Heterogeneity in the tyrosine sulfation Chinese hamster ovary cell produced recombinant FVIII

Mikkelsen, J; Thomsen, Johannes; Ezban, Mirella

doi:10.1021/bi00220a013

Cited by 32 publications

(22 citation statements)

References 28 publications

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“…The nearly complete tyrosine sulfation of the recombinant HCII molecules was unexpected, as several reports showed incomplete sulfate ester modification of recombinant proteins expressed in CHO cell lines [42,43]. Such individual differences indicate that the efficiency of tyrosine sulfation may depend on additional signals [44] and/or accessability to the modifying enzyme.…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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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Section: Discussionmentioning

confidence: 99%

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

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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“…Although several features of consensus sites for tyrosine sulfation have been described (43), tyrosine sulfation in some proteins is incomplete (44). Further studies of the substrate specificity of the tyrosine protein sulfotransferase are needed to define the features required for optimal tyrosine sulfation.…”

Section: Psgl-1 Residues Required For P-selectin Bindingmentioning

confidence: 99%

Identification of N-terminal Residues on P-selectin Glycoprotein Ligand-1 Required for Binding to P-selectin

Liu¹,

Ramachandran²,

Kang

et al. 1998

Journal of Biological Chemistry

103

126

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The major high affinity ligand for P-selectin on human leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). To bind P-selectin, PSGL-1 must be modified with tyrosine sulfate and sialylated, fucosylated, core-2 O-glycan(s). The required sites for these modifications on full-length PSGL-1 have not been defined. The N-terminal region of mature PSGL-1, which begins at residue 42, includes tyrosines at residues 46, 48, and 51, plus potential sites for Thr-linked O-glycans at residues 44 and 57. We expressed full-length PSGL-1 constructs with substitutions of these residues in transfected Chinese hamster ovary cells. The cells were co-transfected with cDNAs for the glycosyltransferases required to construct sialylated and fucosylated, core-2 O-glycans on PSGL-1. The transfected cells were assayed for their abilities to bind fluid-phase P-selectin and to support rolling adhesion of pre-B cells expressing P-selectin under hydrodynamic flow. In both assays, substitution of Thr-57 with alanine eliminated binding of PSGL-1 to P-selectin without affecting sulfation of PSGL-1, whereas substitution with serine, to which an O-glycan might also be attached, did not affect binding. Binding was not altered by substituting alanines for the two amino acids on either side of Thr-57, or by substituting alanine for Thr-44. Substitution of all three tyrosines with phenylalanines markedly reduced sulfation and prevented binding to P-selectin. However, all constructs in which one or two tyrosines were replaced with phenylalanines bound P-selectin. These results suggest that full-length PSGL-1 requires an O-glycan attached to Thr-57 plus sulfation of any one of its three clustered tyrosines to bind P-selectin.The selectins are Ca 2ϩ

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“…Sulfation of the tyrosine residues in this region was originally reported to have no effect on the biochemical properties of CHO-derived rFVIII, including procoagulant activity, vWF binding and thrombin activation. 4 However, the authors later proposed that lack of sulfation of FVIII in this region was associated with a decrease in FVIII activity.5 Variation in retention behavior in reversed-phase (RP) liquid chromatography has been reported as evidence for heterogeneity in the sulfation in this region for CHO-derived FVIII. 4 Here, we report peptide mapping studies of tyrosine sulfation of recombinant human Factor VIII derived from baby hamster kidney (BHK) cells using liquid chromato-…”

mentioning

confidence: 99%

“…4 However, the authors later proposed that lack of sulfation of FVIII in this region was associated with a decrease in FVIII activity.…”

mentioning

confidence: 99%

Characterization of tyrosine sulfate residues in antihemophilic recombinant factor VIII by liquid chromatography electrospray ionization tandem mass spectrometry and amino acid analysis

Severs

Carnine

Eguizabal

et al. 1999

Rapid Commun. Mass Spectrom.

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Recombinant Factor VIII (rFVIII) is involved in the cascade of biochemical reactions leading to blood coagulation and is used for the treatment of haemophilia A. Plasma-derived FVIII (pdFVIII) has been reported to be post-translationally modified by sulfation of tyrosine residues at positions 346, 1664, 1680, 718, 719 and 723. This report describes the quantitation of tyrosine sulfate residues in BHK-derived, human rFVIII by amino acid composition analysis and the identification of their positions in the polypeptide sequence using a combination of liquid chromatography and electrospray ionization mass spectrometry in the analysis of proteolytic digests of the protein.

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Heterogeneity in the tyrosine sulfation Chinese hamster ovary cell produced recombinant FVIII

Cited by 32 publications

References 28 publications

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

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

Identification of N-terminal Residues on P-selectin Glycoprotein Ligand-1 Required for Binding to P-selectin

Characterization of tyrosine sulfate residues in antihemophilic recombinant factor VIII by liquid chromatography electrospray ionization tandem mass spectrometry and amino acid analysis

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