Advisory Committee Chair
Matthew B Renfrow
Advisory Committee Members
Stephen Barnes
William J Placzek
Mark R Walter
Bruce A Julian
Document Type
Dissertation
Date of Award
2018
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
The autoimmune disease IgA nephropathy (IgAN) is a common cause of primary glo-merulonephritis, often resulting in end-stage renal disease. IgAN patients have elevated serum levels of immunoglobulin A1 (IgA1) with autoantigenic galactose-deficient (gd) O-glycans. The gd-O-glycans occur at specific sites within IgA1 hinge-region. We hy-pothesized that GalNAc-Ts play a role in the formation of gd-IgA1, due to their role in determining glycosylation sites and densities. To better understand how GalNAc-Ts may change O-glycans of IgA1 in IgAN we characterized the complex glycosylation mecha-nism involved in mucin-type O-glycan synthesis of IgA1 hinge-region. In the first section of this work, we explore the utility of using nanoLC-MS cou-pled with glycoform-specific relative quantification analysis, to track the activities and progression of enzymes involved in the clustered mucin-type glycosylation of IgA1 hinge-region. We demonstrate the progressive addition of GalNAc to IgA1 hinge-region at glycoform-specific resolution and compare the profiles for GalNAc-T2 and GalNAc-T2 ΔLD. By fitting the progressive reaction curves to reaction-scheme models, we de-rived rates for each glycoforms and show glycoform specific differences for the two en-zyme variants. We then demonstrate the utility of the method for optimization of glyco-sylation reactions by C1GalT1, and for analyzing ST6GalNAc-II and ST3Gal-I competi-tive activity towards native IgA1. In the second section of this work, we show the mechanisms by which GalNAc-T2 initiates glycosylation of IgA1 hinge-region. We characterize a semi-ordered path-way of GalNAc addition that plays a role in glycosylation-site selection and glycosyla-tion density. We define two glycosylation motifs recognized by GalNAc-T2 as the pre-dominant two of the four initial sites of glycosylation. Our data demonstrates a role for both the catalytic domain and the lectin domain of GalNAc-T2 during the process of semi-ordered glycosylation of IgA1 hinge-region. We define a role for glycopeptide isomeric diversity in increasing glycan density and identify features of the catalytic do-main and lectin domain that either increase or decrease glycopeptide isomeric diversity. Together, this work uncovers roles for GalNAc-Ts during initiation of IgA1 hinge-region glycosylation and establish methods for better understanding altered syn-thesis of IgA1 hinge-region O-glycans in IgAN.
Recommended Citation
Stewart, Tyler, "The Role Of Glycosylation Initiation By Galnac-Ts In Iga Nephropathy" (2018). All ETDs from UAB. 3046.
https://digitalcommons.library.uab.edu/etd-collection/3046