N-terminal vacuolar sorting signal at the mouse antibody alters the N-linked glycosylation pattern in suspension-cultured tobacco BY2 cells.

Recombinant DNA technology enables the use of plants as the host for the production of pharmaceutical proteins, such as antibodies. The glycosylation of recombinant proteins plays physiological and biological roles. However, because glycosylation in plants is different from that in human cells, the development of glycoengineering is required. In plant cells, glycan structures are shown to correlate with the localization of the recombinant protein produced. In this study, the vacuolar sorting signal (VSS) of sporamin was fused to the heavy (H) and light (L) chains of a mouse monoclonal antibody (mAb), and the mAb was produced in suspension-cultured tobacco BY2 cells. The sugar chain structures were determined by high-performance liquid chromatography, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Typical plant glycans with α1,3-fucosylation and/or ß1,2-xylosylation derived from mAb with the VSS-fused H-chain (mIgG1000) and mAb with the VSS-fused H- and L-chain (mIgG1010) occupied the large amount of the total N-glycans, 72.1% and 85.0%, respectively, such as those derived from mAb without VSS (mIgG0000), 74.6% (Fujiyama et al., J. Biosci. Bioeng., 101, 212-218, 2006). In contrast, the typical plant glycan structure Man3FucXylGlcNAc2 particularly in vacuoles accounted for 37.8% of the total sugar chains derived from mIgG1000 and 58.5% of those derived from mIgG1010 compared with 24.3% of those derived from mIgG0000. These results suggest that the sporamin signal peptide fused to mAb acts as a VSS and leads to the increase in the amount of Man3FucXylGlcNAc2, which is the main N-glycan structure in vacuoles.

Stable coexpression of two human sialylation enzymes in plant suspension-cultured tobacco cells.

Human CMP-N-acetylneuraminic acid (NeuAc) synthase (hCSS) and α2,6-sialyltransferase (hST) participate in the sialylation of N-linked glycans in mammalian cells. hCSS synthesizes CMP-NeuAc, which hST uses as a donor substrate to transfer NeuAc to the terminal position of N-linked glycans. In plant cells, the presence of NeuAc has not yet been substantiated and the identification of the genes involved in the sialylation of N-glycan has not been carried out. In this study, we introduced hCSS and hST genes into suspension-cultured tobacco BY2 cells to provide the machinery for the sialylation pathway in plants. hCSS and hST stably expressed in the plant cells showed activity. In addition, CMP-NeuAc produced by hCSS in the transformed plant cells functioned as a donor substrate to hST. An in vitro coupled hCSS and hST reaction resulted in the production of mammalian-type sialoglycoproteins bearing terminal NeuAc residues. Furthermore, the results of the purification of the coupled-reaction products by Sambucus sieboldian lectin column chromatography and digestion with linkage-specific neuraminidase revealed that the modified terminal residue was α2,6-linked NeuAc. Here, we demonstrate that the in vitro sialylation of N-linked glycans on mammalian proteins can be achieved using plant cell extracts stably expressing hCSS and hST, providing proof-of-principle that a sialylated human therapeutic protein can be produced in plants.

Arabidopsis thaliana ALG3 mutant synthesizes immature oligosaccharides in the ER and accumulates unique N-glycans.

The core oligosaccharide Glc(3)Man(9)GlcNAc(2) is assembled by a series of membrane-bound glycosyltransferases as the lipid carrier dolichylpyrophosphate-linked glycan in the endoplasmic reticulum (ER). The first step of this assembly pathway on the ER luminal side is mediated by ALG3 (asparagine-linked glycosylation 3), which is a highly conserved reaction among eukaryotic cells. Complementary genetics compared with Saccharomyces cerevisiae ALG gene families and bioinformatic approaches have enabled the identification of ALG3 from other species. In Arabidopsis thaliana, AtALG3 (At2g47760) was identified as alpha1,3-mannosyltransferase. Complementation analysis showed that AtALG3 rescued the temperature-sensitive phenotype, that lipid-linked oligosaccharide assemblies and that protein underglycosylation of S. cerevisiae ALG3-deficient mutant. In Arabidopsis ALG3 mutant, an immature lipid-linked oligosaccharide structure, M5(ER), was synthesized, and used for protein N-glycosylation, resulting in the blockade of subsequent maturation with the concanavalin A affinoactive and Endo H-insensitive structure. N-Glycan profiling of total proteins from alg3 mutants exhibited a unique structural profile, alg3 has rare N-glycan structures including Man(3)GlcNAc(2), M4(ER), M5(ER) and GlcM5(ER), which are not usually detected in Arabidopsis, and a much less amount of complex-type N-glycan than that in wild type. Interestingly, despite protein N-glycosylation differences compared with wild type, alg3 showed no obvious phenotype under normal and high temperature or salt/osmotic stress conditions. These results indicate that AtALG3 is a critical factor for mature N-glycosylation of proteins, but not essential for cell viability and growth in Arabidopsis.

Two Arabidopsis thaliana Golgi alpha-mannosidase I enzymes are responsible for plant N-glycan maturation.

N-Glycosylation is an important post-translational modification that occurs in many secreted and membrane proteins in eukaryotic cells. Golgi alpha-mannosidase I hydrolases (MANI) are key enzymes that play a role in the early N-glycan modification pathway in the Golgi apparatus. In Arabidopsis thaliana, two putative MANI genes, AtMANIa (At3g21160) and AtMANIb (At1g51590), were identified. Biochemical analysis using bacterially produced recombinant AtMANI isoforms revealed that both AtMANI isoforms encode 1-deoxymannojirimycin-sensitive alpha-mannosidase I and act on Man(8)GlcNAc(2) and Man(9)GlcNAc(2) structures to yield Man(5)GlcNAc(2). Structures of hydrolytic intermediates accumulated in the AtMANI reactions indicate that AtMANIs employ hydrolytic pathways distinct from those of mammalian MANIs. In planta, AtMANI-GFP/DsRed fusion proteins were detected in the Golgi stacks. Arabidopsis mutant lines manIa-1, manIa-2, manIb-1, and manIb-2 showed N-glycan profiles similar to that of wild type. On the other hand, the manIa manIb double mutant lines produced Man(8)GlcNAc(2) as the predominant N-glycan and lacked plant-specific complex and hybrid N-glycans. These data indicate that either AtMANIa or AtMANIb can function as the Golgi alpha-mannosidase I that produces the Man(5)GlcNAc(2) N-glycan structure necessary for complex N-glycan synthesis.

Cloning and characterization of cytidine monophosphate-3-deoxy-d-manno-octulosonate synthetase from Arabidopsis thaliana.

The function and metabolic pathway of 3-deoxy-d-manno-octulosonate (KDO) are unclear in plants although it is an essential component in plant cell wall. Here we cloned and characterized a putative Arabidopsis thaliana cytidine monophosphate-KDO synthetase to understand synthetic pathways of KDO. It showed a ubiquitous expression, the activity at an optimal pH of 8.0, and a requirement of Mg2+.

Change in glycosylation pattern with extension of endoplasmic reticulum retention signal sequence of mouse antibody produced by suspension-cultured tobacco BY2 cells.

The production of antibodies using plants as bioreactors has been realized. Because sugar chain structures on recombinant proteins are a cause of concern, remodeling technology is highly promising. Localizing recombinant proteins in the endoplasmic reticulum (ER) affects the glycosylation pattern in transgenic plants. Previously, a mouse antibody produced by suspension-cultured tobacco BY2 cells has sugar chains with possible glycoepitopes as the predominant structures. In this study, we extended the Lys-Asp-Glu-Leu (KDEL) ER retention signal sequence over the heavy (H) and light (L) chains of the antibody and expressed the altered antibody in tobacco BY2 cells to study the effect of the KDEL sequence on glycosylation. For the antibody with the KDEL-extended H-chains, glycans with beta(1,2)-xylose or alpha(1,3)-fucose residues accounted for 49% of the total glycans. Meanwhile, for the antibody with the KDEL-extended H- and L-chains, glycans with xylose or fucose accounted for 38% of the total glycans. Although the addition of an ER retention signal shifted the dominant glycan structures of the KDEL-extended antibody to high-mannose-type structures, some of the antibodies escaped the retrieval system during intracellular traffic and were then modified by xylosylation or fucosylation.

Bradyrhizobium iriomotense sp. nov., isolated from a tumor-like root of the legume Entada koshunensis from Iriomote Island in Japan.

A polyphasic study was performed to determine the taxonomic position of strain EK05(T) isolated from a root-outgrowth of Entada koshunensis, a legume available in Okinawa, Japan. Phylogenetic analysis of the 16S rRNA gene showed that the strain belongs to the genus Bradyrhizobium. Subsequent multilocus sequence analysis with ITS, glnII, recA, gyrB, and atpD sequences revealed that the isolate represents a distinct evolutionary lineage within the genus Bradyrhizobium. DNA-DNA hybridization indicated that strain EK05(T) shares <61% DNA relatedness with the type strains of all six recognized species of Bradyrhizobium, confirming that this strain is a novel species within the genus. Phylogenetic trees based on symbiotic loci, nifH and nodC, also placed strain EK05(T) clearly in a novel branch. On the basis of its phylogenetic distinctiveness, we propose Bradyrhizobium iriomotense sp. nov. for strain EK05(T). The type strain is EK05(T) (= NBRC 102520(T) = LMG 24129(T)).

Candida phangngensis sp. nov., an anamorphic yeast species in the Yarrowia clade, isolated from water in mangrove forests in Phang-Nga Province, Thailand.

Two yeast strains (TM2-16 and PT1-17(T)) were isolated by membrane filtration from samples of estuarine water collected from two mangrove forests, in Khao Lumpee-Haad Thaimueang National Park and Mu Ko Ra-Ko Prathong National Park, Phang-Nga Province, Thailand. Analysis of the D1/D2 domain of the large-subunit rDNA sequences revealed that the sequences of the two strains were identical. The closest species in terms of pairwise sequence similarity was Candida galli, but the level of nucleotide substitutions (13.2 %) was sufficient to justify the description of a separate species. Phylogenetic analysis demonstrated that the two strains occupy a basal position with respect to Yarrowia lipolytica and C. galli of the Yarrowia clade, supported by a high bootstrap value. The two strains showed identical phenotypic characteristics, including proliferation by multilateral budding, absence of ascospores and ballistoconidia and negative Diazonium blue B and urease reactions. The major ubiquinone was Q-9. On the basis of the above findings, these two strains were assigned to a single novel species of the genus Candida, for which the name Candida phangngensis sp. nov. is proposed. The type strain is PT1-17(T) (=BCC 21306(T) =NBRC 101970(T) [corrected] =CBS 10407(T)).

Molecular cloning and expression of two novel beta-N-acetylglucosaminidases from silkworm Bombyx mori.

Beta-N-acetylglucosaminidase is a major glycosidase involved in several physiological processes, such as fertilization, metamorphosis, glycoconjugate degradation, and glycoprotein biosynthesis in insects. A search using the Bombyx mori cDNA database revealed the existence of two putative beta-N-acetylglucosaminidase genes. Their full-length cDNAs were cloned by rapid amplification of cDNA ends and polymerase chain reaction using specific primers, and named BmGlcNAcase1 and BmGlcNAcase2. A BLAST search revealed that BmGlcNAcase1 and BmGlcNAcase2 are homologous to a beta-subunit homolog encoded by Drosophila melanogaster HEXO2 and the Spodoptera frugiperda beta-N-acetylglucosaminidase gene respectively. The recombinant proteins of BmGlcNAcase1 and BmGlcNAcase2 without putative transmembrane domains were expressed in the yeast Pichia pastoris. Both enzymes showed broad substrate specificity, and cleaved terminal N-acetylglucosamine residues from the alpha-3 and alpha-6 branches of a biantennary N-glycan substrate, and also hydrolyzed chitotriose to chitobiose.

Production of dengue 2 envelope domain III in plant using TMV-based vector system.

The envelope protein of dengue virus is the major protein involved in host cell receptor binding for viral entry and induction of immunity. A gene fragment encoding domain III of the dengue 2 envelope protein (D2EIII, amino acids 298-400) was successfully expressed in Nicotinana benthamiana plant using a tobacco mosaic virus (TMV)-based transient expression system. The N-terminal 5' untranslated region-omega sequence located upstream of D2EIII increased protein production in infected plant tissues. The recombinant protein was reactive with anti-D2EIII polyclonal and anti-His tag antibodies. The intramuscular immunization of mice with D2EIII induced the production of the anti-dengue virus antibody. The induced antibody demonstrated neutralizing activity against dengue type 2 virus. The result indicates that the TMV expression system produces the dengue virus antigen in plant, which possesses appropriate antigenicity and immunogenicity.

Production of mouse monoclonal antibody with galactose-extended sugar chain by suspension cultured tobacco BY2 cells expressing human beta(1,4)-galactosyltransferase.

Previously, we developed a transgenic tobacco BY2 cell line (GT6) in which glycosylation was modified by expressing human beta(1,4)-galactosyltransferase (betaGalT). In this study, we produced a mouse monoclonal antibody in GT6 cells, and determined the sugar chain structures of plant-produced antibodies. Galactose-extended N-linked glycans comprised 16.7%, and high-mannose-type and complex-type glycans comprised 38.5% and 35.0% of the total number of glycans, respectively. N-linked glycans with the plant-specific sugars beta(1,2)-xylose and alpha(1,3)-fucose comprised 9.8%. The introduction of human betaGalT into suspension cultured tobacco cells resulted in the production of recombinant proteins with galactose-extended glycans and decreased contents of beta(1,2)-xylose and alpha(1,3)-fucose.

Labrys okinawensis sp. nov. and Labrys miyagiensis sp. nov., budding bacteria isolated from rhizosphere habitats in Japan, and emended descriptions of the genus Labrys and Labrys monachus.

Three strains, MAFF 210191(T), G24103(T) and G24116, assumed to be members of two novel species, were isolated from several rhizosphere habitats in different parts of Japan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolates formed a distinct monophyletic group together with the two known species of the genus Labrys, suggesting that the isolates have a close affiliation with this genus. In this study, a polyphasic approach was used to characterize and compare the three isolates with the two species of the genus Labrys, Labrys monachus and Labrys methylaminiphilus. All three isolates were aerobic, Gram-negative, motile and non-sporulating and they ranged in shape from spherical to short rods. The cells multiplied by budding and utilized a wide variety of monosaccharides, disaccharides and sugar alcohols as sole carbon and energy sources, but they did not utilize C(1) compounds, salicin or d-melezitose. The strains were inhibited by dl-alpha-alanine and glycine (both at 10 mM). The major cellular fatty acids were C(19 : 0) cyclo omega8c, C(16 : 0), C(18 : 0) and C(18 : 1)omega7c. The three isolates shared <12 % and <11 % DNA-DNA relatedness with L. monachus DSM 5896(T) and L. methylaminiphilus DSM 16812(T), respectively. The G+C content of the isolates (61-62 mol%) was also significantly lower than those of the two previously characterized species. In spite of many morphological, physiological and chemotaxonomic similarities among the three isolates, strain MAFF 210191(T) could be differentiated from strains G24103(T) and G24116 on the basis of 16S rRNA gene sequence divergence, DNA-DNA relatedness (<46 %) and gelatin hydrolysis. Two novel species are therefore proposed, namely Labrys okinawensis sp. nov., with the type strain MAFF 210191(T) (=DSM 18385(T)), and Labrys miyagiensis sp. nov., with the type strain G24103(T) (=NBRC 101365(T)=NCIMB 14143(T)) and also including strain G24116 (=NBRC 101366=NCIMB 14144). Emended descriptions of the genus Labrys and Labrys monachus are also presented.

Candida thaimueangensis sp. nov., an anamorphic yeast species from estuarine water in a mangrove forest in Thailand.

Four yeast strains (TM1-01(T), TM1-07, TM3-47 and TM3-49) were isolated by membrane filtration from estuarine water collected from a mangrove forest in Phang-Nga province, southern Thailand. Analysis of the D1/D2 domains of the large-subunit rDNA sequence revealed that the sequences of the four strains were identical. The closest recognized species in terms of pairwise sequence similarity was Pichia deserticola, but the level of nucleotide substitution (4.8 %) was sufficient to justify the description of a separate species. Phylogenetic analysis demonstrated that the four strains occupy a basal position with respect to Pichia membranifaciens and close relatives. The four strains showed identical phenotypic characteristics, including proliferation by multilateral budding, absence of ascospores, arthrospores and ballistospores and negative reactions for Diazonium blue B and urease. The major ubiquinone was Q-7. On the basis of the above findings, these four strains were assigned to a single novel species of the genus Candida, for which the name Candida thaimueangensis sp. nov. is proposed. The type strain is TM1-01(T) (=CBS 10360(T)=NBRC 101967(T)=BCC 21229(T)).

Kazachstania siamensis sp. nov., an ascomycetous yeast species from forest soil in Thailand.

Two strains (S-34T and S-35) of a novel ascomycetous yeast species belonging to the genus Kazachstania were isolated from soil from a mixed deciduous forest in Amphoe Wang Nam Khiao, Nakhon Ratchasima Province, Thailand. The D1/D2 domains of the large-subunit rDNA sequences of the two strains were identical and also indicated a close relationship with respect to Kazachstania aquatica, Kazachstania unispora, Kazachstania aerobia, Kazachstania servazzii and Kazachstania solicola. The most closely related species, K. aquatica, has 14 nucleotide substitutions and three gaps in 566 nt. The phenotypic characteristics of the two strains were typical of those of members of the genus Kazachstania. These characteristics include the formation of a single globose ascospore in an unconjugated and persistent ascus, multilateral budding, the absence of arthrospores and ballistospores, the fermentation of glucose, the inability to assimilate nitrate, negative diazonium blue B and urease reactions, and the presence of ubiquinone Q-6. The novel strains can be distinguished from K. aquatica on the basis of a number of phenotypic characteristics and represent a novel species in the genus Kazachstania, for which the name Kazachstania siamensis sp. nov. is proposed. The type strain is S-34T (=CBS 10361T=NBRC 101968T=BCC 21230T).

Molecular diversity of bamboo-associated fungi isolated from Japan.

Bamboos are common and useful plants in Japan but little information is available about their endophytes. In this study, 257 fungal strains were isolated from bamboo tissues, and 71 representative strains were characterized by 18S rRNA gene and internal transcriber spacer region sequence analysis. Phylogenetic analysis showed that the fungal isolates were located in Sordariomycetes and Dothideomycetes. Xylariales was the dominant group within bamboos. Several rRNA gene sequences were not similar to any current sequence in the database and might be a novel species or genera that could represent sources of novel biological compounds. These findings reveal that bamboos are a huge reservoir of microorganisms that should be extensively investigated.

Enhancement of recombinant soluble dengue virus 2 envelope domain III protein production in Escherichia coli trxB and gor double mutant.

The dengue virus is currently the most important flavivirus causing human diseases in the tropical and subtropical regions of the world. The envelope protein domain III of dengue virus type 2 (D2EIII), which induces protective and neutralizing antibodies, was expressed as an N-terminal fusion to a hexa-histidine tag in Escherichia coli. The expression of recombinant D2EIII of 103 amino acids in the soluble form can be achieved using suitable host strains, such as Origami, at a low induction temperature of 18 degrees C. The enhanced production of the soluble protein could be attributed to the thioredoxin reductase (trxB) and glutathione reductase (gor) double mutations in the Origami genome. The soluble and refolded D2EIII proteins were recognized by different antibodies including human patient antiserum. The immunization of rats with soluble D2EIII protein elicited the production of antibodies that could recognize the D2EIII protein in the D2EIII precursor protein and in C-terminal truncated dengue envelope protein type 1-4. Thus, this protein production system is suitable for the production of authentic recombinant dengue proteins that may be used in the diagnosis of the dengue virus infection or in vaccine development.

Cloning and characterization of a novel gene involved in nitrogen fixation in Heliobacterium chlorum: a possible regulatory gene.

In the present study, the transcriptional properties of the nitrogen fixation gene cluster of Hbt. chlorum, a strictly anaerobic, gram-positive, phototrophic bacterium, were explored. The cluster consisted of eleven genes in the same orientation in the order nifI ( 1 ) , nifI ( 2 ) , nifH, nifD, nifK, nifE, nifN, nifX, fdx, nifB, and nifV as detected previously. An open reading frame (orf1) preceding these genes was revealed by further cloning. The orf1 was co-transcribed with downstream nif genes in a single polycistronic transcript, the transcription start site (TSS) was located upstream of the orf1, and a putative promoter was identified 10 bp preceding the TSS. Unlike most diazotrophs which have a sigma(54)-dependent -24/-12 promoter, the promoter was similar to the -35/-10 E. coli promoter. The orf1 had no nif homolog in DNA databases, and the highest level of identity (27% at amino acid level) was found with hutP, a positive regulatory gene of the histidine utilization (hut) operon in B. subtilis. Analogous to the regulatory mechanism of the hut operon in B. subtilis, it is conceivable that the orf1 product interacts with the terminator-like structure located downstream of the orf1 during N-deficient condition and prevents transcription termination; thus, the transcription continues into the nif structural genes.

In Planta production of immunogenic poliovirus peptide using tobacco mosaic virus-based vector system.

The tobacco mosaic virus (TMV) provides an attractive means of producing foreign peptides in plants. In this study, a TMV-based vector was designed such that a fragment encoding 15 amino acids of the poliovirus peptide (PVP) derived from the viral capsid proteins VP3 and VP1 of poliovirus type 1 Sabin was inserted downstream of the six-base 3' context nucleotide sequence of the TMV coat protein (CP) gene. This design allowed readthrough at the amber stop codon, thereby producing the chimeric TMV particle with both intact CP and CP-fusion protein (CP-PVP) in Nicotiana tabacum cv. Samsun infected with the TMV vector. The TMVCP-PVP virus particle induced antibodies against PVP as well as TMVCP in mice after intraperitoneal immunization. These data illustrate the potential of the readthrough translation system with TMVCP for antigen presentation and vaccine production.

N-linked glycan structures of a mouse monoclonal antibody produced from tobacco BY2 suspension-cultured cells.

cDNA encoding H- and L-chains from a mouse monoclonal antibody was introduced into tobacco BY2 cells, and the resulting sugar chain structures of plant-produced antibodies were analyzed by a combination of HPLC, exoglucosidase digestion and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The glycan structures determined were Man(5-6)GlcNAc2 (22.3%), GlcNAcMan5GlcNAc2 (3.1%), GlcNAcMan3FucXylGlcNAc2 (24.4%), GlcNAcMan3XylGlcNAc2 (17.8%), Man3FucXylGlcNAc2 (24.3%), and Man3XylGlcNAc2 (8.1%). The major glycan structures of the antibodies produced by transgenic suspension-cultured cells contain typical plant bisecting beta(1,2)-xylose and alpha(1,3)-fucose residues, suggesting the posttranslational modification of a recombinant antibody in the late Golgi apparatus.

Molecular evolution of the nif gene cluster carrying nifI1 and nifI2 genes in the Gram-positive phototrophic bacterium Heliobacterium chlorum.

A major nif cluster was detected in the strictly anaerobic, Gram-positive phototrophic bacterium Heliobacterium chlorum. The cluster consisted of 11 genes arranged within a 10 kb region in the order nifI1, nifI2, nifH, nifD, nifK, nifE, nifN, nifX, fdx, nifB and nifV. The phylogenetic position of Hbt. chlorum was the same in the NifH, NifD, NifK, NifE and NifN trees; Hbt. chlorum formed a cluster with Desulfitobacterium hafniense, the closest neighbour of heliobacteria based on the 16S rRNA phylogeny, and two species of the genus Geobacter belonging to the Deltaproteobacteria. Two nifI genes, known to occur in the nif clusters of methanogenic archaea between nifH and nifD, were found upstream of the nifH gene of Hbt. chlorum. The organization of the nif operon and the phylogeny of individual and concatenated gene products showed that the Hbt. chlorum nif operon carrying nifI genes upstream of the nifH gene was an intermediate between the nif operon with nifI downstream of nifH (group II and III of the nitrogenase classification) and the nif operon lacking nifI (group I). Thus, the phylogenetic position of Hbt. chlorum nitrogenase may reflect an evolutionary stage of a divergence of the two nitrogenase groups, with group I consisting of the aerobic diazotrophs and group II consisting of strictly anaerobic prokaryotes.