All ETDs from UAB

Advisory Committee Chair

Matthew B Renfrow

Advisory Committee Members

Stephen Barnes

William J Placzek

Mark R Walter

Bruce A Julian

Document Type


Date of Award


Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine


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.



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