Reducing-end Residue Research Articles

Improved pharmacokinetics and stability properties of catalase by chemical glycosidation with end‐group activated dextran

AbstractCatalase was chemically modified with a monoactivated dextran derivative having a carboxylate group at its reducing end residue. The modified enzyme retained 77% of its initial specific activity and was 3‐fold more resistant to tryptic degradation. The plasma half‐life time was increased to 7.3‐fold after glycosidation. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 4573–4576, 2006

Rice chitinases: sugar recognition specificities of the individual subsites

Sugar recognition specificities of class III (OsChib1a) and class I (OsChia1cDeltaChBD) chitinases from rice, Oryza sativa L., were investigated by analyzing (1)H- and (13)C-nuclear magnetic resonance spectra of the enzymatic products from partially N-acetylated chitosans. The reducing end residue of the enzymatic products obtained by the class III enzyme was found to be exclusively acetylated, whereas both acetylated and deacetylated units were found at the nearest neighbor to the reducing end residue. Both acetylated and deacetylated units were also found at the nonreducing end residue and its nearest neighbor of the class III enzyme products. Thus, only subsite (-1) among the contiguous subsites (-2) to (+2) of the class III enzyme was found to be specific to an acetylated residue. For the class I enzyme, the reducing end residue was preferentially acetylated, although the specificity was not absolute. The nearest neighbor to the acetylated reducing end residue was specifically acetylated. Moreover, the nonreducing end residue produced by the class I enzyme was exclusively acetylated, although there was a low but significant preference for deacetylated units at the nearest neighbor to the nonreducing end. These results suggest that the three contiguous subsites (-2), (-1), and (+1) of the class I enzyme are specific to three consecutive GlcNAc residues of the substrate. In rice plants, the target of the class I enzyme might be a consecutive GlcNAc sequence probably in the cell wall of fungal pathogen, whereas the class III enzyme might act toward an endogenous complex carbohydrate containing GlcNAc residue.

Acceptor specificity of trehalose phosphorylase from Thermoanaerobacter brockii: Production of novel nonreducing trisaccharide, 6-O-α-D-galactopyranosyl trehalose

We investigated the acceptor specificity of a thermostable trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047 (TbTP) was examined using beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor and oligosaccharides as the acceptor. Oligosaccharides with a reducing-end glucose residue as the C-6 substituent (e.g., isomaltose, gentiobiose, melibiose, isomaltotriose, and isopanose) were found to be successful acceptors. The transfer products of isomaltose, gentiobiose, and melibiose were isolated and characterized as 6-O-alpha-D-glucopyranosyl trehalose (alpha-GlcTre), 6-O-beta-D-glucopyranosyl trehalose (beta-GlcTre), and 6-O-alpha-D-galactopyranosyl trehalose (alpha-GalTre), respectively. To produce alpha-GalTre, a novel nonreducing trisaccharide, the reaction conditions of alpha-GalTre were examined using trehalose as a glucosyl donor. As a result, the yield of alpha-GalTre reached 40.5%.

A Single-Step Method for the Production of Sugar Hydrazides: Intermediates for the Chemoselective Preparation of Glycoconjugates

The ability to selectively conjugate carbohydrate molecules to a protein is a key step in the preparation of conjugate vaccines, while facile methods for linking carbohydrates to polymers or solid surfaces to produce diagnostic probes and functional microarrays are also sought. Here, we describe a simple, single-step method of producing glycosylhydrazides from unprotected sugars, which were then linked in a controlled manner to a desired carrier, through an appropriate linker. The method was chemoselective and did not require coupling reagents, and the native pyranose form of the reducing end residue was retained. Initially, mono- and disaccharide hydrazides were produced from the corresponding reducing sugars and linked to BSA through a bifunctional linker. Final exemplification of the procedure was demonstrated by the preparation of a LewisY tetrasaccharide protein conjugate, which was recognized by a LewisY monoclonal antibody indicating the preservation of the natural conformation of the tetrasaccharide in the final construct. It is envisaged that this method will have general applicability to a variety of functionally diverse reducing sugars and provide a route to highly defined glycoconjugates, without the need for elaborate synthetic strategies.

Synthetic disaccharide analogs as potential substrates and inhibitors of a mycobacterial polyprenol monophosphomannose-dependent α-(1→6)-mannosyltransferase

Analogs of the α- d-Man p-(1→6)-α- d-Man p-O(CH 2) 7CH 3 disaccharide 4, a known substrate for a polyprenol monophosphomannose-dependent α-(1→6)-mannosyltransferase involved in mycobacterial LAM biosynthesis, have been synthesized and screened as potential substrates and inhibitors of the enzyme. In the disaccharides synthesized, the hydroxyl groups at C-2 and C-6 on the reducing end residue have been replaced by combinations of amino, fluoro, and methoxy functionalities 9– 14. In addition, a disaccharide in which the nonreducing mannopyranose residue was replaced with a 3,6-anhydromannopyranose residue 34 was synthesized from a byproduct formed during one of the reactions leading to 14. When tested against the enzyme, none were active as substrates, as would be expected as all lack the C-6′ hydroxyl group to which an additional sugar residue would be transferred. Evaluation of these compounds as inhibitors of the enzyme revealed that only three, 11, 12, and 13, all of which contain one or more amino groups, inhibited the enzyme. The most potent inhibitor was the diamino-disaccharide, 11.

Analysis of fluorogenic Smith degradation products of 7-(1,3-disulfonaphtyl)amino-disaccharides for linkage position analysis of carbohydrates.

The linkage position of a glycosidic bond to the reducing-end residue of a pyridylamino (PA-) sugar can be determined sensitively by Smith degradation and HPLC [K. Omichi and S. Hase, (1994) J. Biochem. 115, 429-434]. With the aim of enhancing the sensitivity of this method of linkage position analysis to the fmol-level, use of the 7-(1,3-disulfonaphtyl)amino (DSNA-) group instead of the PA-group as a fluorescent tag was examined. Smith degradation of DSNA-disaccharides with a DSNA-hexose, DSNA-N-acetylglucosamine, or DSNA-N-acetylgalactosamine reducing-end residue was carried out. HPLC and FAB-MS of the fluorogenic Smith degradation products showed that the DSNA-group was stable under the Smith degradation reaction conditions, and that the reaction proceeded in a manner similar to that using PA-disaccharides to give the predicted products. Fluorogenic Smith degradation products specific to the glycosidic linkage position were well separated by reversed-phase HPLC, and were easily assignable by comparing the HPLC elution positions with those of standard compounds. The method was successfully applied to analyzing the structure of an N-linked sugar chain.

Processing pathway deduced from the structures of N-glycans in Carica papaya.

A processing The processing pathway of N-glycans in Carica papaya was deduced from the structures of N-glycans. The N-glycans were liberated by hydrazinolysis followed by N-acetylation. Their reducing-end sugar residues were tagged with 2-aminopyridine and the pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Eleven PA-sugar chains were found, and their structures were analyzed by two-dimensional sugar mapping combined with partial acid hydrolysis and exoglycosidase digestion. The structures of the N-glycans were of the highmannose types with xylose and fucose; however, among them two new N-glycans, Manalpha1-6(Manalpha1-3)Manalpha1-6(Xylbeta1-2)+ ++Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-3)GlcNAc and Manalpha1-3Manalpha1-6(Xylbeta1-2)Manbeta1-4G lcNAcbeta1-4(Fucalpha1-3 )GlcNAc, were found. Judging from these structures together with Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc reported previously [Shimazaki, A., Makino, Y., Omichi, K., Odani, S., and Hase, S. (1999) J. Biochem. 125, 560- 565], a processing pathway for N-glycans in C. papaya is inferred in which the activity of Golgi alpha-mannosidase II is incomplete.

Mode of action of chitin deacetylase from Mucor rouxii on N-acetylchitooligosaccharides.

The mode of action of chitin deacetylase from the fungus Mucor rouxii on N-acetylchitooligosaccharides with a degree of polymerization 1-7 has been elucidated. Identification of the sequence of chitin oligomers following enzymatic deacetylation was verified by the alternative use of two specific exo-glycosidases in conjunction with HPLC. The results were further verified by 1H-NMR spectroscopy. It was observed that the length of the oligomer is important for enzyme action. The enzyme cannot effectively deacetylate chitin oligomers with a degree of polymerization lower than three. Tetra-N-acetylchitotetraose and penta-N-acetylchitopentaose are fully deacetylated by the enzyme, while in the case of tri-N-acetylchitotriose, hexa-N-acetylchitohexaose and hepta-N-acetylchitoheptaose the reducing-end residue always remains intact. Furthermore, the enzyme initially removes an acetyl group from the nonreducing-end residue of all chitin oligomers with a degree of polymerization higher than 2, and further catalyses the hydrolysis of the following acetamido groups in a processive fashion. The results are in agreement with the mode of action that the same enzyme exhibits on partially deacetylated water soluble chitosan polymers.

A new sugar chain of the proteinase inhibitor from latex of Carica papaya.

The structure of a sugar chain of the proteinase inhibitor from the latex of Carica papaya was studied. Sugar chains liberated on hydrazinolysis were N-acetylated, and their reducing-end residues were tagged with 2-aminopyridine. One major sugar chain was detected on size-fractionation and reversed-phase HPLC analyses. The structure of the PA-sugar chain was determined by two-dimensional sugar mapping combined with sequential exoglycosidase digestion and partial acid hydrolysis, and by 750 MHz 1H-NMR spectroscopy. The structure found was Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc. This sugar chain represents a new plant-type sugar chain with five mannose residues.

Mechanism of Heparin Activation of Antithrombin: ROLE OF INDIVIDUAL RESIDUES OF THE PENTASACCHARIDE ACTIVATING SEQUENCE IN THE RECOGNITION OF NATIVE AND ACTIVATED STATES OF ANTITHROMBIN

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of approximately 15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.

Structures of the sugar chains of recombinant macrophage colony-stimulating factor produced in Chinese hamster ovary cells.

The structures of the N- and O-linked sugar chains of recombinant human macrophage colony-stimulating factor (rhM-CSF) from Chinese hamster ovary (CHO) cells were studied. rhM-CSF is a homodimeric glycoprotein. Sugar composition analysis revealed that rhM-CSF contained 4.1 mol N-acetylgalactosamine, 10.3 mol N-acetylglucosamine, 5.0 mol mannose, 10.0 mol galactose, 1.4 mol fucose, and 11.8 mol sialic acid per mol of the monomer. The N- and O-linked sugar chains liberated by hydrazinolysis were N-acetylated, and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. The structures of the PA-sugar chains were analyzed by a combination of reversed-phase and size-fractionation HPLC, and exoglycosidase digestions, from which the structures of the rhM-CSF sugar chains were estimated to be as follows: monosialo biantennary sugar chain (9 mol%), monosialo fucosylbiantennary sugar chain (10 mol%), disialo biantennary sugar chain (30 mol%), disialo fucosylbiantennary sugar chain (28 mol%), disialo triantennary sugar chain (7 mol%), trisialo triantennary sugar chain (11 mol%), and trisialo fucosyltriantennary sugar chain (5 mol%) for the N-linked sugar chains, and asialo (27 mol%), monosialo (51 mol%), and disialo (22 mol%) Galbeta1-3GalNAc for the O-linked sugar chains. Sialic acid residues were linked to the N-linked sugar chains through an alpha2-3 linkage.

Determination of the Linkage Positions of Reducing-End Residues of Oligosaccharides by Partial Periodate Oxidation of Pyridylaminated Derivatives

Determination of the Linkage Positions of Reducing-End Residues of Oligosaccharides by Partial Periodate Oxidation of Pyridylaminated Derivatives

The action of Bacillus circulans WL-12 chitinases on partially N-acetylated chitosan.

Both chitinase A1 and D from Bacillus circulans WL-12 specifically hydrolyzed the N-acetyl-beta-D-glucosaminidic bonds in 50% N-acetylated chitosan molecules to produce hetero-oligosaccharides with GlcNAc at the reducing end residues, together with GlcNAc and (GlcNAc)2. GlcN-GlcNAc and GlcN-GlcNAc-GlcNAc were produced as major hydrolysis products with chitinase A1 and D, respectively, but GlcN-GlcNAc was not detected in the digest of 50% N-acetylated chitosan with chitinase D.

Purification and characterization of novel chitinases from Streptomyces griseus HUT 6037

Two chitinases, C-1 and C-2, were purified from the culture supernatant of Streptomyces griseus HUT 6037 by ammonium sulfate precipitation, Butyl-Toyopearl 650M chromatography, and chromatofocusing. Both enzymes had a molecular weight estimated to be 27,000 by SDS polyacrylamide gel electrophoresis, while their p Is were 7.7 and 7.3, respectively. The enzymes were active from pH 4.5 to 6.0 and their optimum temperature was 55°C. They were stable between pH 6.5 and 10.0 and at temperatures below 50°C. Chitinases C-1 and C-2 hydrolyzed chitin, colloidal chitin, glycol chitin, carboxymethyl chitin, 53% deacetylated chitosan, and (GlcNAc) 3–6, but did not hydrolyze 96% deacetylated chitosan and (GlcN) n. The hydrolyzates of 53% deacetylated chitosan by these enzymes were separated by CM-Sephadex C-25 column chromatography. The products were analyzed structurally to elucidate the specificity of the chitinases. The oligosaccharides isolated had GlcNAc as the nonreducing end and GlcNAc or GlcN as the reducing end residues. These results indicate that the enzymes cleave both the N-acetyl-β- d-glucosaminidic and the β-glucosaminidic linkages in partially N-acetylated chitosan molecules.

Specificity of chitosanase from Bacillus pumilus

Partially (25–35%) N-acetylated chitosan was digested by chitosanase from Bacillus pumilus BN-262, and structures of the products, partially N-acetylated chitooligosaccharides, were analyzed in order to investigate the specificity of the chitosanase. The chitosanase produced glucosamine (GlcN) oligosaccharides abundantly, indicating that the chitosanase splits the β-l,4-glycosidic linkage of GlcN-GlcN. The chitosanase also produced hetero-oligosaccharides consisting of glucosamine and N- acetyl- dglucosamine (GlcNAc). Three types of the hetero-oligosaccharides purified by cation-exchange chromatography and HPLC were found to have GIcNAc residue at their reducing end and GIcN residue at their non-reducing end, indicating that the chitosanase can also split the linkage of GlcNAc-GlcN. The determination of the mode of action toward partially N-acetylated chitosan enables a classification of chitosanases according to their specificities and a more precise definition of chitosanases.

Substrate recognition at the binding site in mammalian pancreatic .alpha.-amylases

Porcine and human pancreatic alpha-amylases (PPA and HPA, respectively) have five binding sites for hexose residues of substrates. As previously reported, when a substrate is too small to occupy subsite 5 of the alpha-amylases, the optimum pH for catalytic activity changes from neutral to acidic [Ishikawa, K., Matsui, I., Honda, K., & Nakatani, H. (1990) Biochemistry 29, 7119-7123]. We studied the mechanism by which the enzyme recognizes the substrate by using the synthetic substrates maltopentaose (G5) and its analogs 4-O-alpha-maltotetraosyl-D-xylose (G4-X), 4-O-alpha-maltotetraosyl-2-deoxy-D-glucose (G4-D), 3-O-alpha-maltotetraosyl-L- sorbose (G4-S), 4-O-beta-maltotetraosyl-D-glucose (G4 beta-G), alpha-maltotetraosyl-beta-D-fructose (G4-F), p-nitrophenyl alpha-maltotetraoside (G4-phi), and maltopentaitol (G4-GOH). The reducing-end residues of these substrates, relevant to subsite 5, are D-xylose (X), 2-deoxy-D-glucose (D), L-sorbose (S), D-glucose (G), alpha-D-fructofuranose (F), p-nitrophenyl (phi), and D-sorbitol (GOH), respectively. The optimum pH for catalytic activity on the substrates G5, G4-X, G4-D, G4-S, G4 beta-G, and G4-phi was neutral, while that for G4-GOH and G4-F was acidic. These results indicate that only six-membered-ring residues and a phenyl group are recognizable by subsite 5. The neutral pH profile for G4-phi suggests that steric compatibility of the substrates at subsite 5 is also important for the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Structures of sugar chains of hen egg yolk riboflavin-binding protein.

The structures of the sugar chains of hen yolk riboflavin-binding protein (RBP) were established. Asparagine-linked sugar chains of yolk-RBP were liberated by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. Fluorescent pyridylamino (PA-) derivatives of the sugar chains were purified by gel-filtration and reversed-phase HPLC. Seven PA-sugar chains were isolated, and the structure of each was determined by composition analysis, sequential exoglycosidase digestion, methylation analysis, and 500-mHz 1H-NMR spectroscopy. These analyses showed that the main sugar chains had sialylbiantenna and sialyltriantenna structures. PA-sugar chains of plasma-RBP were also isolated, and the structures of the PA-sugar chains of yolk- and plasma-RBPs were compared as to their elution patterns on anion-exchange chromatography and reversed-phase HPLC. The plasma RBP had almost the same sugar chains as the yolk RBP did, indicating that sugar chains are not modified during incorporation into the oocyte.

Maltodextrin acceptor reactions of Streptococcus mutans 6715 glucosyltransferases

The maltodextrin (maltose through maltoheptaose) acceptor reactions of two Streptococcus mutans 6715 glucosyltransferases (GTF-1 and GTF-S) were studied. The acceptor product structures were determined by comparing them with the known structures of the acceptor products of Leuconostoc mesenteroides B-512FM dextransucrase (EC 2.4.1.5) and L. mesenteroides B-1355 alternansucrase (EC 2.4.1.140). When reacted with maltose (G2), both GTF-1 and GTF-S transferred a d-glucopyranose from sucrose to the nonreducing glucosyl residue to give panose (6 2-α- d-glucopyranosyl maltose). Panose then served as an acceptor to give two further acceptor products, 6 2-α- d-isomaltosyl maltose and 6 2-α-nigerosyl maltose. 6 2-α-Isomaltosyl maltose then went on to serve as an acceptor to give a series of homologous acceptor products with isomaltodextrin chains attached to C-6 of the nonreducing-end residue of maltose, while 6 2-α-nigerosyl maltose did not further react. When reacted with other maltodextrins (G3–G7), both GTF-I and GTF-S transferred a d-glucopyranose to C-6 of either the nonreducing-end or the reducing-end residues of the maltodextrins, forming α(1→6) linkages. When d-glucopyranose was transferred to the nonreducing-end residue by GTF-I or GTF-S, the first product was also an acceptor to give the second product, which then served as an acceptor to give the third product, etc., to give a homologous series of products. When d-glucopyranose was transferred to the reducing-end residue, the acceptor product that formed did not readily serve as an acceptor, or served only as a very poor acceptor, to give a small amount of the next homologue, as was the case for G7 with GTF-S. In addition, GTF-I also transferred d-glucopyranose to the reducing-end or to the nonreducing-end residue of maltotriose, forming α(1→3) linkages, to give 3 3-α- d-glucopyranosyl maltotriose and 3 1-α- d-glucopyranosyl maltotriose. Neither of these acceptor products further served as acceptors to give a homologous series. Under equivalent conditions of equimolar amounts of acceptor and sucrose, maltose and maltotriose are much better acceptors with GTF-I than they are with GTF-S, which is better than L. mesenteroides B-512M dextransucrase. The three enzymes display significantly different efficiencies for the different maltodextrin acceptor reactions, GTF-I and GTF-S having much higher efficiencies than L. mesenteroides B-512FM dextransucrase.

Acceptor reactions of maltodextrins with Leuconostoc mesenteroides B-512FM dextransucrase

The acceptor products of maltose with Leuconostoc mesenteroides B-512FM dextransucrase are panose (6 2-α- d-glucopyranosyl maltose) and a homologous series of 6 2-isomaltodextrinosyl maltoses. The structures of the acceptor products of dextransucrase with other maltodextrins, maltotriose to maltooctaose (G3–G8), were determined by using the known specificities of α-glucosidase and porcine pancreatic α-amylase, and by methylation analysis. It has been found that dextransucrase transfers a d-glucopyranosyl residue to C-6 of either the nonreducing end or the reducing end residues of the maltodextrins, G3–G8, forming an α(1 → 6) linkage. When a d-glucose was transferred to the nonreducing residue, the first product was also an acceptor to give the second product, which served as an acceptor to give the third product, etc. to give a homologous series. When d-glucose was transferred to the reducing residue, the first product did not readily serve as an acceptor to give products or it served only as a very poor acceptor to give a small amount of the next homologue. The effectiveness of maltodextrins as acceptors decreased as the size of the maltodextrin chain increased. Maltotriose was 40% as effective as maltose and maltooctaose was only 6% as effective.

Teichuronic acid reducing terminal N-acetylglucosamine residue linked by phosphodiester to peptidoglycan of Micrococcus luteus

Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. 31P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB3H4 followed by complete acid hydrolysis yielded [3H] glucosaminitol from the true reducing end residue of teichuronic acid and [3H]glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue.