Construction of Stable BHK-21 Cells Coexpressing Human Secretory Glycoproteins and Human Gal(beta1-4)GlcNAc-R alpha2,6-Sialyltransferase. alpha2,6-Linked NeuAc is Preferentially Attached to the Gal(beta1-4)GlcNAc(beta1-2)Man(alpha1-3)-Branch of Diantennary Oligosaccharides from Secreted Recombinant beta-Trace Protein

Grabenhorst, Eckart; Hoffman, Andrea; Nimtz, Manfred; Zettlmeissl, Gerd; Conradt, Harald S.

doi:10.1111/j.1432-1033.1995.tb20866.x

Cited by 14 publications

(20 citation statements)

References 23 publications

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“…On account of the small quantity of OvGST1b and the minute differences between both proteins observed, only two specific glycopeptides of OvGST1b were identified. Glycopeptide 1, consisting of amino acids 4 to 18 (GP1 [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] ) of OvGST1b, could not be detected and analyzed (Table 1). On all four glycosylation sites of OvGST1a, molecular ions with mass increments of 162 Da were detected, suggesting the presence of high-mannose type structures differing Fig.…”

Section: Resultsmentioning

confidence: 99%

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Structural Analysis and Antibody Response to the Extracellular GlutathioneS-Transferases fromOnchocerca volvulus

et al. 2001

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Onchocerca volvulus is a human pathogenic filarial parasite which, like other parasitic nematodes, is capable of surviving in an immunologically competent host by employing a variety of immune evasion strategies and defense mechanisms including the detoxification and repair mechanisms of the glutathione S-transferases (GSTs). In this study we analyzed the glycosylation pattern and the immunological properties of extracellular O. volvulus GST1a and -1b (OvGST1a and -1b). The enzymes differ in only 10 amino acids, and both are glycoproteins that have cleavable signal peptides and unusual N-terminal extensions. These characteristics have not been described for other GSTs so far. Mass spectrometry analyses indicate that both enzymes carry high-mannose type oligosaccharides on at least four glycosylation sites. Glycosylation sites 1 to 3 of OvGST1a

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Section: Resultsmentioning

confidence: 99%

“…The concentration of the analyte mixture was 1 to 10 pmol/l. Determination of the molecular masses was carried out by positive-ion MALDI-TOF MS using a Bruker REFLEX time-of-flight instrument as described by Grabenhorst et al (15).…”

Section: Maldi-tof Msmentioning

confidence: 99%

Structural Analysis and Antibody Response to the Extracellular GlutathioneS-Transferases fromOnchocerca volvulus

et al. 2001

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“…Genetic augmentation of sialyltransferase activity in mammalian cells has not consistently increased the total sialic acid content of glycoproteins. Although Weikert et al (1999) demonstrated that overexpression of ␣2,3-sialyltransferase in CHO cells resulted in increased glycoprotein sialylation (>90% of branches), expression of ␣2,6-sialyltransferase in BHK-21 cells had no such effect, although ␣2,6-linked sialic acids were present (Grabenhorst et al, 1995).…”

Section: Effect Of N-acetylmannosamine On Cell Growth and N-glycosylamentioning

confidence: 96%

Metabolic control of recombinant protein N‐glycan processing in NS0 and CHO cells

Baker

Rendall

Hills

et al. 2001

Biotech & Bioengineering

167

116

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Chinese hamster ovary and murine myeloma NS0 cells are currently favored host cell types for the production of therapeutic recombinant proteins. In this study, we compared N-glycan processing in GS-NS0 and GS-CHO cells producing the same model recombinant glycoprotein, tissue inhibitor of metalloproteinases 1. By manipulation of intracellular nucleotide-sugar content, we examined the feasibility of implementing metabolic control strategies aimed at reducing the occurrence of murine-specific glycan motifs on NS0-derived recombinant proteins, such as Galalpha1,3Galbeta1,4GlcNAc. Although both CHO and NS0-derived oligosaccharides were predominantly of the standard complex type with variable sialylation, 30% of N-glycan antennae associated with NS0-derived TIMP-1 terminated in alpha1,3-linked galactose residues. Furthermore, NS0 cells conferred a greater proportion of terminal N-glycolylneuraminic (sialic) acid residues as compared with the N-acetylneuraminic acid variant. Inclusion of the nucleotide-sugar precursors, glucosamine (10 mM, plus 2 mM uridine) and N-acetylmannosamine (20 mM), in culture media were shown to significantly increase the intracellular pools of UDP-N-acetylhexosamine and CMP-sialic acid, respectively, in both NS0 and CHO cells. The elevated UDP-N-acetylhexosamine content induced by the glucosamine/uridine treatment was associated with an increase in the antennarity of N-glycans associated with TIMP-1 produced in CHO cells but not N-glycans associated with TIMP-1 from NS0 cells. In addition, elevated UDP-N-acetylhexosamine content was associated with a slight decrease in sialylation in both cell lines. The elevated CMP-sialic acid content induced by N-acetylmannosamine had no effect on the overall level of sialylation of TIMP-1 produced by both CHO and NS0 cells, although the ratio of N-glycolylneuraminic acid:N-acetylneuraminic acid associated with NS0-derived TIMP-1 changed from 1:1 to 1:2. These data suggest that manipulation of nucleotide-sugar metabolism can promote changes in N-glycan processing that are either conserved between NS0 and CHO cells or specific to either NS0 cells or CHO cells.

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“…In addition to the identification of tyrosine sulfate residues detected with the ESI technique, we were also able to identify the dominant glycoforms of all three potential glycopeptides [NLSMPLLPADFHK (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42), DFVNASSK(166-173), SMTNR(T) (365-370)] after desialylation by mild acid hydrolysis ( Table 1). As the corresponding unmodified peptides were neither detectable by MALDI nor by ESI-MS, we deduce from these results that all three potential HCII N-glycosylation sites are completely glycosylated.…”

Section: R E S U L T S Expression Of Hcii From Cho Cellsmentioning

confidence: 99%

“…Approximately 60% of the triantennary structures isolated from the serum HCII contained a fucose residue. In order to determine the linkage position of the fucose to the triantennary structure, ESI-MS/MS analysis was performed on the triply charged molecular ion [M + 3Na] 3+ after isolation of the trisialylated oligosaccharide fraction by anion exchange chromatography on a Mono Q column [31]. From the resulting daughter ion spectrum (Fig.…”

Section: Detailed Characterization Of the N-glycans From Serum And Chmentioning

confidence: 99%

Tyrosine sulfation and N‐glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin binding

Böhme

Nimtz²,

Grabenhorst³

et al. 2002

European Journal of Biochemistry

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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 a2 fi 6 linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sLe x motif. Proximal a1 fi 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 a2 fi 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.

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Cited by 14 publications

References 23 publications

Structural Analysis and Antibody Response to the Extracellular GlutathioneS-Transferases fromOnchocerca volvulus

Structural Analysis and Antibody Response to the Extracellular GlutathioneS-Transferases fromOnchocerca volvulus

Metabolic control of recombinant protein N‐glycan processing in NS0 and CHO cells

Tyrosine sulfation and N‐glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin binding

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