Degree Type

Dissertation

Date of Award

2019

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Major

Molecular, Cellular and Developmental Biology

First Advisor

Barb . Adam

Second Advisor

Eric S. Underbakke

Abstract

Asparagine (N) linked glycosylation is one of the most prevalent protein modifications; nearly all cell surface and secreted proteins are N-glycosylated. The N-glycan structure present on a protein depends on cell’s expression levels of glycan remodeling enzymes, interaction of the N-glycan with glycan or polypeptide residues and substrate availability. These factors contribute to a vast diversity of N-glycan structures that can be present on a protein expressed by a specific cell and renders N-glycan characterization difficult.

Identification of N-glycans on endogenous immunological cell surface receptors pose an additional challenge. Availability of surface receptors is relatively limited. Nonetheless, they represent an important class of proteins which regulate vital immune functions. FcγRⅢA (CD16a) is one such receptor expressed by human natural killer cell (NK cell) and monocyte. Engagement with its ligand, immunoglobulin G (IgG), triggers target cell death and cytokine release. This potential of CD16a signaling has been used to develop monoclonal antibody therapies, wherein a higher CD16a-IgG affinity correlates with better efficacy. CD16a has five N-glycosylation sites and in-vitro binding studies have shown that glycans at N45 and N162 are important for high affinity CD16a-IgG1 interactions.

In this study we determined the site-specific N-glycosylation profile of CD16a isolated from NK cells and monocytes isolated from individual donors. An immunoprecipitation strategy was developed to maximize protein yields. Moreover, liquid chromatography coupled mass spectrometry methods were optimized to identify the CD16a glycoforms. Tetraantennary complex type N-glycans with LacNAc repeats were identified at N38 and N74, while biantennary complex type N-glycans at N162 and N169. N45 had higher abundance of relatively under-processed glycoforms. We also identified donor-dependent variability at N162 and N45, the two sites implicated in high affinity interaction with IgG1. There was preferential retention of IgG1 with afucosylated N-glycans on NK cell surface suggesting priming of NK cell with pro-inflammatory IgG1. This approach highlights the importance of determining site- and donor-specific N-glycosylation of cell surface receptors and will aid in developing functional studies elucidating role of N-glycosylation in immune response.

Copyright Owner

Kashyap Rajeshbhai Patel

Language

en

File Format

application/pdf

File Size

128 pages

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