Dataset Information

Expression Profiling and Glycan Engineering of IgG Subclass 1-4 in Nicotiana benthamiana.

ABSTRACT: IgG, the main serum immunoglobulin isotype, exists in four subclasses which selectively appear with distinctive glycosylation profiles. However, very little is known about the biological consequences mainly due to the difficulties in the generation of distinct IgG subtypes with targeted glycosylation. Here, we show a comprehensive expression and glycan modulation profiling of IgG variants in planta that are identical in their antigen binding domain but differ in their subclass appearance. While IgG1, 2, and 4 exhibit similar expression levels and purification yields, IgG3 is generated only at low levels due to the in planta degradation of the heavy chain. All IgG subtypes are produced with four distinct N-glycosylation profiles, differing in sugar residues previously shown to impact IgG activities, i.e., galactosylation, sialylation and core fucosylation. Affinity purified IgG variants are shown to be fully assembled to heterodimers but display different biochemical/physical features. All subtypes are equally well amenable to targeted glycosylation, except sialylated IgG4 which frequently accumulates substantial fractions of unusual oligo-mannosidic structures. IgG variants show significant differences in aggregate formation and endotoxin contamination which are eliminated by additional polishing steps (size exclusion chromatography, endotoxin removal treatments). Collectively we demonstrate the generation of 16 IgG variants at high purity and large glycan homogeneity which constitute an excellent toolbox to further study the biological impact of the two main Fc features, subclass and glycosylation.

SUBMITTER: Kallolimath S

PROVIDER: S-EPMC7393800 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Expression Profiling and Glycan Engineering of IgG Subclass 1-4 in <i>Nicotiana benthamiana</i>.

Kallolimath Somanath S Hackl Thomas T Gahn Raphaela R Grünwald-Gruber Clemens C Zich Wilhelm W Kogelmann Benjamin B Lux Anja A Nimmerjahn Falk F Steinkellner Herta H

Frontiers in bioengineering and biotechnology 20200724

IgG, the main serum immunoglobulin isotype, exists in four subclasses which selectively appear with distinctive glycosylation profiles. However, very little is known about the biological consequences mainly due to the difficulties in the generation of distinct IgG subtypes with targeted glycosylation. Here, we show a comprehensive expression and glycan modulation profiling of IgG variants <i>in planta</i> that are identical in their antigen binding domain but differ in their subclass appearance. ...[more]

PMID: 32793574

Similar Datasets

Project description:Human rotaviruses are the main cause of severe gastroenteritis in children and are responsible for over 500 000 deaths annually. There are two live rotavirus vaccines currently available, one based on human rotavirus serotype G1P[8], and the other a G1-G4 P[8] pentavalent vaccine. However, the recent emergence of the G9 and other novel rotavirus serotypes in Africa and Asia has prompted fears that current vaccines might not be fully effective against these new varieties.We report an effort to develop an affordable candidate rotavirus vaccine against the new emerging G9P[6] (RVA/Human-wt/ZAF/GR10924/1999/G9P[6]) strain. The vaccine is based on virus-like particles which are both highly immunogenic and safe. The vaccine candidate was produced in Nicotiana benthamiana by transient expression, as plants allow rapid production of antigens at lower costs, without the risk of contamination by animal pathogens. Western blot analysis of plant extracts confirmed the successful expression of two rotavirus capsid proteins, VP2 and VP6. These proteins assembled into VLPs resembling native rotavirus particles when analysed by transmission electron microscopy (TEM). Expression of the rotavirus glycoprotein VP7 and the spike protein VP4 was also tried. However, VP7 expression caused plant wilting during the course of the time trial and expression could never be detected for either protein. We therefore created three fusion proteins adding the antigenic part of VP4 (VP8*) to VP6 in an attempt to produce more appropriately immunogenic particles. Fusion protein expression in tobacco plants was detected by western blot using anti-VP6 and anti-VP4 antibodies, but no regular particles were observed by TEM, even when co-expressed with VP2.Our results suggest that the rotavirus proteins produced in N. benthamiana are candidates for a subunit vaccine specifically for the G9P[6] rotavirus strain. This could be more effective in developing countries, thereby possibly providing a higher overall efficacy for the existing vaccines. The production of rotavirus proteins in plants would probably result in lower manufacturing costs, making it more affordable for developing countries. Further investigation is required to evaluate the immunogenic potential of the VLPs and fusion proteins created in this study.

Dataset Information

Expression Profiling and Glycan Engineering of IgG Subclass 1-4 in Nicotiana benthamiana.

Publications

Expression Profiling and Glycan Engineering of IgG Subclass 1-4 in <i>Nicotiana benthamiana</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets