title: Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans.
abstract: Understanding the biosynthetic pathway of protein glycosylation in various expression cell lines is important for controlling and modulating the glycosylation profiles of recombinant glycoproteins. We found that expression of erythropoietin (EPO) in a HEK293S N-acetylglucosaminyltransferase I (GnT I)(-/-) cell line resulted in production of the Man5GlcNAc2 glycoforms, in which more than 50% were core-fucosylated, implicating a clear GnT I-independent core fucosylation pathway. Expression of GM-CSF and the ectodomain of FcγIIIA receptor led to ∼30% and 3% core fucosylation, suggesting that the level of core fucosylation also depends on the nature of the recombinant proteins. To elucidate the GnT I-independent core fucosylation pathway, we generated a stable HEK293S GnT I(-/-) cell line with either knockdown or overexpression of FUT8 by a highly efficient lentivirus-mediated gene transfer approach. We found that the EPO produced from the FUT8 knockdown cell line was the pure Man5GlcNAc2 glycoform, whereas that produced from the FUT8-overexpressing cell line was found to be fully core-fucosylated oligomannose glycan (Man5GlcNAc2Fuc). These results provide direct evidence that FUT8, the mammalian α1,6-fucosyltransferase, is the sole enzyme responsible for the GnT I-independent core fucosylation pathway. The production of the homogeneous core-fucosylated Man5GlcNAc2 glycoform of EPO in the FUT8-overexpressed HEK293S GnT I(-/-) cell line represents the first example of production of fully core-fucosylated high-mannose glycoforms.
authors.affiliation: From the Department of Chemistry and Biochemistry, University of Maryland College Park, College Park, Maryland 20742.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Yang
authors.affiliation: From the Department of Chemistry and Biochemistry, University of Maryland College Park, College Park, Maryland 20742 wang518@umd.edu.
authors.forename: Lai-Xi
authors.initials: LX
authors.lastname: Wang
mesh_terms: D019943:Amino Acid Substitution; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005643:Fucose / Q000378:metabolism; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008358:Mannose / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin; D005643:Fucose; C058642:mannosyl(5)-N-acetyl(2)-glucose; D005647:Fucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; C020559:Glycoprotein 6-alpha-L-fucosyltransferase; D008358:Mannose
keywords: erythropoietin; fucosyltransferase; glycoprotein; glycoprotein biosynthesis; glycosylation