Deep Mutational Scanning to Identify IgG1 Fc Mutations Affecting its Affinity to EndoS2 Öffentlichkeit

Saltzman, Abigail (Spring 2023)

Permanent URL: https://etd.library.emory.edu/concern/etds/t435gf52z?locale=de
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Abstract

Endoglycosidases are an important group of enzymes that hydrolyze oligosaccharides from glycoproteins. Because IgG antibodies use N-linked glycan on their fragment crystallizable (Fc) region to promote immune responses via effector functions, they can be inactivated by endoglycosidases. Streptococcus pyogenes, a gram-positive bacterial species, produces two novel endoglycosidases, EndoS and EndoS2 to evade host immune responses. EndoS and EndoS2 recognize both the IgG glycan and the Fc of the IgG. Specificity for the Fc glycoprotein backbone, rather than only the glycan, makes EndoS and EndoS2 unique among carbohydrate active enzymes and attractive targets to the field of immunotherapy. To take advantage of these enzymes or to counteract their natural pathogenic activity, their mechanism of substrate recognition must be understood. Consequently, describing the binding modes of EndoS2 and EndoS to the IgG Fc is an area of active research. The binding modes of the two enzymes to the Fc region are known to be quite similar. However, EndoS2 is of particular interest due to its ability to hydrolyze a wider range of glycans than EndoS. In this paper, we tested the effect of every single point mutation in the Fc and hinge sequence on Endo-S2 binding affinity using deep mutational scanning (DMS) and a mammalian surface display platform. We employed flow cytometry and cell sorting to collect the Fc mutants with the highest and lowest affinity for EndoS2 (top and bottom 15 %). Our data can help confirm which portions of the Fc are likely not involved with EndoS2 contact, thus narrowing down which residues may be most central to the complex formation. The scan also provides preliminary insight into which specific mutations at the relevant residues would affect EndoS2 binding. Identifying these mutations would be beneficial to the development of monoclonal antibodies with a different EndoS2 affinity, which could be used clinically.

Table of Contents

Table of contents

1. Introduction……….……………………………………………………1

2. Materials and Methods……….………………………………………..6

Expression and purification of two inactive EndoS2 mutants………..…………..............6

Biotinylation of EndoS2 mutants ......................................................................................6

Mammalian cells transfection…………….………………………………............…7

EndoS2 binding to Fc …………..……………………......................................... ...8

Deep mutational surface-display Fc library generation……………………………….…8

Fluorescence-activated cell sorting of Fc library to select mutants with different affinity to EndoS2…………………………………………………………………………………...9

High-throughput sequencing of sorted cell populations…………………………..……..9

Site directed mutagenesis of Fc wildtype to introduce mutations with different affinity for EndoS2……………………………………………………………………………….…10

Plasmid amplification and retrieval of Fc mutants……………………………………...11

3. Results…………………………………………………………………12

Expression, purification and biotinylation of EndoS2 mutants………………......….......12

A mammalian surface display platform for IgG1 Fc and hinge domains.........................12

A deep mutational scanning library of IgG1 Fc and hinge regions…………………...…14

Identification of Fc mutants with different affinity for EndoS2 ………….....................14

Validation of DMS experiments………………………………………………….…..….15

Comparing EndoS2 binding affinity and EndoS hydrolysis rates of Fc mutants…….….16

4. Discussion……………………………………………………………17

5. Figures……………………………………………………………….22

6. References…………………………………………………………...26 

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