Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality

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Abstract

Introduction: Prolyl-4-hydroxylases ( P4H) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization.

Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana. Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality.

Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1.

Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants.

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MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability

Kazutaka Katoh, Daron Standley (2013)

We report a major update of the MAFFT multiple sequence alignment program. This version has several new features, including options for adding unaligned sequences into an existing alignment, adjustment of direction in nucleotide alignment, constrained alignment and parallel processing, which were implemented after the previous major update. This report shows actual examples to explain how these features work, alone and in combination. Some examples incorrectly aligned by MAFFT are also shown to clarify its limitations. We discuss how to avoid misalignments, and our ongoing efforts to overcome such limitations.

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SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Markus Hoffmann, Hannah Kleine-Weber, Simon Schroeder … (2020)

Summary The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.

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J Banchereau, R Steinman (1998)

B and T lymphocytes are the mediators of immunity, but their function is under the control of dendritic cells. Dendritic cells in the periphery capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses. They not only activate lymphocytes, they also tolerize T cells to antigens that are innate to the body (self-antigens), thereby minimizing autoimmune reactions. Once a neglected cell type, dendritic cells can now be readily obtained in sufficient quantities to allow molecular and cell biological analysis. With knowledge comes the realization that these cells are a powerful tool for manipulating the immune system.

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Author and article information

Contributors

Pia Uetz: URI : https://loop.frontiersin.org/people/2569441/overviewRole: Role: Role: Role: Role: Role:

Kathrin Göritzer: URI : https://loop.frontiersin.org/people/909558/overviewRole: Role: Role: Role: Role:

Emil Vergara: URI : https://loop.frontiersin.org/people/2427621/overviewRole: Role: Role:

Stanislav Melnik: URI : https://loop.frontiersin.org/people/180626/overviewRole: Role: Role: Role:

Clemens Grünwald-Gruber: Role: Role: Role:

Rudolf Figl: URI : https://loop.frontiersin.org/people/1127430/overviewRole: Role:

Ala-Eddine Deghmane: URI : https://loop.frontiersin.org/people/2561242/overviewRole: Role: Role:

Elisabetta Groppelli: Role: Role: Role:

Rajko Reljic: URI : https://loop.frontiersin.org/people/155662/overviewRole: Role: Role: Role:

Julian K.-C. Ma: URI : https://loop.frontiersin.org/people/572501/overviewRole: Role: Role: Role: Role:

Eva Stöger: URI : https://loop.frontiersin.org/people/125688/overviewRole: Role: Role: Role: Role: Role: Role:

Richard Strasser: URI : https://loop.frontiersin.org/people/27404/overviewRole: Role: Role: Role: Role: Role: Role:

Journal

Journal ID (nlm-ta): Front Bioeng Biotechnol

Journal ID (iso-abbrev): Front Bioeng Biotechnol

Journal ID (publisher-id): Front. Bioeng. Biotechnol.

Title: Frontiers in Bioengineering and Biotechnology

Publisher: Frontiers Media S.A.

ISSN (Electronic): 2296-4185

Publication date (Electronic): 06 March 2024

Publication date Collection: 2024

Volume: 12

Electronic Location Identifier: 1329018

Affiliations

[1] ¹ Department of Applied Genetics and Cell Biology , Institute of Plant Biotechnology and Cell Biology , University of Natural Resources and Life Sciences , Vienna, Austria

[2] ² Institute for Infection and Immunity , St George’s University of London , London, United Kingdom

[3] ³ Core Facility Mass Spectrometry , University of Natural Resources and Life Sciences , Vienna, Austria

[4] ⁴ Invasive Bacterial Infections Unit , Institut Pasteur , Université Paris Cité , Paris, France

Author notes

Edited by: Balamurugan Shanmugaraj, Chulalongkorn University, Thailand

Reviewed by: Sezer Okay, Hacettepe University, Türkiye

Somen Nandi, University of California, Davis, United States

*Correspondence: Richard Strasser, richard.strasser@ 123456boku.ac.at

Article

Publisher ID: 1329018

DOI: 10.3389/fbioe.2024.1329018

PMC ID: 10953500

PubMed ID: 38511130

SO-VID: 7c0d7122-2aeb-46ec-a44b-8226c61a5f84

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

History

Date received : 27 October 2023

Date accepted : 12 February 2024

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Austrian Science Fund (FWF) (W1224-B09).