β1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human β1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of bi-antennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat α2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.
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We thank Dr. Aart Lammers (Animal Sciences Group, Wageningen UR) and Dr. Joop Arts (Animal Sciences Group, Wageningen UR) for chicken and zebrafish cDNA, respectively, and Pim Vergeer, Koen Varossieau and Anne Miltenburg for carrying out plant transformations.
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Hesselink, T., Rouwendal, G.J.A., Henquet, M.G.L. et al. Expression of natural human β1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans. Transgenic Res 23, 717–728 (2014). https://doi.org/10.1007/s11248-014-9806-z
- MALDI-TOF MS