Reductive, alkaline degradation of the proteoglycan indicated that the polysaccharide chains were partly linked through O -glycosyl linkages to the threonine residues of the polypeptide chains. The proteoglycans from radish leaves and seeds appeared to share common antigenic determinant(s). The radish-seed arabinogalactan had a high content (81%) of l -arabinose and its basic structure seemed to be similar to that of the polysaccharide component of the proteoglycan.

Purification and characterization of a β-glucuronidase from Aspergillus niger ☆

Abstract A β-glucuronidase from Pectinex Ultra SP-L, a commercial pectolytic enzyme preparation from Aspergillus niger, was purified 170-fold by ion-exchange chromatography and gel filtration. Apparent Mr of the purified enzyme, estimated by denaturing gel electrophoresis and size-exclusion chromatography, were 68,000 and 71,000, respectively, indicating that the enzyme is a monomeric protein. It released uronic acids not only from p-nitrophenyl β-glucosiduronic acid (PNP-GlcA) but also from acidic galactooligosaccharides carrying either β- d -glucosyluronic or 4-O-methyl-β- d -glucosyluronic residues at the nonreducing termini through β-(1→6)-glycosidic linkages. The enzyme exhibited a maximal activity toward these substrates at pH 3.0. A regioisomer, 3-O-β-glucosyluronic acid-galactose, was unsusceptible to the enzyme. The enzyme did act on a polymer substrate, releasing uronic acid from the carbohydrate portion of a radish arabinogalactan–protein modified by treatment with fungal α- l -arabinofuranosidase. The enzyme produced acidic oligosaccharides by transglycosylation, catalyzing the transfer of uronic acid residues of PNP-GlcA and 6-O-β-glucosyluronic acid-galactose to certain exogenous acceptor sugars such as Gal, N-acetylgalactosamine, Glc, and xylose.

Purification and characterization of an endo-β-(1 → 6)-galactanase from Trichoderma viride

An endo-beta-(1-->6)-galactanase from Onozuka R-10, a commercial cellulase preparation from Trichoderma viride, was purified 57-fold. Apparent Mr values of the purified enzyme, estimated by denaturing gel electrophoresis and gel filtration, were 47,000 and 17,000, respectively. The enzyme was assayed with a galactan from Prototheca zopfii, which has a high proportion of beta-(1-->6)-linked galactosyl residues. It exhibited maximal activity toward the galactan at pH 4.3. The enzyme hydrolyzed specifically beta-(1-->6)-galactooligosaccharides with a degree of polymerization higher than 3 and their acidic derivatives with 4-O-methyl-glucosyluronic or glucosyluronic groups at the nonreducing terminals. The methyl beta-glycoside of beta-(1-->6)-galactohexaose was degraded to reducing galactooligomers with a degree of polymerization 2-5 as the products at the initial stage of hydrolysis, and galactose and galactobiose at the final stage, indicating that the enzyme can be classified as an endo-galactanase. The extent of hydrolysis of the carbohydrate portion of a radish root arabinogalactan-protein (AGP) increased when alpha-L-arabinofuranosyl residues attached to beta-(1-->6)-linked galactosyl side chains of the AGP were removed in advance. The enzyme released galactose, beta-(1-->6)-galactobiose, and 4-O-methyl-beta-glucuronosyl-(1-->6)-galactose as major hydrolysis products when allowed to act exhaustively on the modified AGP.

Inhibition of high-molecular-weight-(1 → 3)-β-d-glucan-dependent activation of a limulus coagulation factor G by laminaran oligosaccharides and curdlan degradation products

Extensive surveys for the effects of various beta-D-glucans on the coagulation cascade in horseshoe crab amebocyte lysates showed that low-mol-wt-(1-->3)-beta-D-glucans and laminaran oligosaccharides inhibit the activation of a limulus coagulation factor G by high-mol-wt-(1-->3)-beta-D-glucans. The inhibitory properties are exclusively dependent upon their number-average mol wt (Mn) in a range of 342-58,100, which correspond to a degree of polymerization (dp) range of 2-359. The most effective is a laminaran dextrin of Mn 5800 (dp of 35-36), which causes 50% inhibition of factor G activation at a concentration of 3.16 ng/mL. The inhibition of the activation of factor G proportional to the concentration of the inhibitor, and the adsorption of factor G by inhibitory beta-D-glucan-conjugated cellulose suggested a high affinity of the inhibitory saccharides for the activator-recognition site of factor G. Branched (1-->6), (1-->3)-beta-D-glucans, laminarans, mixed linkage (1-->3), (1-->4)-beta-D-glucans, and partially substituted curdlan and laminaran were found to be inhibitory, possibly owing to clusters of consecutive (1-->3)-beta-D-glucopyranosyl residues as intrachain units. The inhibition appears to be related to the inability of the inhibitory (1-->3)-beta-D-glucans to form ordered conformations and to their tendency to take a random-coil structure in aqueous solution.

[alpha]-L-Fucosyltransferases from Radish Primary Roots

A novel [alpha]-L-fucosyltransferase capable of transferring L-fucose (L-Fuc) from GDP-L-Fuc to the O-2 of [alpha]-L-arabinofuranosyl residue (GDP-L-Fuc:[alpha]-L-arabinofuranoside 2-[alpha]-L-fucosyltransferase) has been found in the microsomal fraction of primary roots from 6-d-old radish (Raphanus sativus L.) seedlings. Enzyme activity was measured fluorometrically at 25[deg]C using a pyridylaminated trisaccharide, L-arabinofuranosylf[alpha](1->3)D-galactopyranosyl[beta](1->6)D-galac tose (AraGalGal-PA) as the acceptor. This enzyme found in the microsomal fraction is maximally active at pH 6.8 and requires 0.1% (w/v) Zwittergent 3–16 and 5 mM Mn2+. Chemical and enzymatic analyses of fucosylated AraGalGal-PA confirmed the attachment of L-Fuc to the L-arabinofuranosyl (L-Araf) residue at O-2 by [alpha]-glycosidic linkage. Radiolabeling was used to assay L-Fuc transfer to L-Araf-containing galacto-oligomers and tamarind xyloglucan. The enzyme specific for the L-Araf residue undergoes development- and organ-specific expression in root tissue, whereas the L-Fuc transfer to tamarind xyloglucan can be detected in microsomal fractions from various organs in developing radish plants. Enzyme assays of membranes fractionated from microsomal fractions revealed that two distinct [alpha]-L-fucosyltransferases with different acceptor specificity are associated with Golgi membranes from primary roots, whereas hypocotyl Golgi membranes completely lack the enzyme specific for the L-Araf residue.

Production and characterization of antibodies to the β-(1→6)-galactotetraosyl group and their interaction with arabinogalactan-proteins

Rabbit antisera were raised against β-(1→6)-galactotetraose coupled to bovine serum albumin (Gal4-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of α-l-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, β-(1→4)-galactan, and β-(1→3)-galactan, indicating their high specificity toward the consecutive β-(1→6)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immobilized β-(1→6)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (1→6)-linked β-galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal4-BSA as antigen, which revealed that β-(1→6)-galactotriose and-tetraose were potent inhibitors, while β-(1→3)-or β-(1→4)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or β-(1→6)-galactotetraose.

Human serum dipeptidyl peptidase IV (DPPIV) and its unique properties.

Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) has been purified 18,000‐fold in a yield of 2.2% from human serum. Serum DPPIV, a serine enzyme with an apparent mass of 250 kDa, consists of two identical subunits with an apparent mass of 100 kDa and is inhibited by DPPIV‐specific inhibitor Diprotin A and also by p‐chloromercuribenzoate (p‐CMB), 2‐mercaptoethanol, HgCl2, CdCl2, SrCl2, and ZnCl2. One of the remarkable properties of DPPIV is that its activity is greatly enhanced by Gly‐X (X: especially, Gly, Gln, Glu and Ser) dipeptides. Gly‐X dipeptides increase not only an apparent Km of serum DPPIV for glycyl‐L‐proline 3,5‐dibromo‐4‐hydroxyanilide nearly 10‐fold, but also an apparent kcat nearly 4‐fold. This mechanism is unclear, but one possibility is that Gly‐Pro from substrate might bind amino acids or dipeptides instead of water molecules as DPPIV transpeptidyl activity reported previously. Another remarkable property of DPPIV is the ability to bind adenosine deaminase‐I and ‐II, as is the case with recombinant soluble CD26 (rsCD26). This probably indicates that DPPIV purified from human serum by our method originates from T‐lymphocytes.