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81.
Xinqiao Zhan Qingwen Shen Jie Chen Pei Yang Xuemin Wang Yueyun Hong 《Plant, cell & environment》2019,42(7):2215-2230
Sulfoquinovosyltransferase 2 (SQD2) catalyses the final step in the sulfoquinovosyldiacylglycerol (SQDG) biosynthetic pathway. It is involved in the phosphate starvation response. Here, we show that rice SQD2.1 has dual activities catalysing SQDG synthesis and flavonoid glycosylation. SQD2.1 null mutants (sqd2.1) in rice had decreased levels of glycosidic flavonoids, particularly apigenin 7‐O‐glucoside (A7G), whereas these metabolites were increased in rice plants overexpressing SQD2.1. The sqd2.1 mutants and SQD2.1 overexpressing lines showed reduced and enhanced, respectively, tolerance to salinity and drought. Treating the sqd2.1 mutants with A7G decreased oxidative damage and restored stress tolerance to the wild‐type levels. These findings demonstrate that SQD2.1 has a novel function in the glycosylation of flavonoids that is required for osmotic stress tolerance in rice. The novel activity of SQD2.1 in the production of glycosidic flavonoids improves scavenging of reactive oxygen species and protects against excessive oxidation. 相似文献
82.
Paul J. Hensbergen Arnoud H. de Ru Annemieke H. Friggen Jeroen Corver Wiep Klaas Smits Peter A. van Veelen 《The Journal of biological chemistry》2022,298(3)
The type A glycan modification found in human pathogen Clostridioides difficile consists of a monosaccharide (GlcNAc) that is linked to an N-methylated threonine through a phosphodiester bond. This structure has previously been described on the flagellar protein flagellin C of several C. difficile strains and is important for bacterial motility. The study of post-translational modifications often relies on some type of enrichment strategy; however, a procedure for enrichment of this modification has not yet been demonstrated. In this study, we show that an approach that is commonly used in phosphoproteomics, Fe3+-immobilized metal affinity chromatography, also enriches for peptides with this unique post-translational modification. Using LC–MS/MS analyses of immobilized metal affinity chromatography–captured tryptic peptides, we observed not only type A-modified C. difficile flagellin peptides but also a variety of truncated/modified type A structures on these peptides. Using an elaborate set of mass spectrometry analyses, we demonstrate that one of these modifications consists of a type A structure containing a phosphonate (2-aminoethylphosphonate), a modification that is rarely observed and has hitherto not been described in C. difficile. In conclusion, we show that a common enrichment strategy results in reliable identification of peptides carrying a type A glycan modification, and that the results obtained can be used to advance models about its biosynthesis. 相似文献
83.
Tetsuya Hirata Atsushi Kobayashi Tamio Furuse Ikuko Yamada Masaru Tamura Hiroyuki Tomita Yuko Tokoro Akinori Ninomiya Yoshitaka Fujihara Masahito Ikawa Yusuke Maeda Yoshiko Murakami Yasuhiko Kizuka Taroh Kinoshita 《The Journal of biological chemistry》2022,298(3)
Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrPC, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)−galactose−sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrPC GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases. 相似文献
84.
Tejeshwar C. Rao Reena R. Beggs Katherine E. Ankenbauer Jihye Hwang Victor Pui-Yan Ma Khalid Salaita Susan L. Bellis Alexa L. Mattheyses 《The Journal of biological chemistry》2022,298(4)
Heterogeneity within the glycocalyx influences cell adhesion mechanics and signaling. However, the role of specific glycosylation subtypes in influencing cell mechanics via alterations of receptor function remains unexplored. It has been shown that the addition of sialic acid to terminal glycans impacts growth, development, and cancer progression. In addition, the sialyltransferase ST6Gal-I promotes epidermal growth factor receptor (EGFR) activity, and we have shown EGFR is an ‘allosteric mechano-organizer’ of integrin tension. Here, we investigated the impact of ST6Gal-I on cell mechanics. Using DNA-based tension gauge tether probes of variable thresholds, we found that high ST6Gal-I activity promotes increased integrin forces and spreading in Cos-7 and OVCAR3, OVCAR5, and OV4 cancer cells. Further, employing inhibitors and function-blocking antibodies against β1, β3, and β5 integrins and ST6Gal-I targets EGFR, tumor necrosis factor receptor, and Fas cell surface death receptor, we validated that the observed phenotypes are EGFR-specific. We found that while tension, contractility, and adhesion are extracellular-signal-regulated kinase pathway-dependent, spreading, proliferation, and invasion are phosphoinositide 3-kinase-Akt serine/threonine kinase dependent. Using total internal reflection fluorescence microscopy and flow cytometry, we also show that high ST6Gal-I activity leads to sustained EGFR membrane retention, making it a key regulator of cell mechanics. Our findings suggest a novel sialylation-dependent mechanism orchestrating cellular mechanics and enhancing cell motility via EGFR signaling. 相似文献
85.
Xiaodong Liu Rui Liu Yongheng Bai Heya Jiang Xinxin Fu Shumei Ma 《Cell biochemistry and function》2020,38(3):283-289
Based on central dogma of genetics, protein is the embodiment and executor of genetic function, post-translational modifications (PTMs) of protein are particularly important and involved in almost all aspects of cell biology and pathogenesis. Studies have shown that ionizing radiation (IR) alters gene expression much more profoundly and a broad variety of cell-process pathways, lots of proteins are modified and activated. Our understanding of the protein in response to ionizing radiation is steadily increasing. Among the various biological processes known to induce radioresistance, PTMs have attracted marked attention in recent years. The present review summarizes the latest knowledge about how PTMs response to ionizing radiation and pathway analysis were conducted. The data provided insights into biological effects of IR and contributing to the development of novel IR-based strategies. 相似文献
86.
Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation. 相似文献
87.
Gagneux P Amess B Diaz S Moore S Patel T Dillmann W Parekh R Varki A 《American journal of physical anthropology》2001,115(2):99-109
Most blood plasma proteins are glycosylated. These glycoproteins typically carry sialic acid-bearing sugar chains, which can modify the observed molecular weights and isoelectric points of those proteins during electrophoretic analyses. To explore changes in protein expression and glycosylation that occurred during great ape and human evolution, we subjected multiple blood plasma samples from all these species to high-resolution proteomic analysis. We found very few species-specific differences, indicating a remarkable degree of conservation of plasma protein expression and glycosylation during approximately 12 million years of evolution. A few lineage-specific differences in protein migration were noted among the great apes. The only obvious differences between humans and all great apes were an apparent decrease in transthyretin (prealbumin) and a change in haptoglobin isoforms (the latter was predictable from prior genetic studies). Quantitative studies of transthyretin in samples of blood plasma (synthesized primarily by the liver) and of cerebrospinal fluid (synthesized locally by the choroid plexus of the brain) confirmed approximately 2-fold higher levels in chimpanzees compared to humans. Since transthyretin binds thyroid hormones, we next compared plasma thyroid hormone parameters between humans and chimpanzees. The results indicate significant differences in the status of thyroid hormone metabolism, which represent the first known endocrine difference between these species. Notably, thyroid hormones are known to play major roles in the development, differentiation, and metabolism of many organs and tissues, including the brain and the cranium. Also, transthyretin is known to be the major carrier of thyroid hormone in the cerebrospinal fluid, likely regulating delivery of this hormone to the brain. A potential secondary difference in retinoid (vitamin A) metabolism is also noted. The implications of these findings for explaining unique features of human evolution are discussed. 相似文献
88.
IgG carries bi-antennary N-linked glycans which differ in degrees of galactosylation, core fucosylation and bisecting N-acetyl glucosamine. The majority of these are non-sialyated closely related neutral structures which can be resolved by HPLC analysis, but which are difficult to separate in techniques such as fluorophore-coupled carbohydrate electrophoresis. Derivatisation with the singly charged fluorophore, 2-amino benzoic acid and separation in gels with a 30% monomer content in tris/glycine buffer enabled separation of neutral glycans. In particular, agalactosyl glycans with either a core fucose substitution or bisecting N-acetyl galactosamine could be resolved. Good separation of mono- and di-galactosylated glycans was also achieved with this system. It was shown that IgG can be separated from serum by size-exclusion and anion exchange chromatography with minimal contamination, with complete glycan release accomplished by the enzyme peptide-N-glycosidase F (F. meningosepticum). This method of resolving IgG glycans could be used to monitor patients in which glycosylation changes may have a diagnostic value, as in rheumatoid arthritis. It could also be used to monitor recombinant IgG glycosylation where routine screening is required in the biotechnology industry. 相似文献
89.
Mazhari-Tabrizi R Blank M Mumberg D Funk M Schwarz RT Eckert V 《Glycoconjugate journal》1999,16(11):673-679
Heterologous complementation in yeast has been a successful tool for cloning and characterisation of genes from various organisms. Therefore we constructed conditionally lethal Saccharomyces cerevisiae strains by replacing the endogenous promoter from the genes of interest (glycosyltransferases) by the stringently regulated GAL1-promoter, by a technique called chromosomal promoter replacement. Such yeast strains were constructed for the genes Alg 1, Alg7, Sec59, Wbp1 involved in N-Glycosylation, the genes Gpi2, Gpi3/Spt14, Gaal, Pis1, involved in GPI-anchor biosynthesis and Dpm involved in both pathways. All strains show the expected conditionally lethal phenotype on glucose-containing medium when expression of the respective gene is turned off. 相似文献
90.