BGAL1 depletion boosts the level of β-galactosylation of N- and O-glycans in N. benthamiana

Glyco-design of proteins is a powerful tool in fundamental studies of structure-function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advan...

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Main Authors: Kriechbaum, R, Ziaee, E, Grünwald-Gruber, C, Buscaill, P, van der Hoorn, RAL, Castilho, A
Format: Journal article
Language:English
Published: Wiley 2020
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author Kriechbaum, R
Ziaee, E
Grünwald-Gruber, C
Buscaill, P
van der Hoorn, RAL
Castilho, A
author_facet Kriechbaum, R
Ziaee, E
Grünwald-Gruber, C
Buscaill, P
van der Hoorn, RAL
Castilho, A
author_sort Kriechbaum, R
collection OXFORD
description Glyco-design of proteins is a powerful tool in fundamental studies of structure-function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco-engineering facilitate customized N-glycosylation of plant-derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human-like β1,4-galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4-galactosyltransferase, many plant-derived glycoproteins still exhibit incomplete processed N-glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β-galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4- and β1,3-galactose residues on both N- and O-glycans. Transient BGAL1 down-regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β-galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N-glycans on several plant-produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N-glycans of plant-produced glycoproteins.
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spelling oxford-uuid:424d6ca3-b13c-4096-86a4-ba96c8da28bb2022-03-26T14:48:43ZBGAL1 depletion boosts the level of β-galactosylation of N- and O-glycans in N. benthamianaJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:424d6ca3-b13c-4096-86a4-ba96c8da28bbEnglishSymplectic ElementsWiley2020Kriechbaum, RZiaee, EGrünwald-Gruber, CBuscaill, Pvan der Hoorn, RALCastilho, AGlyco-design of proteins is a powerful tool in fundamental studies of structure-function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco-engineering facilitate customized N-glycosylation of plant-derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human-like β1,4-galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4-galactosyltransferase, many plant-derived glycoproteins still exhibit incomplete processed N-glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β-galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4- and β1,3-galactose residues on both N- and O-glycans. Transient BGAL1 down-regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β-galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N-glycans on several plant-produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N-glycans of plant-produced glycoproteins.
spellingShingle Kriechbaum, R
Ziaee, E
Grünwald-Gruber, C
Buscaill, P
van der Hoorn, RAL
Castilho, A
BGAL1 depletion boosts the level of β-galactosylation of N- and O-glycans in N. benthamiana
title BGAL1 depletion boosts the level of β-galactosylation of N- and O-glycans in N. benthamiana
title_full BGAL1 depletion boosts the level of β-galactosylation of N- and O-glycans in N. benthamiana
title_fullStr BGAL1 depletion boosts the level of β-galactosylation of N- and O-glycans in N. benthamiana
title_full_unstemmed BGAL1 depletion boosts the level of β-galactosylation of N- and O-glycans in N. benthamiana
title_short BGAL1 depletion boosts the level of β-galactosylation of N- and O-glycans in N. benthamiana
title_sort bgal1 depletion boosts the level of β galactosylation of n and o glycans in n benthamiana
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