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N-linked Glycan (N-link + glycan)
Selected AbstractsEngineering of a monomeric and low-glycosylated form of human butyrylcholinesteraseFEBS JOURNAL, Issue 2 2002Expression, characterization, crystallization, purification Human butyrylcholinesterase (BChE; EC 3.1.1.8) is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine, organophosphorus pesticides and nerve agents. The relative contribution of each N-linked glycan for the solubility, the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. The purified recombinant BChE showed catalytic properties similar to those of the native enzyme. Tetragonal crystals suitable for X-ray crystallography studies were obtained; they were improved by recrystallization and found to diffract to 2.0 Å resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a = b = 154.7 Å, c = 124.9 Å, giving a Vm of 2.73 Å3 per Da (estimated 60% solvent) for a single molecule of recombinant BChE in the asymmetric unit. The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general. [source] Glycosylation status of haptoglobin in sera of patients with prostate cancer vs. benign prostate disease or normal subjectsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2008Tsutomu Fujimura Abstract We studied chemical level and glycosylation status of haptoglobin in sera of patients with prostate cancer, as compared to benign prostate disease and normal subjects, with the following results. (i) Haptoglobin level was enhanced significantly in sera of prostate cancer. (ii) Sialylated bi-antennary glycans were the dominant structures in haptoglobins from all 3 sources, regardless of different site of N-linked glycan. The N-linked glycans at N184 were exclusively bi-antennary, and showed no difference between prostate cancer vs. benign prostate disease. (iii) Tri-antennary, N-linked, fucosylated glycans, carrying at least 1 sialyl-Lewisx/a antenna, were predominantly located on N207 or N211 within the amino acid 203-215 sequence of the ,-chain of prostate cancer, and were minimal in benign prostate disease. Fucosylated glycans were not observed in normal subjects. A minor tri-antennary N-linked glycan was observed at N241 of the ,-chain in prostate cancer, which was absent in benign prostate disease. (iv) None of these N-linked structures showed the expected presence of disialylated antennae with GalNAc,4(NeuAc,3)Gal,3(NeuAc,6)GlcNAc,Gal, or its analogue, despite cross-reactivity of prostate cancer haptoglobin with monoclonal antibody RM2. (v) Minor levels of O -glycosylation were identified in prostate cancer haptoglobin for the first time. Mono- and disialyl core Type 1 O-linked structures were identified after reductive ,-elimination followed by methylation and mass spectrometric analysis. No evidence was found for the presence of specific RM2 or other tumor-associated glycosyl epitopes linked to this O -glycan core. In summary, levels of haptoglobin are enhanced in sera of prostate cancer patients, and the N -glycans attached to a defined peptide region of its ,-chain are characterized by enhanced branching as well as antenna fucosylation. © 2007 Wiley-Liss, Inc. [source] Protein N-glycosylation determines functionality of the Saccharomyces cerevisiae cell wall integrity sensor Mid2pMOLECULAR MICROBIOLOGY, Issue 6 2008Franziska Hutzler Summary The fungal cell wall is a highly dynamic structure that is essential to maintain cell shape and stability. Hence in yeasts and fungi cell wall integrity is tightly controlled. The Saccharomyces cerevisiae plasma membrane protein Mid2p is a putative mechanosensor that responds to cell wall stresses and morphological changes during pheromone induction. The extracellular domain of Mid2p, which is crucial to sensing, is highly O- and N-glycosylated. We showed that O-mannosylation is determining stability of Mid2p. If and how N-glycosylation is linked to Mid2p function was unknown. Here we demonstrate that Mid2p contains a single high mannose N-linked glycan at position Asn-35. The N -glycan is located close to the N-terminus and is exposed from the plasma membrane towards the cell wall through a highly O-mannosylated domain that is predicted to adopt a rod-like conformation. In contrast to O-mannosylation, lack of the N-linked glycan affects neither, stability of Mid2p nor distribution at the plasma membrane during vegetative and sexual growth. However, non-N-glycosylated Mid2p fails to perceive cell wall challenges. Our data further demonstrate that both the extent of the N-linked glycan and its distance from the plasma membrane affect Mid2p function, suggesting the N -glycan to be directly involved in Mid2p sensing. [source] Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N-linked glycosylation, protein maturation, and stabilityPROTEIN SCIENCE, Issue 9 2010Lynn Ziegler Abstract Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N-linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high-molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N , A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high-affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N-linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N-linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex. [source] Tyrosine sulfation and N-glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin bindingFEBS JOURNAL, Issue 3 2002Christoph Böhme The structure of post-translational modifications of human heparin cofactor II isolated from human serum and from recombinant Chinese hamster ovary cells and their effects on heparin binding have been characterized. Oligosaccharide chains were found attached to all three potential N-glycosylation sites in both protein preparations. The carbohydrate structures of heparin cofactor II circulating in blood are complex-type diantennary and triantennary chains in a ratio of 6 : 1 with the galactose being > 90% sialylated with ,2,6 linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sLex motif. Proximal ,1,6 fucosylation of oligosacharides from Chinese hamster ovary cell-derived HCII was detected in >,90% of the diantennary and triantennary glycans, the latter being slightly less sialylated with exclusively ,2,3-linked N -acetylneuraminic acid units. Applying the ESI-MS/ MS-MS technique, we demonstrate that the tryptic peptides comprising tyrosine residues in positions 60 and 73 were almost completely sulfated irrespective of the protein's origin. Treatment of transfected Chinese hamster ovary cells with chlorate or tunicamycin resulted in the production of heparin cofactor II molecules that eluted with higher ionic strength from heparin,Sepharose, indicating that tyrosine sulfation and N-linked glycans may affect the inhibitor's interaction with glycosaminoglycans. [source] Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorphaFEMS YEAST RESEARCH, Issue 7 2007Jens Klabunde Abstract The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for supertransformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-, and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L,1 range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MF,1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha. [source] Synthesis of enzymatically active human ,- l -iduronidase in Arabidopsis cgl (complex glycan-deficient) seedsPLANT BIOTECHNOLOGY JOURNAL, Issue 2 2006Willa L. Downing Summary As an initial step to develop plants as systems to produce enzymes for the treatment of lysosomal storage disorders, Arabidopsis thaliana wild-type (Col-0) plants were transformed with a construct to express human ,- l -iduronidase (IDUA; EC 3.2.1.76) in seeds using the promoter and other regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene. IDUA protein was easily detected on Western blots of extracts from the T2 seeds, and extracts contained IDUA activity as high as 2.9 nmol 4-methylumbelliferone (4 MU)/min/mg total soluble protein (TSP), corresponding to approximately 0.06 µg IDUA/mg TSP. The purified protein reacted with an antibody specific for xylose-containing plant complex glycans, indicating its transit through the Golgi complex. In an attempt to avoid maturation of the N-linked glycans of IDUA, the same IDUA transgene was introduced into the Arabidopsis cgl background, which is deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of complex glycan biosynthesis. IDUA activity and protein levels were significantly higher in transgenic cgl vs. wild-type seeds (e.g. maximum levels were 820 nmol 4 MU/min/mg TSP, or 18 µg IDUA/mg TSP). Affinity-purified IDUA derived from cgl mutant seeds showed a markedly reduced reaction with the antibody specific for plant complex glycans, despite transit of the protein to the apoplast. Furthermore, gel mobility changes indicated that a greater proportion of its N-linked glycans were susceptible to digestion by Streptomyces endoglycosidase H, as compared to IDUA derived from seeds of wild-type Arabidopsis plants. The combined results indicate that IDUA produced in cgl mutant seeds contains glycans primarily in the high-mannose form. This work clearly supports the viability of using plants for the production of human therapeutics with high-mannose glycans. [source] Application of the StrOligo algorithm for the automated structure assignment of complex N-linked glycans from glycoproteins using tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2003Martin Ethier Oligosaccharides associated with proteins are known to give these molecules specific conformations and functions. Analysis of proteins would not be complete without studying the glycans. However, high-throughput techniques in proteomics will soon overwhelm the current capacity of methods if no automation is incorporated into glycomics. New capabilities of the StrOligo algorithm introduced for this purpose (Ethier et al., Rapid Commun. Mass Spectrom., 2002; 16: 1743) will be discussed here. Experimental tandem mass spectra were acquired to test the algorithm using a hybrid quadrupole-time-of-flight (QqTOF) instrument with a matrix-assisted laser desorption/ionization (MALDI) source. The samples were N-linked oligosaccharides from monoclonal antibody IgG, beta interferon and fetuin, detached by enzymatic deglycosylation and labeled at the reducing end. Improvements to the program were made in order to reduce the need for user intervention. StrOligo strips the spectra down to monoisotopic peaks only. The algorithm first builds a relationship tree, accounting for each observed loss of a monosaccharide moiety, and then analyzes the tree and proposes possible structures from combinations of adducts and fragment ion types. A score, which reflects agreement with experimental results, is then given to each proposed structure. The program then decides which combination is the best one and labels relevant peaks in the experimental mass spectrum using a modified nomenclature. The usefulness of the algorithm has been demonstrated by assigning structures to several glycans released from glycoproteins. The analysis was completed in less than 2 minutes for any glycan, which is a substantial improvement over manual interpretation. Copyright © 2003 John Wiley & Sons, Ltd. [source] Structural analysis of oligosaccharides by atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 3 2002Colin S. Creaser An ion source incorporating a fibre optic interface has been constructed for atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap mass spectrometry. The configuration has been applied to the study of linear and complex oligosaccharides. Multi-stage tandem mass spectrometry (MSn, n,=,2,4) experiments carried out in the ion trap enable extended fragmentation pathways to be investigated that yield structural information. Collisional activation of sodiated oligosaccharides, as demonstrated on the model compound maltoheptaose, produces primarily B and Y fragments resulting from cleavage of glycosidic bonds; fragments from cross-ring cleavages are also observed following further stages of tandem mass spectrometry, providing additional linkage information. The analyses of mixtures of complex oligosaccharides are demonstrated for N-linked glycans from chicken egg glycoproteins and a ribonuclease glycan mixture. Mass spectrometric and tandem mass spectrometric data for sugars with molecular weights up to 4000,Da is shown for mixtures of linear dextrans and N-linked glycans. The use of MSn (n,=,3,,4) on these complex molecules enabled structural information to be elucidated that confirms data observed in the MS/MS spectra. Copyright © 2001 John Wiley & Sons, Ltd. [source] | |||||||||||||