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1.
N-Glycosylation of eukaryotic membrane proteins is a co-translational event that occurs in the lumen of the endoplasmic reticulum (ER). This process is catalyzed by a membrane-associated oligosaccharyl transferase (OST) complex that transfers a preformed oligosaccharide (Glc3Man9GlcNAc2-) to an asparagine (Asn) side-chain acceptor located within the sequon (-Asn-X-Ser/Thr-). Scanning N-glycosylation mutagenesis experiments, where novel acceptor sites are introduced at unique sites within membrane proteins, have shown that the acceptor sites must be located a minimum distance (12–14 amino acids) away from the luminal membrane surface of the ER in order to be efficiently N-glycosylated. Scanning N-glycosylation mutagenesis can therefore be used to determine membrane protein topology and it can also serve as a molecular ruler to define the ends of transmembrane (TM) segments. Furthermore, since N-glycosylation is a co-translational event, N-glycosylation mutagenesis can be used to identify folding intermediates in membrane proteins that may expose segments to the ER lumen transiently during biosynthesis. 相似文献
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Hiroyuki Kajiura Masashi Wasai Saori Kasahara Fumio Takaiwa Kazuhito Fujiyama 《Molecular biotechnology》2013,54(3):784-794
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. 相似文献
3.
Kieran F. Geoghegan Xi Song Lise R. Hoth Xidong Feng Suman Shanker Amira Quazi Deborah P. Luxenberg Jill F. Wright Matthew C. Griffor 《Protein expression and purification》2013,87(1):27-34
The T helper cell-derived cytokine interleukin-17A (IL-17A) is a variably glycosylated disulfide-linked homodimer of 34–38 kDa. Its polypeptide monomer contains one canonical N-glycosylation site at Asn68, and human recombinant IL-17A was partly N-glycosylated when expressed in human kidney (HEK293) cells as a fusion protein with a melittin signal sequence and an N-terminal hexahistidine tag. Orbitrap mass analyses of the tryptic N-glycopeptide 63–69 indicated that the N-glycosylation was of the GalNAc-terminated type characteristic of cultured kidney cells. The mass spectrum of IL-17A monomer also included peaks shifted by +948 Da from the respective masses of unglycosylated and N-glycosylated polypeptides. These were caused by unpredicted partial O-glycosylation of Thr26 with the mucin-like structure -GalNAc(-NeuNAc)-Gal-NeuNAc. Identical O-glycosylation occurred in commercially sourced recombinant IL-17A also expressed in HEK293 cells but with a different N-terminal sequence. Therefore, the kidney host cell line not only imposed its characteristic pattern of N-glycosylation on recombinant IL-17A but additionally created an O-glycosylation not known to be present in the T cell-derived cytokine. Mammalian host cell lines for recombinant protein expression generally impose their characteristic patterns of N-glycosylation on the product, but this work exemplifies how a host may also unpredictably O-glycosylate a protein that is probably not normally O-glycosylated. 相似文献
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M. M. Harmsen C. B. van Solt H. P. D. Fijten 《Applied microbiology and biotechnology》2009,84(6):1087-1094
Single-domain antibody fragments (VHHs) have several beneficial properties as compared to conventional antibody fragments.
However, their small size complicates their toxin- and virus-neutralizing capacity. We isolated 27 VHHs binding Escherichia coli heat-labile toxin and expressed these in Saccharomyces cerevisiae. The most potent neutralizing VHH (LT109) was N-glycosylated, resulting in a large increase in molecular mass. This suggests that N-glycosylation of LT109 improves its neutralizing capacity. Indeed, deglycosylation of LT109 decreased its neutralizing capacity
three- to fivefold. We also studied the effect of glycosylation of two previously isolated VHHs on their ability to neutralize
foot-and-mouth disease virus. For this purpose, these VHHs that lacked potential N-glycosylation sites were genetically fused to another VHH that was known to be glycosylated. The resulting fusion proteins
were also N-glycosylated. They neutralized the virus at at least fourfold-lower VHH concentrations as compared to the single, non-glycosylated
VHHs and at at least 50-fold-lower VHH concentrations as compared to their deglycosylated counterparts. Thus, we have shown
that N-glycosylation of VHHs contributes to toxin- and virus-neutralizing capacity. 相似文献
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Canfang Niu Huiying Luo Pengjun Shi Huoqing Huang Yaru Wang Peilong Yang Bin Yao 《Applied and environmental microbiology》2016,82(4):1004-1014
N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin''s accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed. 相似文献
6.
Multigene families are features of most eukaryotic genomes, which evolve through a variety of mechanisms. This study describes
the structure, expression, and evolution of a novel family in the oomycete Phytophthora. In the heterothallic species P. infestans, M96 is expressed specifically during sexual sporogenesis, and encodes a low-complexity extracellular protein that may be a component
of oospore walls. Intriguingly, M96 exists in P. infestans as 22 relatively homogeneous loci tandemly repeated at a single site, which is partitioned by inversions and retroelements
into subclusters exhibiting semi-independent evolution. M96 relatives were detected in other heterothallic and homothallic oomycetes including species closely (P. mirabilis, P. phaseoli) or distantly (P. ramorum, P. sojae) related to P. infestans. Those M96 relatives also exhibit oosporogenesis-specific expression and are arrayed multigene families. Nucleotide changes and repeat
expansion diversify M96 in each species, however, paralogues are more related than orthologues. Concerted evolution through gene conversion and not
strong purifying selection appears to be the major contributor to intraspecific homogenization. Divergence and concerted evolution
was also detected between isolates of P. infestans. The divergence of M96 proteins between P. infestans, P. ramorum, and P. sojae exceeds that of typical proteins, reflecting trends in reproductive proteins from other kingdoms.
Sequence data from this article have been deposited with the GenBank Data Libraries under accessions DQ196155 to DQ196175. 相似文献
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PsHint1, associated with the G‐protein α subunit PsGPA1, is required for the chemotaxis and pathogenicity of Phytophthora sojae 下载免费PDF全文
Xin Zhang Chunhua Zhai Chenlei Hua Min Qiu Yujuan Hao Pingping Nie Wenwu Ye Yuanchao Wang 《Molecular Plant Pathology》2016,17(2):272-285
Zoospore chemotaxis to soybean isoflavones is essential in the early stages of infection by the oomycete pathogen Phytophthora sojae. Previously, we have identified a G‐protein α subunit encoded by PsGPA1 which regulates the chemotaxis and pathogenicity of P. sojae. In the present study, we used affinity purification to identify PsGPA1‐interacting proteins, including PsHint1, a histidine triad (HIT) domain‐containing protein orthologous to human HIT nucleotide‐binding protein 1 (HINT1). PsHint1 interacted with both the guanosine triphosphate (GTP)‐ and guanosine diphosphate (GDP)‐bound forms of PsGPA1. An analysis of the gene‐silenced transformants revealed that PsHint1 was involved in the chemotropic response of zoospores to the isoflavone daidzein. During interaction with a susceptible soybean cultivar, PsHint1‐silenced transformants displayed significantly reduced infectious hyphal extension and caused a strong cell death in plants. In addition, the transformants displayed defective cyst germination, forming abnormal germ tubes that were highly branched and exhibited apical swelling. These results suggest that PsHint1 not only regulates chemotaxis by interacting with PsGPA1, but also participates in a Gα‐independent pathway involved in the pathogenicity of P. sojae. 相似文献
10.
Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and β-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15. 相似文献
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Nobutake Akiyama Yuji Ohno Yosinobu Manome 《Biochemical and biophysical research communications》2009,381(4):612-618
Several fusion proteins of mouse Interleukins (mILs) and the enhanced green fluorescent protein (EGFP) were expressed in fibroblast and epithelial cells. Among these proteins, the mIL-31 derivative was the most efficiently secreted into the medium in a N-glycosylation-dependent manner. From the analysis of deletion mutants, the minimal structure for constitutive secretions consisted of a signal peptide and N-glycosylation. Introduction of the signal sequence from mIL-31 to human p53 protein failed to secrete the products, but further addition of the N-glycosylation site resulted in constitutive secretion of biologically active p53 protein into the medium in the N-glycosylated form. In this report, we showed the importance of N-glycosylation for constitutive protein secretions, especially using non-polarized cells. 相似文献
14.
O-mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein 总被引:2,自引:0,他引:2
Secretory proteins in yeast are N- and O-glycosylated while they enter the endoplasmic reticulum. N-glycosylation is initiated by the oligosaccharyl transferase complex and O-mannosylation is initiated by distinct O-mannosyltransferase complexes of the protein mannosyl transferase Pmt1/Pmt2 and Pmt4 families. Using covalently linked cell-wall protein 5 (Ccw5) as a model, we show that the Pmt4 and Pmt1/Pmt2 mannosyltransferases glycosylate different domains of the Ccw5 protein, thereby mannosylating several consecutive serine and threonine residues. In addition, it is shown that O-mannosylation by Pmt4 prevents N-glycosylation by blocking the hydroxy amino acid of the single N-glycosylation site present in Ccw5. These data prove that the O- and N-glycosylation machineries compete for Ccw5; therefore O-mannosylation by Pmt4 precedes N-glycosylation. 相似文献
15.
PsMPK7, a stress‐associated mitogen‐activated protein kinase (MAPK) in Phytophthora sojae,is required for stress tolerance,reactive oxygenated species detoxification,cyst germination,sexual reproduction and infection of soybean 下载免费PDF全文
Jian Gao Mingna Cao Wenwu Ye Haiyang Li Liang Kong Xiaobo Zheng Yuanchao Wang 《Molecular Plant Pathology》2015,16(1):61-70
16.
Xavier Herman Johann Far Marie Peeters Loïc Quinton François Chaumont Catherine Navarre 《Plant biotechnology journal》2023,21(9):1773-1784
Production of recombinant pharmaceutical glycoproteins has been carried out in multiple expression systems. However, N-glycosylation, which increases heterogeneity and raises safety concerns due to the presence of non-human residues, is usually not controlled. The presence and composition of N-glycans are also susceptible to affect protein stability, function and immunogenicity. To tackle these issues, we are developing glycoengineered Nicotiana tabacum Bright Yellow-2 (BY-2) cell lines through knock out and ectopic expression of genes involved in the N-glycosylation pathway. Here, we report on the generation of BY-2 cell lines producing deglycosylated proteins. To this end, endoglycosidase T was co-expressed with an immunoglobulin G or glycoprotein B of human cytomegalovirus in BY-2 cell lines producing only high mannose N-glycans. Endoglycosidase T cleaves high mannose N-glycans to generate single, asparagine-linked, N-acetylglucosamine residues. The N-glycosylation profile of the secreted antibody was determined by mass spectrometry analysis. More than 90% of the N-glycans at the conserved Asn297 site were deglycosylated. Likewise, extensive deglycosylation of glycoprotein B, which possesses 18 N-glycosylation sites, was observed. N-glycan composition of gB glycovariants was assessed by in vitro enzymatic mobility shift assay and proven to be consistent with the expected glycoforms. Comparison of IgG glycovariants by differential scanning fluorimetry revealed a significant impact of the N-glycosylation pattern on the thermal stability. Production of deglycosylated pharmaceutical proteins in BY-2 cells expands the set of glycoengineered BY-2 cell lines. 相似文献
17.
Delong Li Zhijian Zhao Yidan Huang Zhaojun Lu Meng Yao Yujuan Hao Chunhua Zhai Yuanchao Wang 《PloS one》2013,8(3)
Plant pathogens secrete effector proteins to suppress plant immunity. However, the mechanism by which oomycete pathogens deliver effector proteins during plant infection remains unknown. In this report, we characterized a Phytophthora sojae vps1 gene. This gene encodes a homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene vps1 that mediates budding of clathrin-coated vesicles from the late Golgi, which are diverted from the general secretory pathway to the vacuole. PsVPS1-silenced mutants were generated using polyethylene glycol-mediated protoplast stable transformation and were viable but had reduced extracellular protein activity. The PsVPS1-silenced mutants showed impaired hyphal growth, and the shapes of the vacuoles were highly fragmented. Silencing of PsVPS1 affected cyst germination as well as the polarized growth of germinated cysts. Silenced mutants showed impaired invasion of susceptible soybean plants regardless of wounding. These results suggest that PsVPS1 is involved in vacuole morphology and cyst development. Moreover, it is essential for the virulence of P. sojae and extracellular protein secretion. 相似文献
18.
《Molecular membrane biology》2013,30(4):331-337
The effect of N-glycosylation on the assembly of N-methyl-D-aspartate (NMDA) heteromeric cloned receptors was studied. Thus human embryonic kidney (HEK) 293 cells were cotransfected with N-methyl-D-aspartate R1 (NR1) and N-methyl-d-aspartate R2A (NR2A) clones and the cells grown post-transfection in the presence of tunicamycin (TM). TM treatment resulted in a decrease of the NR1 subunit with Mr 117 000 with a concomitant increase in a Mr 97 000 immunoreactive species previously identified as the non-N-glycosylated NR1 subunit. In parallel, TM caused a dose-dependent inhibition of [3H]MK801 binding to the expressed receptor which was a result of an approximate four-told reduction in the Dissociation Constant (KD) but with no change in the number of binding sites (BMAX)-NMDA receptor cell surface expression was unchanged following TM treatment but it did result in a decrease in the percentage cell death post-transfection compared to control samples. The removal of TM from the cell culture media resulted in a return to the control KD value for [3H]MK801 binding and partial reglycosylation of newly synthesized NR1 subunit. These results demonstrate that N-glycosylation is requisite for the efficient expression of functional NR1/NR2A receptors. Furthermore, they suggest that N-glycosylation may be important for the correct formation of the channel domain of the NR1/NR2A receptor. 相似文献
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Shuchen Wang Xiaoyi Zhang Zhichao Zhang Yun Chen Qing Tian Dandan Zeng Miao Xu Yan Wang Suomeng Dong Zhonghua Ma Yuanchao Wang Xiaobo Zheng Wenwu Ye 《植物学报(英文版)》2023,65(9):2204-2217
Plants can be infected by multiple pathogens concurrently in natural systems. However,pathogen–pathogen interactions have rarely been studied. In addition to the oomycete Phytophthora sojae, fungi such as Fusarium spp. also cause soybean root rot. In a 3-year field investigation, we discovered that P. sojae and Fusarium spp. frequently coexisted in diseased soybean roots. Out of 336 P. sojae–soybean–Fusarium combinations,more than 80% aggravated disease. Different Fusarium species all enhanced P... 相似文献
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Ban Sato Yohko U. Katagiri Kazutoshi Iijima Hiroyuki Yamada Satsuki Ito Nana Kawasaki Hajime Okita Junichiro Fujimoto Nobutaka Kiyokawa 《Biochimica et Biophysica Acta (BBA)/General Subjects》2012