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 Man 5 GlcNAc 2 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 Fc␥IIIA receptor led to ϳ30% 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 Man 5 GlcNAc 2 glycoform, whereas that produced from the FUT8-overexpressing cell line was found to be fully core-fucosylated oligomannose glycan (Man 5 GlcNAc 2 Fuc). These results provide direct evidence that FUT8, the mammalian ␣1,6-fucosyltransferase, is the sole enzyme responsible for the GnT I-independent core fucosylation pathway. The production of the homogeneous core-fucosylated Man 5 GlcNAc 2 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.

Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation

“…Recently, we observed unexpected high corefucosylation of Man5GlcNAc2 (M5) glycan (more than 50%) of EPO produced from a HEK293S GnTI knockout cell line 33 . To characterize the underlying mechanism and to increase the fucosylation level of EPO-M5 as a potential starting material for glycan remodeling, we have generated a 293S GnTĪFUT8↑ cell line by lentiviral-mediated gene transfer, and found that expression of EPO from this cell line yielded almost 100% homogenous M5F glycan 33 . This successful generation of this unusual glycoform prompted us to explore it to obtain the Fucα1,6GlcNAc-EPO as the key precursor for EPO glycan remodeling.…”

“…The cells were passaged every 3-4 days with the initial seeding density of 3 ×10 5 cells per ml. The culture of HEK293S GnTĪFUT8↑followed the procedures previously reported by Yang et al 33 .…”

“…The PNGaseF-released N-glycan was analyzed with an Autoflex III Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker Daltonics). The release, clean-up, and analysis of glycan followed the previously reported procedures 33 .…”

“…Nevertheless, the complexity and multi-step manipulations including the construction of complex glycopeptide building blocks, the often low efficiency in native chemical ligation, and the folding of synthetic glycoproteins still pose great challenge in producing significant amount of pure glycoproteins. We have recently demonstrated that expression of EPO in an engineered HEK293 cell line with overexpression of FUT8 (HEK293S GnT ĪFUT8↑) resulted in the production of an unusual, fully fucosylated Man 5 GlcNAc 2 glycoform of EPO (EPO-M5F, 4 ) 33 . We report in this paper that the fucosylated Man 5 GlcNAc 2 glycoform of EPO could serve as an excellent starting material for endoglycosidase-catalyzed glycan remodeling to provide sialylated or azide-tagged glycoforms of EPO.…”

Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

“…In animals and humans, core-fucosylation is catalyzed solely by the mammalian α1,6-fucosyltransferase, FUT8 27,28 . However, FUT8 has a very strict substrate specificity, requires the presence of a free GlcNAc at the α1,3-linked mannose arm in the N-glycan as the substrate and usually is unable to fucosylate full-size mature N-glycans 29–32 .…”