Remodeling of sugar chain structures of human interferon- 

Abstract

Natural human interferon (IFN)-gamma has mainly biantennary complex-type sugar chains and scarcely has multiantennary structures. We attempted to remodel the sugar chain structures using IFN-gamma as a model glycoprotein. To obtain the branching glycoforms of IFN-gamma, we introduced the genes for GnT-IV (UDP-N-acetylglucosamine:alpha-1,3-D-mannoside beta-1, 4-N-acetylglucosaminyltransferase) and/or GnT-V (UDP-N-acetylglucosamine:alpha-1,6-D-mannoside beta-1, 6-N-acetylglucosaminyltransferase) into Chinese hamster ovary (CHO) cells producing human IFN-gamma. The parental CHO cells produced IFN-gamma with biantennary sugar chains mainly. When the GnT-IV activity was increased, triantennary sugar chains with a branch produced by GnT-IV increased up to 66.9% of the total sugar chains. When the GnT-V activity was increased, triantennary sugar chains with a corresponding branch increased up to 55.7% of the total sugar chains. When the GnT-IV and -V activities were increased at a time, tetraantennary sugar chains increased up to 56.2% of the total sugar chains. The proportion of these multiantennary sugar chains corresponded to the intracellular activities of GnT-IV and -V. What is more, lectin blot and flow cytometric analysis indicated that the multi-branch structure of the sugar chains was increased not only on IFN-gamma, one of the secretory glycoproteins, but also on almost CHO cellular proteins by introducing either or both of the GnT genes. The results suggest that the branching structure of sugar chains of glycoproteins could be controlled by cellular GnT-IV and GnT-V activities. This technology can produce glycoforms out of natural occurrence, which should enlarge the potency of glycoprotein therapeutics.

Enhancing recombinant glycoprotein sialylation through CMP‐sialic acid transporter over expression in Chinese hamster ovary cells

“…Potential demand for sialylation can vary from 4 moles of sialic acid per mole of IFN-g if predominantly complex biantennary structures are considered (Gu et al, 1997), to 14 moles of sialic acid per mole of EPO if predominantly complex tetraantennary structures are considered (Koury, 2003). When IFN-g branching was increased through over expression of GnT-IV and GnT-V (Fukuta et al, 2000b), increased sialylation also resulted (Fukuta et al, 2000a).…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update covering the period 1999–2000

“…Methods, including MALDI, for analyzing these glycosylation differences have recently been reviewed (Hooker & James, 1998). In experiments aimed at remodeling the carbohydrates attached to human interferon-g, Fukuta et al (2000) have introduced the genes for GlcNAc transferases-IVand -V into Chinese hamster ovary (CHO) cells producing the glycoprotein. The largely biantennary structures normally found attached to this glycoprotein were found to be augmented by additional tri-and tetra-antennary glycans, as revealed by MALDI analysis of their 2-AP derivatives, opening the possibility of engineering glycoproteins to provide improved function.…”

Control of Bisecting GlcNAc Addition to N-Linked Sugar Chains

“…GnT-III and ␤-1,4-GalT activities were measured using pyridylaminated agalactosyl biantennary sugar chain as a substrate as described in our preceding paper (36). The substrate was prepared as reported previously (31).…”

“…Next, the whole PA-sugar chains were desialylated with sialidase from Arthrobacter ureafaciens in 0.2 M ammonium acetate buffer, pH 5.0, for 20 h at 37°C. The desialylated sugar chains were separated as a neutral fraction by anion-exchange HPLC using a Mono Q column (5 ϫ 50 mm, Amersham Pharmacia Biotech) as described in our preceding paper (36).…”