The Experts below are selected from a list of 252 Experts worldwide ranked by ideXlab platform
Toshiyoshi Araki - One of the best experts on this subject based on the ideXlab platform.
-
Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module
Marine Biotechnology, 2010Co-Authors: Yoshiaki Umemoto, Toshiyoshi ArakiAbstract:The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-Mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra . In this study, we visualized β-Mannan in the regenerating cell walls of B . atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A Mannan-binding family 27 CBM (CBM27) of β-1,4-Mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed in Escherichia coli . Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-Mannans, while normal GFP could not bind to β-Mannans. Protoplasts were isolated from the fronds of B . atropurpurea by using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-Mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-Mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of β-Mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.
-
Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module
Marine Biotechnology, 2009Co-Authors: Yoshiaki Umemoto, Toshiyoshi ArakiAbstract:The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-Mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized β-Mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A Mannan-binding family 27 CBM (CBM27) of β-1,4-Mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-Mannans, while normal GFP could not bind to β-Mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-Mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-Mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of β-Mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.
Shigeo Suzuki - One of the best experts on this subject based on the ideXlab platform.
-
Structural analysis of cell wall Mannan of Candida sojae, a new yeast species isolated from defatted soybean flakes
Archives of Microbiology, 2008Co-Authors: Hiroko Oyamada, Yoshio Okawa, Nobuyuki Shibata, Shigeo Suzuki, Yukiko Ogawa, Hidemitsu KobayashiAbstract:We investigated the structural and immunochemical characteristics of cell wall Mannan obtained from Candida sojae JCM 1644, which is a new yeast species isolated from defatted soybean flakes. The results of a slide-agglutination test and of an enzyme-linked immunosorbent assay using anti-factor sera to the pathogenic Candida species indicated that the cells and the C. sojae Mannan were cross-reactive to the specific anti-factor sera against Candida albicans serotype A (FAb 6) and Candida guilliermondii (FAb 9). Two-dimensional homonuclear Hartmann–Hahn analysis indicated that the Mannan consisted of various linked oligomannosyl side chains containing α-1,2-, α-1,3-, α-1,6- and β-1,2-linked mannose residues. However, although the determinants of antigenic factors 6 and 9 could be not found in this Mannan, branched side chains, Manβ1-2Manα1-3[Manα1-6]Manα1-(2Manα1-)n2Man and a linear α-1,6-linked polymannosyl backbone, which are cross-reacted by FAbs 6 and 9, respectively, were identified. The Mannan was subjected to acetolysis in order to determine the polymerization length of the α-1,2-linked oligomannosyl residue in the side chains. The result of ^1H-nuclear magnetic resonance analysis of the released oligosaccharides showed that the remarkable regularity in the length of α-1,2-linked oligomannosyl side chains, which were previously found in Mannans of other Candida species, is not observed in this Mannan.
-
distribution of antigenic oligomannosyl side chains in the cell wall Mannans of several strains of candida tropicalis
Archives of Microbiology, 2003Co-Authors: Hidemitsu Kobayashi, Hiroko Oyamada, Nobuyuki Shibata, Kyoko Matsuda, Shigeo SuzukiAbstract:In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall Mannans of the pathogenic yeast Candida tropicalis, the chemical structure of Mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the Mannans. The linear side chain Manα1–3Manα1–(2Manα1–)n2Man [n≥2] is present in the Mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the Mannans from IFO 0199 and IFO 1647. The Mannan of IFO 0589 is the only Mannan with the branched side chains, Manα1–3[Manα1–6]Manα1–(2Manα1-)n2Man and Manα1–2Manα1–3[Manα1–6]Manα1–(2Manα1-)n2Man [n≥2]. However, this Mannan lacked the phosphate group and the β-1,2-linked oligomannosyl side chain which are features of this group. The Mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an α-1,3-linked mannose residue previously observed in Candida albicans.
-
existence of novel branched side chains containing 1 2 and 1 6 linkages corresponding to antigenic factor 9 in the Mannan of candida guilliermondii
Journal of Biological Chemistry, 1996Co-Authors: Nobuyuki Shibata, Yoshio Okawa, Hidemitsu Kobayashi, Akifumi Suzuki, Kyoko Ikuta, Kanehiko Hisamichi, Rieko Akagi, Tomoko Hosoya, Kumi Kawahara, Shigeo SuzukiAbstract:Abstract Isolation of β-linkage-containing side chain oligosaccharides from the Mannan of Candida gilliermondii IFO 10279 strain has been conducted by acetolysis under mild conditions. A structural study of these oligosaccharides by one- and two-dimensional NMR and methylation analyses indicated the presence of extended oligosaccharide side chains with two consecutive β-1,2-linked mannose units at the nonreducing terminal of α-linked oligosaccharides. The linkage sequence present in this Mannan, Manβ12Manα13Manα, has also been found in the Mannan of Saccharomyces kluyveri but not in the Mannan of Candida species. Furthermore, these oligosaccharides are branched at position 6 of the 3-O-substituted mannose units as follows. and The H-1 signals of the mannose units substituted by a 3,6-di-O-substituted unit showed a significant upfield shift (Δ = 0.04-0.08 ppm) due to a steric effect. The inhibition of an enzyme-linked immunosorbent assay between the Mannan of C. guilliermondii and factor 9 serum with oligosaccharides obtained from several Mannans indicated that only the oligosaccharides with the above structure were active, suggesting that these correspond to the epitope of antigenic factor 9.
-
STRUCTURE AND ANTIGENICITY OF THE MannanS OF CANDIDA FAMATA AND CANDIDA SAITOANA : COMPARATIVE STUDY WITH THE Mannan OF CANDIDA GUILLIERMONDII
Archives of Biochemistry and Biophysics, 1996Co-Authors: Nobuyuki Shibata, Hidemitsu Kobayashi, Akifumi Suzuki, Shigeo Suzuki, Masako Onozawa, Norihiko Tadano, Yasuko Hinosawa, Kyoko Ikuta, Yoshio OkawaAbstract:Abstract The chemical structure of the Mannans of antigenic factor 9-expressing yeast, Candida famata and Candida saitoana, was analyzed by acetolysis and NMR. The structural study of the oligosaccharides and Mannans using one- and two-dimensional NMR indicated that the Mannan of C. saitoana contains a third type of β-1,2-linked mannose unit. On the other hand, the Mannan of C. famata does not contain any β-1,2-linked mannose units. The Mannan of C. saitoana gave two groups of β-1,2 linkage-containing oligosaccharides by acetolysis. One contains one β-1,2-linked mannose unit and the other contains two consecutive β-1,2-linked mannose units at the nonreducing terminal. The inhibition of the reactivity of factor 9 serum on an enzyme-linked immunosorbent assay (ELISA) with these oligosaccharides indicated that the inhibition activity of the former oligosaccharide is 1/20 of that of the latter ones. The ELISA of the Mannans of Candida guilliermondii, C. saitoana, and Saccharomyces kluyveri, all of which contain the third type of β-1,2-linked mannose unit, indicated that Manβ1 → 2Manβ1 → 2Manα1 → 3Manα1 → works as the antigenic factor 9 but Manβ1 → 2Manα1 → 3Manα1 → weakly behaves as both antigenic factors 6 and 9. The epitope structures of the side chain oligosaccharides agreed well with that proposed from the 2D-HOHAHA spectra of the Mannans. This result demonstrates the usefulness of the H-1–H-2-correlated cross-peak pattern, which was reported in a preceding paper (Shibata, N., Akagi, R., Hosoya, T., Kawahara, K., Suzuki, A., Ikuta, K., Kobayashi, H., Hisamichi, K., Okawa, Y., and Suzuki, S. (1996) J. Biol. Chem. 271, 9259–9266) for the determination of the epitope structure of Candida Mannans without any chemical fragmentation.
-
existence of branched side chains in the cell wall Mannan of pathogenic yeast candida albicans structure antigenicity relationship between the cell wall Mannans of candida albicans and candida parapsilosis
Journal of Biological Chemistry, 1995Co-Authors: Nobuyuki Shibata, Hidemitsu Kobayashi, Kyoko Ikuta, Tomonori Imai, Yohko Satoh, Richi Satoh, Atsuko Suzuki, Chizuko Kojima, Kanehiko Hisamichi, Shigeo SuzukiAbstract:Abstract Isolation of side chain oligosaccharides from Mannans of Candida albicans NIH B-792 (serotype B) and Candida parapsilosis IFO 1396 strains has been conducted by acetolysis under mild conditions. Structural study of these oligosaccharides by 1H and C NMR and methylation analyses indicated the presence of novel branched side chains with the following structures in C. albicans Mannan.
Nobuyuki Shibata - One of the best experts on this subject based on the ideXlab platform.
-
Structural analysis of cell wall Mannan of Candida sojae, a new yeast species isolated from defatted soybean flakes
Archives of Microbiology, 2008Co-Authors: Hiroko Oyamada, Yoshio Okawa, Nobuyuki Shibata, Shigeo Suzuki, Yukiko Ogawa, Hidemitsu KobayashiAbstract:We investigated the structural and immunochemical characteristics of cell wall Mannan obtained from Candida sojae JCM 1644, which is a new yeast species isolated from defatted soybean flakes. The results of a slide-agglutination test and of an enzyme-linked immunosorbent assay using anti-factor sera to the pathogenic Candida species indicated that the cells and the C. sojae Mannan were cross-reactive to the specific anti-factor sera against Candida albicans serotype A (FAb 6) and Candida guilliermondii (FAb 9). Two-dimensional homonuclear Hartmann–Hahn analysis indicated that the Mannan consisted of various linked oligomannosyl side chains containing α-1,2-, α-1,3-, α-1,6- and β-1,2-linked mannose residues. However, although the determinants of antigenic factors 6 and 9 could be not found in this Mannan, branched side chains, Manβ1-2Manα1-3[Manα1-6]Manα1-(2Manα1-)n2Man and a linear α-1,6-linked polymannosyl backbone, which are cross-reacted by FAbs 6 and 9, respectively, were identified. The Mannan was subjected to acetolysis in order to determine the polymerization length of the α-1,2-linked oligomannosyl residue in the side chains. The result of ^1H-nuclear magnetic resonance analysis of the released oligosaccharides showed that the remarkable regularity in the length of α-1,2-linked oligomannosyl side chains, which were previously found in Mannans of other Candida species, is not observed in this Mannan.
-
distribution of antigenic oligomannosyl side chains in the cell wall Mannans of several strains of candida tropicalis
Archives of Microbiology, 2003Co-Authors: Hidemitsu Kobayashi, Hiroko Oyamada, Nobuyuki Shibata, Kyoko Matsuda, Shigeo SuzukiAbstract:In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall Mannans of the pathogenic yeast Candida tropicalis, the chemical structure of Mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the Mannans. The linear side chain Manα1–3Manα1–(2Manα1–)n2Man [n≥2] is present in the Mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the Mannans from IFO 0199 and IFO 1647. The Mannan of IFO 0589 is the only Mannan with the branched side chains, Manα1–3[Manα1–6]Manα1–(2Manα1-)n2Man and Manα1–2Manα1–3[Manα1–6]Manα1–(2Manα1-)n2Man [n≥2]. However, this Mannan lacked the phosphate group and the β-1,2-linked oligomannosyl side chain which are features of this group. The Mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an α-1,3-linked mannose residue previously observed in Candida albicans.
-
existence of novel branched side chains containing 1 2 and 1 6 linkages corresponding to antigenic factor 9 in the Mannan of candida guilliermondii
Journal of Biological Chemistry, 1996Co-Authors: Nobuyuki Shibata, Yoshio Okawa, Hidemitsu Kobayashi, Akifumi Suzuki, Kyoko Ikuta, Kanehiko Hisamichi, Rieko Akagi, Tomoko Hosoya, Kumi Kawahara, Shigeo SuzukiAbstract:Abstract Isolation of β-linkage-containing side chain oligosaccharides from the Mannan of Candida gilliermondii IFO 10279 strain has been conducted by acetolysis under mild conditions. A structural study of these oligosaccharides by one- and two-dimensional NMR and methylation analyses indicated the presence of extended oligosaccharide side chains with two consecutive β-1,2-linked mannose units at the nonreducing terminal of α-linked oligosaccharides. The linkage sequence present in this Mannan, Manβ12Manα13Manα, has also been found in the Mannan of Saccharomyces kluyveri but not in the Mannan of Candida species. Furthermore, these oligosaccharides are branched at position 6 of the 3-O-substituted mannose units as follows. and The H-1 signals of the mannose units substituted by a 3,6-di-O-substituted unit showed a significant upfield shift (Δ = 0.04-0.08 ppm) due to a steric effect. The inhibition of an enzyme-linked immunosorbent assay between the Mannan of C. guilliermondii and factor 9 serum with oligosaccharides obtained from several Mannans indicated that only the oligosaccharides with the above structure were active, suggesting that these correspond to the epitope of antigenic factor 9.
-
STRUCTURE AND ANTIGENICITY OF THE MannanS OF CANDIDA FAMATA AND CANDIDA SAITOANA : COMPARATIVE STUDY WITH THE Mannan OF CANDIDA GUILLIERMONDII
Archives of Biochemistry and Biophysics, 1996Co-Authors: Nobuyuki Shibata, Hidemitsu Kobayashi, Akifumi Suzuki, Shigeo Suzuki, Masako Onozawa, Norihiko Tadano, Yasuko Hinosawa, Kyoko Ikuta, Yoshio OkawaAbstract:Abstract The chemical structure of the Mannans of antigenic factor 9-expressing yeast, Candida famata and Candida saitoana, was analyzed by acetolysis and NMR. The structural study of the oligosaccharides and Mannans using one- and two-dimensional NMR indicated that the Mannan of C. saitoana contains a third type of β-1,2-linked mannose unit. On the other hand, the Mannan of C. famata does not contain any β-1,2-linked mannose units. The Mannan of C. saitoana gave two groups of β-1,2 linkage-containing oligosaccharides by acetolysis. One contains one β-1,2-linked mannose unit and the other contains two consecutive β-1,2-linked mannose units at the nonreducing terminal. The inhibition of the reactivity of factor 9 serum on an enzyme-linked immunosorbent assay (ELISA) with these oligosaccharides indicated that the inhibition activity of the former oligosaccharide is 1/20 of that of the latter ones. The ELISA of the Mannans of Candida guilliermondii, C. saitoana, and Saccharomyces kluyveri, all of which contain the third type of β-1,2-linked mannose unit, indicated that Manβ1 → 2Manβ1 → 2Manα1 → 3Manα1 → works as the antigenic factor 9 but Manβ1 → 2Manα1 → 3Manα1 → weakly behaves as both antigenic factors 6 and 9. The epitope structures of the side chain oligosaccharides agreed well with that proposed from the 2D-HOHAHA spectra of the Mannans. This result demonstrates the usefulness of the H-1–H-2-correlated cross-peak pattern, which was reported in a preceding paper (Shibata, N., Akagi, R., Hosoya, T., Kawahara, K., Suzuki, A., Ikuta, K., Kobayashi, H., Hisamichi, K., Okawa, Y., and Suzuki, S. (1996) J. Biol. Chem. 271, 9259–9266) for the determination of the epitope structure of Candida Mannans without any chemical fragmentation.
-
existence of branched side chains in the cell wall Mannan of pathogenic yeast candida albicans structure antigenicity relationship between the cell wall Mannans of candida albicans and candida parapsilosis
Journal of Biological Chemistry, 1995Co-Authors: Nobuyuki Shibata, Hidemitsu Kobayashi, Kyoko Ikuta, Tomonori Imai, Yohko Satoh, Richi Satoh, Atsuko Suzuki, Chizuko Kojima, Kanehiko Hisamichi, Shigeo SuzukiAbstract:Abstract Isolation of side chain oligosaccharides from Mannans of Candida albicans NIH B-792 (serotype B) and Candida parapsilosis IFO 1396 strains has been conducted by acetolysis under mild conditions. Structural study of these oligosaccharides by 1H and C NMR and methylation analyses indicated the presence of novel branched side chains with the following structures in C. albicans Mannan.
Slavomir Bystrický - One of the best experts on this subject based on the ideXlab platform.
-
preparation and characterization of cationic and amphoteric Mannans from candida albicans
Carbohydrate Polymers, 2016Co-Authors: Alžbeta Cižova, Anna Malovikova, Zuzana Nescakova, Slavomir BystrickýAbstract:Cationic and amphoteric Mannans from Candida albicans were prepared by chemical modification with (3-chloro-2-hydroxypropyl)trimethylammonium chloride (CHPTAC) and sodium chloroacetate under aqueous alkaline conditions. The optimal reaction conditions for Mannan cationization were found to be 6h, 60°C, and NaOH/CHPTAC ratio of 1.0. Adjusting the molar ratio of cationization agent to anhydromannose unit, cationic and amphoteric Mannans with degree of substitution ranging from 0.07 to 0.57 were obtained. Their structure was confirmed by elemental analysis as well as FTIR and NMR spectroscopies. Moderate decrease of molecular weight of both cationic and amphoteric Mannans was recorded by size exclusion chromatography. With increasing level of modification, reduction of the antibody-binding capacity was observed by enzyme-linked immunosorbent assay.
-
preparation and characterization of carboxymethyl derivatives of yeast Mannans in aqueous solutions
Carbohydrate Polymers, 2014Co-Authors: Eva Machova, Peter Bystrický, Anna Malovikova, Slavomir BystrickýAbstract:Abstract Novel carboxymethyl derivatives of yeast Mannans of different degrees of substitution (DS) were prepared by optimized reaction of concentrated polysaccharides in alkaline aqueous solution. Mannans from various yeasts differing in size and degree of branching show similar reactivity. Strong alkaline conditions during carboxymethylation caused degradation of the polysaccharides. The degree of substitution (DS) of Candida albicans Mannan and dextran were proportional to the amount of monochloroacetate added. However, degrees of carboxymethylation of Candida albicans Mannan (0.30, 0.41, 0.73) were lower than those of dextran (DS = 0.33, 0.6, 1.1) using the same amounts of monochloroacetate. Evidently the resulted polyanionic derivatives have higher hydrodynamic sizes than the original polysaccharides. Non-uniform, variable position of substitutions results to non-proportional change of optical rotation and increase of complexity of NMR spectra. Basic physico-chemical characteristics of novel carboxymethyl Mannans obtained by potentiometric titration, FT-IR, UV, HPLC, 1 H NMR and optical rotation measurements are presented here.
-
yeast Mannans protect liposomes against peroxidation but do not scavenge free radicals
Carbohydrate Polymers, 2012Co-Authors: Eva Machova, Slavomir BystrickýAbstract:Abstract The Mannans from Candida albicans serotype A, Candida dubliniensis , Candida tropicalis and Saccharomyces cerevisiae protected liposomes against peroxidation by OH radicals in a concentration-dependent manner. The most efficacious antioxidant was Mannan from C. albicans serotype A with antioxidant activity ∼49% comparable with known antioxidant carboxymethylated glucan (∼46%). The natural antioxidant α-tocopherol exhibited 90% protection of liposomes. The Mannans and glucans scavenged negligible amount of free radicals in the common 2,2-diphenyl-1-picrylhydrazyl test. It seems likely that protection of liposomes against OH radicals by polysaccharides is not due to their scavenging properties, but may be caused by a sterical hindrance. The polysaccharides containing flexible β-linkages are more effective protectors of liposome peroxidation than these polysaccharides that contain α-linkages only.
-
functionalization of Mannans from pathogenic yeasts by different means of oxidations preparation of precursors for conjugation reactions with respect to preservation of immunological properties
Carbohydrate Polymers, 2006Co-Authors: Richard ďurana, Igor Lacik, Ema Paulovicova, Slavomir BystrickýAbstract:Abstract Three different reagents (sodium periodate, Dess–Martin periodinane (DMP), 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)/hypochlorite/bromide) were used to prepare oxidized forms of Mannans from the four pathogenic yeasts of Candida genus ( Candida albicans , Candida tropicalis , Candida glabrata, Candida parapsilosis ) in order to prepare glycoconjugate vaccine precursors suitable for reductive amination reactions. A combination of NMR, IR, potentiometric titration, Park–Johnson colorimetric assay and size exclusion chromatography was applied for physicochemical characterization of the oxidized Mannans. Correlation between molecular weight of periodate, DMP and TEMPO-mediated oxidized Mannans and branching frequency as a characteristic structural feature of original cell wall Mannans was found. It indicates that higher branching frequency of original Mannan used in the reaction, lesser molecular weight decrease of oxidized Mannan measured. This dependence determined by SEC upon oxidation agent can be expressed by following relationship: (TEMPO)>original>DMP≫periodate. Immunological characteristics relevant to applications in vaccine technology (i.e. preservation of antigenic structural properties of polysaccharides) were analyzed in detail. It was revealed, that modification of immunological properties or damage of relevant epitopes due to structural changes via DMP and TEMPO-mediated oxidation can be excluded.
Yoshiaki Umemoto - One of the best experts on this subject based on the ideXlab platform.
-
Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module
Marine Biotechnology, 2010Co-Authors: Yoshiaki Umemoto, Toshiyoshi ArakiAbstract:The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-Mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra . In this study, we visualized β-Mannan in the regenerating cell walls of B . atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A Mannan-binding family 27 CBM (CBM27) of β-1,4-Mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed in Escherichia coli . Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-Mannans, while normal GFP could not bind to β-Mannans. Protoplasts were isolated from the fronds of B . atropurpurea by using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-Mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-Mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of β-Mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.
-
Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module
Marine Biotechnology, 2009Co-Authors: Yoshiaki Umemoto, Toshiyoshi ArakiAbstract:The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-Mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized β-Mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A Mannan-binding family 27 CBM (CBM27) of β-1,4-Mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-Mannans, while normal GFP could not bind to β-Mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-Mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-Mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of β-Mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.
