Mass spectrometry as a tool for glycoengineering in yeast

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Abstract

Mass spectrometry as a tool for glycoengineering in yeast

Mari A Piirainen*, Heidi Salminen*, Alexander Frey, Aalto University

Introduction:
Baker’s yeast is a well-known organism that has been widely used as a production host for various products. Yeast is also a potential alternative host for the production of therapeutic proteins such as antibodies. Most of these proteins are N-glycosylated and are currently produced using mammalian cells. Our work utilizes mass spectrometric analysis of N-glycans and glycopeptides for developing yeast as an efficient expression system for full-length IgG production. We focus on protein N-glycosylation in yeast from two aspects: monitoring N-glycan intermediate structures along the secretory pathway, and creating human-compatible N-glycosylation by glycoengineering.

Methods:
In order to investigate the N-glycan processing in the secretory pathway, an anti-CD20 antibody was expressed in S. cerevisiae cells. Intracellular IgG was isolated, digested with trypsin, and analyzed by MALDI-TOF/TOF. For engineering the N-glycosylation pattern in yeast, genes relevant to the glycosylation process, such as glycosyltransferases and nucleotide sugar transporters, were deleted or overexpressed in yeast. The effects on the glycan pattern were assessed by isolating N-glycans from cell wall glycoproteins and analyzing them by MALDI-TOF MS.

Results:
Anti-CD antibody has two N-glycosylation sites, one on each heavy chain and in mass spectrum, signals corresponding to nonglycosylated peptide as well as to different glycoforms of the peptide were detected. Fragmentation of the assumed glycopeptides gave the typical fragment signal pattern, which confirmed the presence of N-glycans.
We have obtained a yeast strain that produces glycoproteins bearing a complex-type GlcNAc2Man3GlcNAc2 N-glycan. In addition, we could increase the relative abundance of this glycan by enhancing the nucleotide sugar transport into Golgi apparatus.

Conclusion:
N-glycosylation is a common posttranslational modification in therapeutic proteins. It has an impact on protein properties such as biological activity and stability, but N-glycans are also involved in protein folding in the secretory pathway. By investigating N-glycans or glycopeptides of IgG molecules with mass spectrometry, we can get information on the antibody’s passage through the secretory pathway and identify possible bottlenecks in the process. Along with humanization of the N-glycan structures, we are on the road to more efficient production of therapeutic glycoprotein in yeast.

Funding:
The work is funded by Academy of Finland project ProFold and Aalto University School of Chemical Engineering.

Details

Original languageEnglish
Pages1
Number of pages1
Publication statusPublished - 2017
EventFinnish Symposium on Biological Mass Spectrometry - Hämeenlinna, Finland
Duration: 31 Oct 20171 Nov 2017
Conference number: 2

Conference

ConferenceFinnish Symposium on Biological Mass Spectrometry
CountryFinland
CityHämeenlinna
Period31/10/201701/11/2017

ID: 16808798