Purification and Characterization of a GDP-fucose:Polypeptide Fucosyltransferase from Chinese Hamster Ovary Cells

Yang Wang,Michael W Spellman

doi:10.1074/jbc.273.14.8112

Abstract

A GDP-fucose:polypeptide fucosyltransferase was purified 5000-fold to homogeneity from Chinese hamster ovary cell extracts in the absence of detergent. The purification procedure included two affinity chromatographic steps using the acceptor substrate, a recombinant factor VII EGF-1 domain, and the donor substrate analog, GDP-hexanolamine, as ligands. The purified enzyme migrates as a single band of 44,000 daltons on SDS-polyacrylamide gel electrophoresis and is itself a glycoprotein with more than one high mannose type oligosaccharide chain with a total molecular weight of 4000. The Km values for factor VII EGF-1 domain and GDP-fucose are 15 and 6 microM, respectively. The Vmax is 2.5 micromol.min-1.mg-1. The presence of 50 mM Mn2+ increased the enzyme activity 17-fold, but Mn2+ was not absolutely required, since the enzyme exhibited some activity even in the presence of EDTA. The acceptor substrate specificity was studied using site-directed mutagenesis of human factor IX EGF domain. Only one of several differently folded species could serve as acceptor substrate, although they all had the same molecular weight as determined by liquid chromatography on-line with mass spectrometry. This indicates that the enzyme requires proper folding of the epidermal growth factor domain for its activity.

Highlights

The identification of O-linked fucose attached to a specific protein was first made by Kentzer et al [1], who found a residue of fucose covalently linked through an O-glycosidic bond to the EGF1 domain of recombinant urokinase
Similar fucosylation was shown to occur on the EGF domain of human factor IX [6], but in this case the fucose residue was at the reducing end of a tetrasaccharide: NeuAc␣236Gal␤134GlcNAc␤133Fuc␣13O-Ser61 [7]
Structural analysis has revealed that O-linked fucose is present on an insect protease inhibitor [11], the flanking amino acids were not represented by the consensus sequence described above, and the protease inhibitor sequence is not homologous to an EGF domain

Summary

Introduction

The identification of O-linked fucose attached to a specific protein was first made by Kentzer et al [1], who found a residue of fucose covalently linked through an O-glycosidic bond to the EGF1 domain of recombinant urokinase. Studies by two other laboratories using the lec mutant cell line indicated that O-fucosylation occurs on many proteins in Chinese hamster ovary cells [9, 10]. The assay uses a recombinant human factor VII EGF-1 domain as acceptor substrate and GDP-fucose as donor substrate Using this assay, we were able to detect activity in extracts of Chinese hamster ovary cells and rat liver. When rat liver was homogenized in the presence of protease inhibitors, 37% of the activity was recovered by Triton X-100 extraction of the membrane particles after extensive aqueous washes These results suggest that the enzyme is probably a membrane protein and, by analogy with other glycosyltransferases, probably has a “stem” region that is very susceptible to proteolysis [13]. This study includes the initial characterization of the purified enzyme and its acceptor substrate specificity

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Results

Conclusion