Glycoform modification of secreted recombinant glycoproteins through kifunensine addition during transient vacuum agroinfiltration

Yongao Xiong, Qiongyu Li, Muchena J. Kailemia, Carlito B Lebrilla, Somen Nandi, Karen A. McDonald

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Kifunensine, a potent and selective inhibitor of class I α-mannosidases, prevents α-mannosidases I from trimming mannose residues on glycoproteins, thus resulting in oligomannose-type glycans. We report for the first time that through one-time vacuum infiltration of kifunensine in plant tissue, N-linked glycosylation of a recombinant protein transiently produced in whole-plants shifted completely from complex-type to oligomannose-type. Fc-fused capillary morphogenesis protein 2 (CMG2-Fc) containing one N-glycosylation site on the Fc domain, produced in Nicotiana benthamiana whole plants, served as a model protein. The CMG2-Fc fusion protein was produced transiently through vacuum agroinfiltration, with and without kifunensine at a concentration of 5.4 μM in the agroinfiltration suspension. The CMG2-Fc N-glycan profile was determined using LC-MS/MS with a targeted dynamic multiple reaction monitoring (MRM) method. The CMG2-Fc expression level in the infiltrated plant tissue and the percentage of oligomannose-type N-glycans for kifunensine treated plants was 874 mg/kg leaf fresh weight (FW) and 98.2%, respectively, compared to 717 mg/kg leaf FW and 2.3% for untreated plants. Oligomannose glycans are amenable to in vitro enzymatic modification to produce more human-like N-glycan structures that are preferred for the production of HIV-1 viral vaccine and certain monoclonal antibodies. This method allows glycan modifications using a bioprocessing approach without compromising protein yield or modification of the primary sequence, and could be expanded to other small molecule inhibitors of glycan-processing enzymes. For recombinant protein targeted for secretion, kifunensine treatment allows collection of glycoform-modified target protein from apoplast wash fluid (AWF) with minimal plant-specific complex N-glycan at higher starting purity and concentration than in whole-leaf extract, thus simplifying the downstream processing.

Original languageEnglish (US)
Article number890
JournalInternational Journal of Molecular Sciences
Volume19
Issue number3
DOIs
StatePublished - Mar 17 2018

Fingerprint

Glycoproteins
Vacuum
Polysaccharides
proteins
Proteins
vacuum
mannosyl-oligosaccharide 1,2-alpha-mannosidase
Glycosylation
Recombinant proteins
leaves
Tissue
inhibitors
Recombinant Proteins
bioprocessing
Monoclonal antibodies
Vaccines
Trimming
Processing
Infiltration
vaccines

Keywords

  • Apoplast wash fluid
  • Fc-fusion protein
  • Kifunensine
  • N-glycosylation
  • Nicotiana benthamiana
  • Transient protein expression

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

Cite this

Glycoform modification of secreted recombinant glycoproteins through kifunensine addition during transient vacuum agroinfiltration. / Xiong, Yongao; Li, Qiongyu; Kailemia, Muchena J.; Lebrilla, Carlito B; Nandi, Somen; McDonald, Karen A.

In: International Journal of Molecular Sciences, Vol. 19, No. 3, 890, 17.03.2018.

Research output: Contribution to journalArticle

Xiong, Yongao ; Li, Qiongyu ; Kailemia, Muchena J. ; Lebrilla, Carlito B ; Nandi, Somen ; McDonald, Karen A. / Glycoform modification of secreted recombinant glycoproteins through kifunensine addition during transient vacuum agroinfiltration. In: International Journal of Molecular Sciences. 2018 ; Vol. 19, No. 3.
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