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Localization, purification and specificity of the full-length membrane-bound form of human recombinant alpha 1,3/4-fucosyltransferase from BHK-21B cells.

ABSTRACT: Fucosyltransferase III [galactoside 3(4)-L-fucosyltransferase; EC 2.4.1.65] (FT3) is a Golgi type II membrane protein that catalyses the synthesis of fucosylated Lewis motifs that are associated with cell-adhesion events and are differentially expressed during cell differentiation. In the present work, the full-length membrane bound form of FT3 has been expressed in baby hamster kidney cells. The enzyme has been found in the trans-Golgi and trans-Golgi network (TGN) of the transfected cells, where it appeared as monomers and dimers, but not as oligomers with high molecular masses. Therefore oligomerization is not the basis for correct localization of FT3 in the Golgi. The enzyme has been purified, with a final yield of 2% and a total purification of 2900-fold, by DEAE-Sepharose, SP-Sepharose, GDP-Fractogel and Superdex 200 chromatography. The purified enzyme showed a clear preference for the Gal beta 3GlcNAc motif in oligosaccharides conjugated with the hydrophobic tail (CH(2))(3)-NHCO-(CH(2))(5)-NH-biotin. Substitution of galactose with alpha 2-linked fucose or alpha 2,3-linked N-acetylneuraminic acid yielded a 1.9-fold increase or a 43% decrease in activity respectively. The enzyme showed no activity towards asialofetuin, a glycoprotein containing the Gal beta 3GlcNAc acceptor motif. Therefore it has been concluded that the membrane-bound form of FT3 is present in the Golgi and the TGN in an equilibrium of monomers<-->dimers, which might fucosylate glycans from glycolipids, but not from glycoproteins.

SUBMITTER: Sousa VL

PROVIDER: S-EPMC1222010 | biostudies-other | 2001 Aug

REPOSITORIES: biostudies-other

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Project description:Backgroundβ-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing β-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of β-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel β-1,3-glucanases with high catalytic efficiency and stability is of particular interest.ResultsA β-1,3-glucanase gene designated PglA was cloned from a newly isolated strain Paenibacillus sp. S09. The gene PglA contained a 2631-bp open reading frame encoding a polypeptide of 876 amino acids which shows 76% identity with the β-1,3-glucanase (BglH) from Bacillus circulans IAM1165. The encoded protein PglA is composed of a signal peptide, an N-terminal leader region, a glycoside hydrolase family 16 (GH16) catalytic domain and a C-terminal immunoglobulin like (Ig-like) domain. The Escherichia coli expression system of PglA and five truncated derivatives containing one or two modules was constructed to investigate the role of catalytic and non-catalytic modules. The pH for optimal activity of the enzymes was slightly affected (pH 5.5-6.5) by the presence of different modules. However, the temperature for optimal activity was strongly influenced by the C-terminal domain and ranged from 50 to 60°C. Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability. Specific activity assay indicated that PglA specifically hydrolyzes β-1,3-glucan. Insoluble β-1,3-glucan binding and hydrolysis were boosted by the presence of N-and C-terminal domains. Kinetic analysis showed that the presence of N-and C-terminus enhances the substrate affinity and catalytic efficiency of the catalytic domain toward laminarin. Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains.ConclusionsThis study provides new insight into the impacts of non-catalytic modules on enzymatic properties of β-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble β-1,3-glucan, and could be classified into CBM families.

Project description:IntroductionBacillus anthracis is the causative agent for the lethal disease anthrax, primarily affecting animals and humans in close contact with an infected host. The pathogenicity of B. anthracis is attributed to the secreted exotoxins and their outer capsule. The host cell-binding exotoxin component "protective antigen" (PA) is reported to be a potent vaccine candidate. The aim of our study is to produce several PA constructs and analyze their vaccine potential.MethodsWe have designed the various subunit, PA-based recombinant proteins, i.e., full-length Protective antigen (PA-FL), C-terminal 63 kDa fragment (PA63), Protective antigen domain 1-domain 4 chimeras (PA-D1-4) and protective antigen domain 4 (PA-D4) and analyzed their vaccine potential with different human-compatible adjuvants in the mouse model. We have optimized the process and successfully expressed our recombinant antigens as soluble proteins, except full-length PA. All the recombinant antigen formulations with three different adjuvants i.e., Addavax, Alhydrogel, and Montanide ISA 720, were immunized in different mouse groups. The vaccine efficacy of the formulations was analyzed by mouse serum antigen-specific antibody titer, toxin neutralization assay, and survival analysis of mouse groups challenged with a lethal dose of B. anthracis virulent spores.ResultsWe have demonstrated that the PA-FL addavax and PA63 addavax formulations were most effective in protecting spore-challenged mice and serum from the mice immunized with PAFL addavax, PA-FL alhydrogel, PA63 addavax, and PA63 alhydrogel formulations were equivalently efficient in neutralizing the anthrax lethal toxin. The higher levels of serum Th1, Th2, and Th17 cytokines in PA-FL addavax immunized mice correspond to the enhanced protection provided by the formulation in challenged mice.DiscussionWe have demonstrated that the PA-FL addavax and PA63 addavax formulations exhibit equivalent efficiency as vaccine formulation both in a mouse model of anthrax and mammalian cell lines. However, PA63 is a smaller antigen than PA-FL and more importantly, PA63 is expressed as a soluble protein in E. coli, which imparts a translational advantage to PA63-based formulation. Thus, the outcome of our study has significant implications for the development of protective antigen-based vaccine formulations for human use against the lethal disease anthrax.

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Localization, purification and specificity of the full-length membrane-bound form of human recombinant alpha 1,3/4-fucosyltransferase from BHK-21B cells.

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