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Find video protocols related to scientific articles indexed in Pubmed.
Improvement of glycosylation structure by suppression of ?-N-acetylglucosaminidases in silkworm.
J. Biosci. Bioeng.
PUBLISHED: 09-02-2014
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The baculovirus-silkworm recombinant protein expression system is an excellent method for achieving high-level expression and post-translational modifications, especially glycosylation. However, the presence of paucimannosidic-type N-glycan in glycoproteins restricts their clinical use. Paucimannosidic-type N-glycan is produced by insect-specific membrane-binding-type ?-N-acetylglucosaminidase (GlcNAcase). In the silkworm, BmGlcNAcase1, BmGlcNAcase2, and BmFDL are membrane-binding-type GlcNAcases. We investigated the localization of these GlcNAcases and found that BmFDL and BmGlcNAcase2 were mainly located in the fat body and hemolymph, respectively. The fat body is the main tissue of recombinant protein expression by baculovirus, and many glycoproteins are secreted into the hemolymph. These results suggest that inhibition of BmFDL and BmGlcNAcase2 could increase GlcNAc-type N-glycan levels. We therefore injected a GlcNAcase inhibitor into silkworms to investigate changes in the N-glycan structure of the glycoprotein expressed by baculovirus; modest levels of GlcNAc-type N-glycan were observed (0.8% of total N-glycan). Next, we generated a transgenic silkworm in which RNA interference (RNAi) reduced the BmFDL transcript level and enzyme activity to 25% and 50%, respectively, of that of the control silkworm. The proportion of GlcNAc-type N-glycan increased to 4.3% in the RNAi-transgenic silkworm. We conclude that the structure of N-glycan can be changed by inhibiting the GlcNAcases in silkworm.
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Cloning of a cDNA encoding the Gly m Bd 28K precursor and its vacuole transport in tobacco BY2 suspension-cultured cells.
J. Nutr. Sci. Vitaminol.
PUBLISHED: 07-01-2014
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Gly m Bd 28K (Gm28K), a soybean allergen, is formed as a preproprotein consisting of a predicted signal peptide, Gm28K, and the 23-kDa peptide (Gm23K). Gm28K and Gm23K are found in the protein-storage vacuoles (PSVs) of developing soybean seeds. However, the complete structure of Gm28K has not yet been identified and its processing and transport to the vacuoles has never been clarified. In the present study, we elucidated the 5'-nucleotide sequence of cDNA encoding the Gm28K precursor and identified a putative signal peptide (SP) with 24 N-terminal amino acid residues. We expressed peptides from the Gm28K precursor as fusion proteins with enhanced green fluorescent protein (EGFP) in tobacco BY2 suspension-cultured cells. BY2 cells transformed by an expression vector for SP-EGFP-Gm28-Gm23K (SP-EGFP-Gm28-Gm23K/BY2 cells) and SP-Gm28-Gm23K-EGFP/BY2 cells produced the EGFP fused-Gm28K precursor, and the EGFP-fluorescence in their vacuoles were recorded. In the experiments with SP-EGFP/BY2 and SP-EGFP-Gm28K/BY2 cells, large amounts of the EGFP segments were secreted into the medium. On the other hand, the fluorescence of EGFP in SP-EGFP-Gm23K/BY2 cells was shown to accumulate only in the endoplasmic reticulum without secretion into the medium. These findings show that the SP signals the precursor to enter the lumen of the endoplasmic reticulum and that both the Gm28K and Gm23K components may be involved in the transport from the endoplasmic reticulum (ER) lumen via the Golgi to the vacuoles in a proprotein form.
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Barnettozyma siamensis f.a., sp. nov., a lipid-accumulating ascomycete yeast species.
Int. J. Syst. Evol. Microbiol.
PUBLISHED: 06-12-2014
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Two strains, DMKU-UbN24(1)(T) and DMKU-CPN24(1), of a novel yeast species were obtained from soil and palm oil fruit, respectively, collected in Thailand by an enrichment isolation technique using a nitrogen-limited medium containing glycerol as the sole source of carbon. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics and sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, the two strains were found to represent a novel species of the genus Barnettozyma although the formation of ascospores was not observed. The novel species was related most closely to the type strain of Candida montana but differed by 5.4?% nucleotide substitutions in the D1/D2 region of the LSU rRNA gene and by 10.3-10.5?% nucleotide substitutions in the ITS region. The name Barnettozyma siamensis f.a., sp. nov. is proposed. The type strain is DMKU-UbN24(1)(T) (?=?BCC 61189(T)?=?NBRC 109701(T)?=?CBS 13392(T)).
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Antibody germline characterization of cross-neutralizing human IgGs against 4 serotypes of dengue virus.
Biochem. Biophys. Res. Commun.
PUBLISHED: 02-12-2014
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Dengue virus (DENV), a re-emerging virus, constitutes the largest vector-borne disease virus, with 50-100 million cases reported every year. Although DENV infection induces lifelong immunity against viruses of the same serotypes, the subsequent infection with the heterologous serotypes can cause more severe form of the disease, such as Dengue Haemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS). However, there is neither approved vaccine nor specific drugs available to treat this disease. In this study, previously developed 19 human monoclonal antibodies (HuMAbs) showing strong to moderate cross neutralizing activity were selected. Most of them (13/19) were targeted to domain II of envelop glycoprotein. To understand and clarify the recognition properties, the maturation mechanisms comprising Variable/Diversity/Joining (VDJ) recombination, Variable Heavy (VH)/Variable Light (VL) chain pairing, variability at junctional site, and somatic hypermutation (SHM) of those antibodies were studied and compared with their predecessor germline sequences. IMGT/V-QUEST database was applied to analyze the isolated VH and VL sequences. To confirm the correction of isolated VH/VL, 3 HuMAbs (1A10H7, 1B3B9, 1G7C2) was transiently expressed in HEK293T cell. All three clones of the expressed recombinant IgG (rIgG) showed the same binding and neutralizing activity as same as those from hybridomas. The data obtained in this study will elucidate the properties of those HuMAbs for further genetic modification, and its binding epitopes.
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Identification of a protein glycosylation operon from Campylobacter jejuni JCM 2013 and its heterologous expression in Escherichia coli.
J. Biosci. Bioeng.
PUBLISHED: 02-01-2014
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Campylobacter jejuni is a human enteropathogenic bacterium possessing an N-glycosylation system. In this work, a protein glycosylation (pgl) operon conferring prokaryotic N-glycosylation in C. jejuni JCM 2013 was cloned and identified. Fourteen open reading frames (ORFs) were found in the pgl operon. The operon organization was similar to that of C. jejuni NCTC 11168, with 98% and 99% identities in overall nucleotide sequence and amino acid sequence, respectively. The pgl operon was heterologously co-expressed with model protein CmeA in the Escherichia coli BL21 ?waaL mutant. The immuno- and lectin-blotting analysis indicated the protein glycosylation on the recombinant CmeA. In addition, to analyze the glycan composition, the recombinant CmeA was purified and subjected to in-gel trypsin digestion followed by mass spectrometry analysis. The mass spectrometry analysis showed the presence of the N-acetylhexosamine residue at the reducing end but not the predicted di-N-acetylbacillosamine (diNAcBac) residue. Further glycan structural study using the conventional fluorophore-labeling method revealed the GalNAc?-GalNAc?-(Hex-)HexNAc-HexNAc-HexNAc-HexNAc structure. Transcriptional analysis showed that UDP-diNAcBac synthases and diNAcBac transferase are transcribed but might not function in the constructed system. In conclusion, a pgl operon from C. jejuni JCM 2013 successfully functioned in E. coli, resulting in the observed prokaryotic glycosylation.
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N-glycosylation and N-glycan moieties of CTB expressed in rice seeds.
Mol. Biotechnol.
PUBLISHED: 11-05-2013
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Cholera toxin B subunit (CTB) is widely used as a carrier molecule and mucosal adjuvant and for the expression of fusion proteins of interest. CTB-fusion proteins are also expressed in plants, but the N-glycan structures of CTB have not been clarified. To gain insights into the N-glycosylation and N-glycans of CTB expressed in plants, we expressed CTB in rice seeds with an N-terminal glutelin signal and a C-terminal KDEL sequence and analyzed its N-glycosylation and N-glycan structures. CTB was successfully expressed in rice seeds in two forms: a form with N-glycosylation at Asn32 that included both plant-specific N-glycans and small oligomannosidic N-glycans and a non-N-glycosylated form. N-Glycan analysis of CTB showed that approximately 50 % of the N-glycans had plant-specific M3FX structures and that almost none of the N-glycans was of high-mannose-type N-glycan even though the CTB expressed in rice seeds contains a C-terminal KDEL sequence. These results suggest that the CTB expressed in rice was N-glycosylated through the endoplasmic reticulum (ER) and Golgi N-glycosylation machinery without the ER retrieval.
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Transgenic rice seeds accumulating recombinant hypoallergenic birch pollen allergen Bet v 1 generate giant protein bodies.
Plant Cell Physiol.
PUBLISHED: 03-28-2013
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A versatile hypoallergenic allergen derivative against multiple allergens is an ideal tolerogen for allergen-specific immunotherapy. Such a tolerogen should exhibit high efficacy, without side effects, when administered at high doses and should be applicable to several allergens. Tree pollen chimera 7 (TPC7), a hypoallergenic Bet v 1 tolerogen against birch pollen allergy, was previously selected by DNA shuffling of 14 types of Fagales tree pollen allergens. In this study, transgenic rice seed accumulating TPC7 was generated as an oral vaccine against birch pollen allergy by expressing this protein as a secretory protein using the N-terminal signal peptide and the C-terminal KDEL tag under the control of an endosperm-specific glutelin promoter. The highest level of TPC7 accumulation was approximately 207 µg grain(-1). Recombinant TPC7 is a glycoprotein with high mannose-type N-glycan, but without ?1,2-xylose or ?1,3-fucose, suggesting that TPC7 is retained in the endoplasmic reticulum (ER). TPC7 is deposited as a novel, giant spherical ER-derived protein body, >20 µm in diameter, which is referred to as the TPC7 body. Removal of the KDEL retention signal or mutation of a cysteine residue resulted in an alteration of TPC7 body morphology, and deletion of the signal peptide prevented the accumulation of TPC7 in rice seeds. Therefore, the novel TPC7 bodies may have formed aggregates within the ER lumen, primarily due to the intrinsic physicochemical properties of the protein.
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Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks.
PLoS Negl Trop Dis
PUBLISHED: 03-07-2013
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Cholera toxin B subunit (CTB) is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale) hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants.
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Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection.
Antiviral Res.
PUBLISHED: 02-13-2013
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Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab)2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection.
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Human monoclonal antibodies broadly neutralizing against influenza B virus.
PLoS Pathog.
PUBLISHED: 02-07-2013
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Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus.
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Molecular cloning and characterization of the ?-glucosidase II from Bombyx mori and Spodoptera frugiperda.
Insect Biochem. Mol. Biol.
PUBLISHED: 01-12-2013
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The ?-glucosidase II (GII) is a heterodimer of ?- and ?-subunits and important for N-glycosylation processing and quality control of nascent glycoproteins. Although high concentration of ?-glucosidase inhibitors from mulberry leaves accumulate in silkworms (Bombyx mori) by feeding, silkworm does not show any toxic symptom against these inhibitors and N-glycosylation of recombinant proteins is not affected. We, therefore, hypothesized that silkworm GII is not sensitive to the ?-glucosidase inhibitors from mulberry leaves. However, the genes for B. mori GII subunits have not yet been identified, and the protein has not been characterized. Therefore, we isolated the B. mori GII ?- and ?-subunit genes and the GII ?-subunit gene of Spodoptera frugiperda, which does not feed on mulberry leaves. We used a baculovirus expression system to produce the recombinant GII subunits and identified their enzyme characteristics. The recombinant GII ?-subunits of B. mori and S. frugiperda hydrolyzed p-nitrophenyl ?-d-glucopyranoside (pNP-?Glc) but were inactive toward N-glycan. Although the B. mori GII ?-subunit was not required for the hydrolysis of pNP-?Glc, a B. mori GII complex of the ?- and ?-subunits was required for N-glycan cleavage. As hypothesized, the B. mori GII ?-subunit protein was less sensitive to ?-glucosidase inhibitors than was the S. frugiperda GII ?-subunit protein. Our observations suggest that the low sensitivity of GII contributes to the ability of B. mori to evade the toxic effect of ?-glucosidase inhibitors from mulberry leaves.
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Emerging antigenic variants at the antigenic site Sb in pandemic A(H1N1)2009 influenza virus in Japan detected by a human monoclonal antibody.
PLoS ONE
PUBLISHED: 01-01-2013
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The swine-origin pandemic A(H1N1)2009 virus, A(H1N1)pdm09, is still circulating in parts of the human population. To monitor variants that may escape from vaccination specificity, antigenic characterization of circulating viruses is important. In this study, a hybridoma clone producing human monoclonal antibody against A(H1N1)pdm09, designated 5E4, was prepared using peripheral lymphocytes from a vaccinated volunteer. The 5E4 showed viral neutralization activity and inhibited hemagglutination. 5E4 escape mutants harbored amino acid substitutions (A189T and D190E) in the hemagglutinin (HA) protein, suggesting that 5E4 recognized the antigenic site Sb in the HA protein. To study the diversity of Sb in A(H1N1)pdm09, 58 viral isolates were obtained during the 2009/10 and 2010/11 winter seasons in Osaka, Japan. Hemagglutination-inhibition titers were significantly reduced against 5E4 in the 2010/11 compared with the 2009/10 samples. Viral neutralizing titers were also significantly decreased in the 2010/11 samples. By contrast, isolated samples reacted well to ferret anti-A(H1N1)pdm09 serum from both seasons. Nonsynonymous substitution rates revealed that the variant Sb and Ca2 sequences were being positively selected between 2009/10 and 2010/11. In 7,415 HA protein sequences derived from GenBank, variants in the antigenic sites Sa and Sb increased significantly worldwide from 2009 to 2013. These results indicate that the antigenic variants in Sb are likely to be in global circulation currently.
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Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of ?2,6-sialyltransferase derived from Chinese hamster ovary cells.
Appl. Microbiol. Biotechnol.
PUBLISHED: 10-14-2011
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Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies.
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Double-knockout of putative endo-?-N-acetylglucosaminidase (ENGase) genes in Arabidopsis thaliana: loss of ENGase activity induced accumulation of high-mannose type free N-glycans bearing N,N-acetylchitobiosyl unit.
Biosci. Biotechnol. Biochem.
PUBLISHED: 05-20-2011
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Endo-?-N-acetylglucosaminidase (ENGase) is involved in the production of high-mannose type free N-glycans during plant development and fruit maturation. In a previous study (K. Nakamura et al. Biosci. Biotechnol. Biochem., 73, 461-464 (2009)), we identified the tomato ENGase gene and found that gene expression remained relatively constant. In the present study, we constructed an Arabidopsis thaliana mutant in which the expression of two putative ENGase genes was suppressed. The mutant showed no ENGase activity, but produced high-mannose type free N-glycans carrying the N,N-acetylchitobiosyl unit that is produced by peptide:N-glycanase, indicating that both these genes encode Arabidopsis ENGase.
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Reduced immunogenicity of Arabidopsis hgl1 mutant N-glycans caused by altered accessibility of xylose and core fucose epitopes.
J. Biol. Chem.
PUBLISHED: 04-08-2011
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Arabidopsis N-glycosylation mutants with enhanced salt sensitivity show reduced immunoreactivity of complex N-glycans. Among them, hybrid glycosylation 1 (hgl1) alleles lacking Golgi ?-mannosidase II are unique, because their glycoprotein N-glycans are hardly labeled by anti-complex glycan antibodies, even though they carry ?1,2-xylose and ?1,3-fucose epitopes. To dissect the contribution of xylose and core fucose residues to plant stress responses and immunogenic potential, we prepared Arabidopsis hgl1 xylT double and hgl1 fucTa fucTb triple mutants by crossing previously established T-DNA insertion lines and verified them by mass spectrometry analyses. Root growth assays revealed that hgl1 fucTa fucTb but not hgl1 xylT plants are more salt-sensitive than hgl1, hinting at the importance of core fucose modification and masking of xylose residues. Detailed immunoblot analyses with anti-?1,2-xylose and anti-?1,3-fucose rabbit immunoglobulin G antibodies as well as cross-reactive carbohydrate determinant-specific human immunoglobulin E antibodies (present in sera of allergy patients) showed that xylose-specific reactivity of hgl1 N-glycans is indeed reduced. Based on three-dimensional modeling of plant N-glycans, we propose that xylose residues are tilted by 30° because of untrimmed mannoses in hgl1 mutants. Glycosidase treatments of protein extracts restored immunoreactivity of hgl1 N-glycans supporting these models. Furthermore, among allergy patient sera, untrimmed mannoses persisting on the ?1,6-arm of hgl1 N-glycans were inhibitory to immunoreaction with core fucoses to various degrees. In summary, incompletely trimmed glycoprotein N-glycans conformationally prevent xylose and, to lesser extent, core fucose accessibility. Thus, in addition to N-acetylglucosaminyltransferase I, Golgi ?-mannosidase II emerges as a so far unrecognized target for lowering the immunogenic potential of plant-derived glycoproteins.
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N-terminal vacuolar sorting signal at the mouse antibody alters the N-linked glycosylation pattern in suspension-cultured tobacco BY2 cells.
J. Biosci. Bioeng.
PUBLISHED: 02-14-2011
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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 ?1,3-fucosylation and/or ?1,2-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 Man?FucXylGlcNAc? 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 Man?FucXylGlcNAc?, which is the main N-glycan structure in vacuoles.
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Improved expression and characterization of recombinant human Golgi ?1,2-mannosidase I isoforms (IA2 and IC) by Escherichia coli.
J. Biosci. Bioeng.
PUBLISHED: 02-02-2011
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Golgi ?1,2-mannosidase I is involved in the N-linked oligosaccharide processing pathway. In this study, two truncated genes encoding for human Golgi ?1,2-mannosidase I (hManIA2: amino acids 127-626 and hManIC: amino acids 118-617) were expressed in Escherichia coli to characterize the enzymes. These genes were fused to a T7 protein tag and a histidine tag at the N- and C-terminal ends, respectively, and purified using Co(2+) affinity chromatography. The properties including optimal temperature, optimal pH, and substrate specificity of the purified enzymes were investigated by HPLC using pyridylamino (PA)-labeled oligosaccharides as substrates. The stability of hManIA2 was dependent on the presence of Ca(2+), which was also required for its activity. On the other hand, hManIC was stable in the absence of Ca(2+), even though Ca(2+) was also effective for the activity of hManIC. While the similarity of the amino acid sequences is over 60%, hManIA2 and hManIC showed different substrate specificities particularly toward M9A and M8C.
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Arabidopsis ?1,2-xylosyltransferase: substrate specificity and participation in the plant-specific N-glycosylation pathway.
J. Biosci. Bioeng.
PUBLISHED: 01-31-2011
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?1,2-Xylosyltransferase (XYLT) is a plant-specific glycosyltransferase that contributes to the biosynthesis of N-glycoproteins in plants. However, the specificity of XYLT for N-glycans has not yet been completely clarified. To gain insights into the function of XYLT in the plant N-glycosylation pathway, we examined the acceptor substrate specificity of recombinant Arabidopsis XYLT (AtXYLT) using 2-aminopyridine-labeled N-glycans as the substrates and confirmed the N-glycans of Arabidopsis xylt mutant. Recombinant AtXYLT expressed in insect cells required the ?1,2-linked N-acetylglucosamine (GlcNAc) residue at the nonreducing terminus of the ?1,3-branched mannose (Man) residue (GlcNAc?1,2-Man?1,3-Man; GNM3B) for activity. However, AtXYLT showed decreased activity with substrates that contained ?1,3-fucose at the chitobiose core-GlcNAc or a terminal GlcNAc at the ?1,6-branched Man residue of GlcNAc?1,2-Man (GlcNAc?1,2-Man?1,6-Man; GNM3A), whose ratios were 10% and 50% of the optimal substrate, GNM3B, respectively. Moreover, AtXYLT did not show any activity in the transfer of the Xyl residue to N-glycans that contained a mammalian-type ?1,4-linked galactose (Gal) residue at the nonreducing terminus of GlcNAc?1,2-Man. These results indicate that a ?1,2-linked GlcNAc residue at the nonreducing terminus of an ?1,3-branched Man residue is necessary for AtXYLT activity and that mammalian-type ?1,4-linked Gal residue(s) on the same branch completely inhibit(s) the activity. Furthermore, N-glycan analysis showed that approximately 30% of the N-glycans carry the Xyl residue in the wild type. These findings suggest that AtXYLT acts on protein-bound N-glycans prior to ?1,3-fucosyltransferase and mannosidase II in planta.
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Jack bean ?-mannosidase digestion profile of hybrid-type N-glycans: effect of reaction pH on substrate preference.
Biochimie
PUBLISHED: 01-13-2011
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Jack bean ?-mannosidase (JBM) is a well-studied plant vacuolar ?-mannosidase, and is widely used as a tool for the enzymatic analysis of sugar chains of glycoproteins. In this study, the JBM digestion profile of hybrid-type N-glycans was examined using pyridylamino (PA-) sugar chains. The digestion efficiencies of the PA-labeled hybrid-type N-glycans Man?1,6(Man?1,3)Man?1,6(GlcNAc?1,2Man?1,3)Man?1,4GlcNAc?1,4GlcNAc-PA (GNM5-PA) and Man?1,6(Man?1,3)Man?1,6(Gal?1,4GlcNAc?1,2Man?1,3)Man?1,4GlcNAc?1,4GlcNAc-PA (GalGNM5-PA) were significantly lower than that of the oligomannose-type N-glycan Man?1,6(Man?1,3)Man?1,6Man?1,4GlcNAc?1,4GlcNAc-PA (M4-PA), and the trimming pathways of GNM5-PA and GalGNM5-PA were different from that of M4-PA, suggesting a steric hindrance to the JBM activity caused by GlcNAc?1-2Man(?) residues of the hybrid-type N-glycans. We also found that the substrate preference of JBM for the terminal Man?1-6Man(?) and Man?1-3Man(?) linkages in the hybrid-type N-glycans was altered by the change in reaction pH, suggesting a pH-dependent change in the enzyme-substrate interaction.
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Stable coexpression of two human sialylation enzymes in plant suspension-cultured tobacco cells.
J. Biosci. Bioeng.
PUBLISHED: 01-08-2011
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Human CMP-N-acetylneuraminic acid (NeuAc) synthase (hCSS) and ?2,6-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 ?2,6-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.
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Arabidopsis thaliana ALG3 mutant synthesizes immature oligosaccharides in the ER and accumulates unique N-glycans.
Glycobiology
PUBLISHED: 03-08-2010
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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.
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Cloning and characterization of a ?-N-acetylglucosaminidase (BmFDL) from silkworm Bombyx mori.
J. Biosci. Bioeng.
PUBLISHED: 01-29-2010
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In insects, ?-N-acetylglucosaminidase (GlcNAcase) participates in critical physiological processes such as fertilization, metamorphosis, and glycoconjugate degradation. Insects produce glycoproteins carrying paucimannosidic-type N-glycans, the terminal GlcNAc residue of which is cleaved by a GlcNAc-linkage specific GlcNAcase, also known as the fused lobes (FDL) protein. To obtain information on the structure of GlcNAcases and insight into their contribution to physiological processes, we cloned Bombyx mori FDL (BmFDL) from silkworm larvae. The full-length cDNA (1.9 kb) encoded a protein of 633 amino acids with 42% amino acid sequence identity to Drosophila melanogaster FDL (DmFDL). Recombinant BmFDL cleaved only ?-1,2-linked GlcNAc residues from the ?-1,3 branch of biantennary N-glycan. This substrate specificity was similar to that of DmFDL. Microsomal FDL activity was inhibited by anti-BmFDL antibodies. Taken together, our results suggest that BmFDL is a N-glycan-processing GlcNAcase in B. mori.
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Two Arabidopsis thaliana Golgi alpha-mannosidase I enzymes are responsible for plant N-glycan maturation.
Glycobiology
PUBLISHED: 11-12-2009
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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.
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Geotrichum siamensis sp. nov. and Geotrichum phurueaensis sp. nov., two asexual arthroconidial yeast species isolated in Thailand.
FEMS Yeast Res.
PUBLISHED: 08-26-2009
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Two asexual arthroconidial yeast strains, TM3-44(T) and LYSM5(T), were isolated, respectively, from estuarine water in a mangrove forest and soil in a terrestrial forest in Thailand. Analysis of the D1/D2 domains of the large-subunit rRNA gene sequences revealed that strain TM3-44(T) differed from the closest species in terms of pairwise sequence similarity, Dipodascus albidus, by 11.7% nucleotide substitutions, while strain LYSM5(T) was closest to Galactomyces geotrichum with only 2.9% nucleotide substitutions. The phylogenetic tree further demonstrated that strain TM3-44(T) was at a distant position from the closest species, D. albidus, and other related species in the Dipodascus clade, while strain LYSM5(T) clustered with G. geotrichum, it closest relative in the Galactomyces clade. The phenotypic characteristics of the two strains were typical of the genus Geotrichum. On the basis of the above findings, strain TM3-44(T) was assigned as a novel species of Geotrichum, for which the name Geotrichum siamensis sp. nov. is proposed. The type strain is TM3-44(T) (BCC 29903(T)=NBRC 104880(T)=CBS 10929(T)). Strain LYSM5(T) represented another novel species of Geotrichum, which was named Geotrichum phurueaensis sp. nov. The type strain is LYSM5(T) (BCC 34756(T)=NBRC 105674(T)=CBS 11418(T)).
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The impact of the overexpression of human UDP-galactose transporter gene hUGT1 in tobacco plants.
J. Biosci. Bioeng.
PUBLISHED: 06-22-2009
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When the human UDP-galactose transporter 1 gene (hUGT1) was introduced into tobacco plants, the plants displayed enhanced growth during cultivation, and axillary shoots had an altered determinate growth habit, elongating beyond the primary shoots and having a sympodial growth pattern similar to that observed in tomatoes at a late cultivation stage. The architecture and properties of tissues in hUGT1-transgenic plants were also altered. The leaves had an increase in thickness, due to an increased amount of spongy tissue, and a higher content of chlorophyll a and b; the stems had an increased number of xylem vessels and accumulated lignin and arabinogalactan proteins (AGPs). Some of these characteristics resembled a gibberellin (GA)-responsive phenotype, suggesting involvement of GA. RT-PCR-based analysis of genes involved in GA biosynthesis suggested that the GA biosynthetic pathway was not activated. However, an increase in the proportion of galactose in polysaccharide side chains of AGPs was detected. These results suggested that because of higher UDP-galactose transport from the cytosol to the Golgi apparatus, galactose incorporation into polysaccharide side chains of AGP is involved in the gibberellin response, resulting in morphological and architectural changes.
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O-glycosylation of protein subpopulations in alcohol-extracted rice proteins.
J. Plant Physiol.
PUBLISHED: 06-11-2009
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Mucin-type O-glycosylation has been well characterized in mammalian systems but not in plants. In this study, the purified alcohol-soluble, non-reduced protein (prolamin) fraction from rice seed was investigated for the occurrence of O-linked oligosaccharides. As storage prolamins are unlikely to be O-glycosylated, any O-glycosylation found was likely to belong to co-extracted proteins, whether because of association with the protein body or solubility. SDS-PAGE and MS analyses revealed 14 and 16kDa protein families in fractions that bound to the lectins peanut agglutinin (PNA), Vicia villosa lectin (VVL) and Jacalin, indicative of the presence of O-linked saccharides. Enzymatic cleavage, fluorescent labeling and high-performance liquid chromatography (HPLC) analysis demonstrated a peak consistent with Gal-beta-(1-->3)-GalNAc, with similar MS/MS fragmentation. Additionally, upon chemical analysis, a GlcNAc-containing O-linked carbohydrate moiety was discovered. Protein blotting with anti-O-GlcNAc antibody (clone CTD110.6) was positive in a subpopulation of the 14kDa alcohol-soluble protein fraction, but a hot capping experiment was negative. Therefore, the GlcNAc residue in this case is unlikely to be terminal. Additionally, a positive reaction with CTD110.6mAb cannot be taken as absolute proof of O-GlcNAc modification and further confirmatory experiments should be employed. We hypothesize that O-glycosylation may contribute to protein functionality or regulation. Further investigation is required to identify the specific proteins with these modifications. This reverse approach could lead to the identification of proteins involved in mRNA targeting, signaling, translation, anchoring or maintenance of translational quiescence and may be applied to germinating rice seed extracts for further elucidation of protein function and regulation.
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Glycosylation pattern of humanized IgG-like bispecific antibody produced by recombinant CHO cells.
Appl. Microbiol. Biotechnol.
PUBLISHED: 06-02-2009
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The glycosylation pattern of a humanized anti-EGFRxanti-CD3 bispecific single-chain diabody with an Fc portion (hEx3-scDb-Fc) produced by recombinant Chinese hamster ovary cells was evaluated and compared with those of a recombinant humanized anti-IL-8 antibody (IgG1) and human serum IgG. N-Linked oligosaccharide structures were estimated by two-dimensional high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. No sialylation was observed with purified hEx3-scDb-Fc and the anti-IL-8 antibody. From the analysis of neutral oligosaccharides, approximately more than 90% of the N-linked oligosaccharides of hEx3-scDb-Fc and the anti-IL-8 antibody were alpha-1,6-fucosylated. The galactosylated biantennary oligosaccharides comprise over 40% of the total N-linked oligosaccharides in both hEx3-scDb-Fc and the anti-IL-8 antibody. The fully galactosylated biantennary oligosaccharides from hEx3-scDb-Fc and the anti-IL-8 antibody accounted for only 10% of the N-linked; however, more than 20% of the N-linked oligosaccharides were fully galactosylated biantennary oligosaccharides in human serum IgG. The glycosylation pattern of hEx3-scDb-Fc was quite similar to that of the anti-IL-8 antibody.
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Recombinant expression and characterization of N-acetylglucosaminyltransferase I derived from Nicotiana tabacum.
J. Biosci. Bioeng.
PUBLISHED: 05-30-2009
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The C-terminal catalytic domain of tobacco N-acetylglucosaminyltransferase I fused to maltose-binding protein was produced in Escherichia coli as a soluble form with significant activity. The protein was affinity-purified using amylose resin, and its enzymatic properties were investigated, including its divalent cation requirements, optimal temperature, optimal pH, and substrate specificity.
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Cloning and characterization of cytidine monophosphate-3-deoxy-d-manno-octulosonate synthetase from Arabidopsis thaliana.
J. Biosci. Bioeng.
PUBLISHED: 05-28-2009
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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+.
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Wickerhamomyces edaphicus sp. nov. and Pichia jaroonii sp. nov., two ascomycetous yeast species isolated from forest soil in Thailand.
FEMS Yeast Res.
PUBLISHED: 02-18-2009
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Four yeast strains were isolated from soil in a mixed deciduous forest in Amphoe Wang Nam Khiao, Nakhon Ratchasima province, Thailand. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analyses of the D1/D2 domain of the large-subunit (LSU) rRNA gene, small-subunit rRNA gene and internal transcribed spacer (ITS) region, the three strains (S-29, S-63 and S-80) were found to represent a single species of the genus Wickerhamomyces, which were named Wickerhamomyces edaphicus sp. nov. The type strain is S-29(T) (BCC 21231(T)=NBRC 101969(T)=CBS 10408(T)). Strain S-75 represented a novel species of the genus Pichia on the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analyses of the D1/D2 domain of the LSU rRNA gene, for which the name Pichia jaroonii sp. nov. is proposed. The type strain is S-75(T) (BCC 23061(T), NRBC 102180(T)=CBS 10930(T)).
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Change in glycosylation pattern with extension of endoplasmic reticulum retention signal sequence of mouse antibody produced by suspension-cultured tobacco BY2 cells.
J. Biosci. Bioeng.
PUBLISHED: 02-17-2009
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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.
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Analysis of CMP-sialic acid transporter-like proteins in plants.
Phytochemistry
PUBLISHED: 02-13-2009
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It is commonly accepted that sialic acids do not exist in plants. However, putative gene homologs of animal sialyltransferases and CMP-sialic acid transporters have been detected in the genomes of some plants. To elucidate the physiological functions of these genes, we cloned 2 cDNAs from Oryza sativa (Japanese rice), each of which encodes a CMP-sialic acid transporter-like protein designated as OsCSTLP1 and OsCSTLP2. To examine the CMP-sialic acid transporter activity of OsCSTLP1 and OsCSTLP2, we introduced their expression vectors into CMP-sialic acid transporter activity-deficient Lec2 cells. Transfection with OsCSTLP1 resulted in recovery of the deficit phenotype of Lec2 cells, but transfection with OsCSTLP2 did not. We also performed an in vitro nucleotide sugar transport assay using a yeast expression system. Among the nucleotide sugars examined, the OsCSTLP1-containing yeast microsomal membrane vesicles specifically incorporated CMP-sialic acid, indicating that OsCSTLP1 has CMP-sialic acid transporter activity. On the other hand, OsCSTLP2 did not exhibit any nucleotide sugar transporter activity. T-DNA insertion lines of Arabidopsis thaliana targeting the homologs of the OsCSTLP1 and OsCSTLP2 genes exhibited a lethal phenotype, suggesting that these proteins play important roles in plant development and may transport important nucleotide sugars such as CMP-Kdo in physiological conditions.
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Biochemical and immunological characterization of the plant-derived candidate human immunodeficiency virus type 1 mucosal vaccine CTB-MPR.
Plant Biotechnol. J.
PUBLISHED: 02-10-2009
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Plants are potentially the most economical platforms for the large-scale production of recombinant proteins. Thus, plant-based expression of subunit human immunodeficiency virus type 1 (HIV-1) vaccines provides an opportunity for their global use against the acquired immunodeficiency syndrome pandemic. CTB-MPR(649-684)[CTB, cholera toxin B subunit; MPR, membrane proximal (ectodomain) region of gp41] is an HIV-1 vaccine candidate that has been shown previously to induce antibodies that block a pathway of HIV-1 mucosal transmission. In this article, the molecular characterization of CTB-MPR(649-684) expressed in transgenic Nicotiana benthamiana plants is reported. Virtually all of the CTB-MPR(649-684) proteins expressed in the selected line were shown to have assembled into pentameric, GM1 ganglioside-binding complexes. Detailed biochemical analyses on the purified protein revealed that it was N-glycosylated, predominantly with high-mannose-type glycans (more than 75%), as predicted from a consensus asparagine-X-serine/threonine (Asn-X-Ser/Thr) N-glycosylation sequon on the CTB domain and an endoplasmic reticulum retention signal attached at the C-terminus of the fusion protein. Despite this modification, the plant-expressed protein retained the nanomolar affinity to GM1 ganglioside and the critical antigenicity of the MPR(649-684) moiety. Furthermore, the protein induced mucosal and serum anti-MPR(649-684) antibodies in mice after mucosal prime-systemic boost immunization. Our data indicate that plant-based expression can be a viable alternative for the production of this subunit HIV-1 vaccine candidate.
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Molecular cloning and gene expression analysis of tomato endo-beta-N-acetylglucosaminidase, an endoglycosidase involved in the production of high-mannose type free N-glycans during tomato fruit ripening.
Biosci. Biotechnol. Biochem.
PUBLISHED: 02-07-2009
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In this study, we identified a gene encoding tomato ENGase (Endo-LE) using the gene information of rice ENGase, and expressed the Endo-LE protein in Escherichia coli. The substrate specificity of the recombinant Endo-LE was the same as that of the native enzyme, showing strong activity towards the high-mannose type N-glycans with the Manalpha1-2Manalpha1-3Manbeta1-4GlcNAcbeta1-4GlcNAc unit. Real-time PCR analysis revealed that the gene expression of Endo-LE did not vary significantly with the tomato ripening process, indicating that Endo-LE activity is ubiquitously expressed.
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Identification of novel ?1,3-galactosyltransferase and elimination of ?-galactose-containing glycans by disruption of multiple ?-galactosyltransferase genes in Schizosaccharomyces pombe.
J. Biol. Chem.
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The oligosaccharides from fission yeast Schizosaccharomyces pombe contain large amounts of D-galactose (Gal) in addition to D-mannose (Man), in contrast to the budding yeast Saccharomyces cerevisiae. Detailed structural analysis has revealed that the Gal residues are attached to the N- and O-linked oligosaccharides via ?1,2- or ?1,3-linkages. Previously we constructed and characterized a septuple ?-galactosyltransferase disruptant (7GalT?) anticipating a complete lack of ?-Gal residues. However, the 7GalT? strain still contained oligosaccharides consisting of ?1,3-linked Gal residues, indicating the presence of at least one more additional unidentified ?1,3-galactosyltransferase. In this study we searched for unidentified putative glycosyltransferases in the S. pombe genome sequence and identified three novel genes, named otg1(+)-otg3(+) (? one, three-galactosyltransferase), that belong to glycosyltransferase gene family 8 in the Carbohydrate Active EnZymes (CAZY) database. Gal-recognizing lectin blotting and HPLC analyses of pyridylaminated oligosaccharides after deletion of these three additional genes from 7GalT? strain demonstrated that the resultant disruptant missing 10 ?-galactosyltransferase genes, 10GalT?, exhibited a complete loss of galactosylation. In an in vitro galactosylation assay, the otg2(+) gene product had Gal transfer activity toward a pyridylaminated Man(9)GlcNAc(2) oligosaccharide and pyridylaminated Man?1,2-Man?1,2-Man oligosaccharide. In addition, the otg3(+) gene product exhibited Gal transfer activity toward the pyridylaminated Man(9)GlcNAc(2) oligosaccharide. Generation of an ?1,3-linkage was confirmed by HPLC analysis, ?-galactosidase digestion analysis, (1)H NMR spectroscopy, and LC-MS/MS analysis. These results indicate that Otg2p and Otg3p are involved in ?1,3-galactosylation of S. pombe oligosaccharides.
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Evolutionarily conserved glycan signal to degrade aberrant brassinosteroid receptors in Arabidopsis.
Proc. Natl. Acad. Sci. U.S.A.
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Asparagine-linked glycans (N-glycans) are crucial signals for protein folding, quality control, and endoplasmic reticulum (ER)-associated degradation (ERAD) in yeast and mammals. Although similar ERAD processes were reported in plants, little is known about their biochemical mechanisms, especially their relationships with N-glycans. Here, we show that a missense mutation in the Arabidopsis EMS-mutagenized bri1 suppressor 3 (EBS3) gene suppresses a dwarf mutant, bri1-9, the phenotypes of which are caused by ER retention and ERAD of a brassinosteroid receptor, BRASSINOSTEROID-INSENSITIVE 1 (BR1). EBS3 encodes the Arabidopsis ortholog of the yeast asparagine-linked glycosylation 9 (ALG9), which catalyzes the ER luminal addition of two terminal ?1,2 mannose (Man) residues in assembling the three-branched N-glycan precursor [glucose(Glc)](3)(Man)(9)[N-acetylglucosamine(GlcNAc)](2). Consistent with recent discoveries revealing the importance of the Glc(3)Man(9)GlcNAc(2) C-branch in generating an ERAD signal, the ebs3-1 mutation prevents the Glc(3)Man(9)GlcNAc(2) assembly and inhibits the ERAD of bri1-9. By contrast, overexpression of EBS4 in ebs3-1 bri1-9, which encodes the Arabidopsis ortholog of the yeast ALG12 catalyzing the ER luminal ?1,6 Man addition, adds an ?1,6 Man to the truncated N-glycan precursor accumulated in ebs3-1 bri1-9, promotes the bri1-9 ERAD, and neutralizes the ebs3-1 suppressor phenotype. Furthermore, a transfer (T)-DNA insertional alg3-T2 mutation, which causes accumulation of an even smaller N-glycan precursor carrying a different exposed ?1,6 Man, promotes the ERAD of bri1-9 and enhances its dwarfism. Taken together, our results strongly suggest that the glycan signal to mark an ERAD client in Arabidopsis is likely conserved to be an ?1,6 Man-exposed N-glycan.
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Production of different glycosylation variants of the tumour-targeting mAb H10 in Nicotiana benthamiana: influence on expression yield and antibody degradation.
Transgenic Res.
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We previously described the expression of a tumour-targeting antibody (mAb H10) in Nicotiana benthamiana by vacuum-agro-infiltration and the remarkable yields of highly pure protein achieved. The objective of the present work was to investigate different strategies for transient overexpression of the mAb H10 in which glycan configuration was modulated and assess how these strategies affect the accumulation yield and stability of the antibody. To this aim, three procedures have been assayed: (1) Site-directed mutagenesis to abolish the glycosylation site; (2) endoplasmic reticulum retention (C-terminal SEKDEL fusion) to ensure predominantly high-mannose type glycans; and (3) expression in a N. benthamiana RNAi down-regulated line in which ?1,2-xylosyltransferase and ?1,3-fucosyltransferase gene expression is silenced. The three antibody variants (H10-Mut) (H10-SEKDEL) (H10(XylT/FucT)) were transiently expressed, purified and characterised for their glycosylation profile, expression/purification yield and antibody degradation pattern. Glycosylation analysis of H10(XylT/FucT) demonstrated the absence of plant complex-type sugars, while H10-SEKDEL, although substantially retained in the ER, revealed the presence of ?1,2-xylose and ?1,3-fucose residues, indicating a partial escape from the ER retrieval system. Antibody accumulation and purification yields were not enhanced by ER retention. All H10 antibody glyco-forms revealed greater degradation compared to the original, resulting mostly in the formation of Fab fragments. In the case of aglycosylated H10-Mut, more than 95% of the heavy chain was cleaved, confirming the pivotal role of the sugar moiety in protein stability. Identification of possible fragile sites in the H10 antibody hinge region could be of general interest for the development of new strategies to reduce antibody degradation and increase the yield of intact IgGs in plants.
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