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91.
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
92.
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
93.
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
94.
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
95.
96.
In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic enzymes responsible for generating the essential donor sugar nucleotide, CMP- N -acetylneuraminic acid, required for sialylation. Inhibition of N -acetylglucosaminidase has also been applied to alter N-glycan processing in insect cells. This review summarizes current knowledge on N-glycan processing in lepidopteran insect cell lines, and recent progress in glycoengineering lepidopteran insect cells to produce glycoproteins containing complex N-glycans. 相似文献
97.
Koles K van Berkel PH Mannesse ML Zoetemelk R Vliegenthart JF Kamerling JP 《Glycobiology》2004,14(11):979-986
The large-scale production of recombinant biopharmaceutical glycoproteins in the milk of transgenic animals is becoming more widespread. However, in comparison with bacterial, plant cell, or cell culture production systems, little is known about the glycosylation machinery of the mammary gland, and hence on the glycosylation of recombinant glycoproteins produced in transgenic animals. Here the influence is presented of several lactation parameters on the N-glycosylation of recombinant C1 inhibitor (rhC1INH), a human serum glycoprotein, expressed in the milk of transgenic rabbits. Enzymatically released N-glycans of series of rhC1INH samples were fluorescently labeled and fractionated by HPLC. The major N-glycan structures on rhC1INH of pooled rabbit milk were similar to those on native human C1 inhibitor and recombinant human C1 inhibitor produced in transgenic mouse milk, with only the degree of sialylation and core fucosylation being lower. Analyses of individual animals furthermore showed slight interindividual differences; a decrease in the extent of sialylation, core fucosylation, and oligomannose-type glycosylation with the progress of lactation; and a positive correlation between expression level and oligomannose-type N-glycan content. However, when large quantities of rhC1INH were isolated for preclinical and clinical studies, highly consistent N-linked glycan profiles and monosaccharide compositions were found. 相似文献
98.
Bobrowicz P Davidson RC Li H Potgieter TI Nett JH Hamilton SR Stadheim TA Miele RG Bobrowicz B Mitchell T Rausch S Renfer E Wildt S 《Glycobiology》2004,14(9):757-766
A significant percentage of eukaryotic proteins contain posttranslationalmodifications, including glycosylation, which are required forbiological function. However, the understanding of the structurefunctionrelationships of N-glycans has lagged significantly due to themicroheterogeneity of glycosylation in mammalian produced proteins.Recently we reported on the cellular engineering of yeast toreplicate human N-glycosylation for the production of glycoproteins.Here we report the engineering of an artificial glycosylationpathway in Pichia pastoris blocked in dolichol oligosaccharideassembly. The PpALG3 gene encoding Dol-P-Man:Man5GlcNAc2-PP-Dolmannosyltransferase was deleted in a strain that was previouslyengineered to produce hybrid GlcNAcMan5GlcNAc2 human N-glycans.Employing this approach, combined with the use of combinatorialgenetic libraries, we engineered P. pastoris strains that synthesizecomplex GlcNAc2Man3GlcNAc2 N-glycans with striking homogeneity.Furthermore, through expression of a Golgi-localized fusionprotein comprising UDP-glucose 4-epimerase and ß-1,4-galactosyltransferase activities we demonstrate that this structure isa substrate for highly efficient in vivo galactose addition.Taken together, these data demonstrate that the artificial invivo glycoengineering of yeast represents a major advance inthe production of glycoproteins and will emerge as a practicaltool to systematically elucidate the structurefunctionrelationship of N-glycans.
1 These authors contributed equally to this work. 2 To whom correspondence should be addressed; e-mail: swildt{at}glycofi.com 相似文献
99.
Absidia coerulea transformed four anthraquinones from rhubarb, chrysophanol, physcion, emodin and aloe-emodin to their corresponding glycosylated metabolites. The structures of the products were characterized as chrysophanol 8-O-beta-D-glucoside, physcion 8-O-beta-D-glucoside, emodin 6-O-beta-D-glucoside, and aloe-emodin 1-O-beta-D-glucoside, respectively. 相似文献
100.
A software tool, Sweet Substitute, is described, which assists tandem mass spectrometry (MS/MS)-based glycosylation characterization from within a tryptic digest. The algorithm creates a virtual nanoelectrospray-quadrupole time-of-flight style-MS/MS spectrum of any user-defined N-linked glycan structure. An empirical peak height modeling routine is implemented in the program. By comparing the theoretical MS/MS data with the deconvoluted and deisotoped experimental MS/MS data, the user is able to quickly assess whether a proposed candidate oligosaccharide structure is a plausible one. 相似文献