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The HIV-1 envelope glycoprotein trimer (Env) is covered by an extensive array of glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints that limit the actions of glycan processing enzymes, such that multiple under-processed structures remain on specific locations. These oligomannose-type glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, perhaps paradoxically, target it. In multiple studies, bNAbs have been shown to be capable of providing passive protection from viral challenge, making Env a focus of antibody-mediated vaccine design. Here, the development of a workflow for the semi-quantitative, site-specific N-glycosylation analysis of a soluble recombinant, native-like trimer mimic (BG505 SOSIP.664) is reported. The resulting data reveal a mosaic of dense clusters on the outer domain of Env and allow mapping the extremes of simplicity and diversity of glycan processing. Although individual sites usually minimally affect the global integrity of the glycan shield, examples are identified of how deleting certain glycans can subtly influence neutralization by bNAbs that bind at distant sites. Env is a trimer of heterodimers of gp120 and gp41, which is generated by cleavage of an endogenous protease. In this thesis, the detailed effect of protease cleavage on glycan processing is examined by comparing the site-specific N-glycosylation profiles of the native-like trimer mimic to the corresponding uncleaved pseudotrimer and the matched gp120 monomer. Trimer-associated glycan remodeling forms a localized subdomain of the native mannose patch. Furthermore, the glycosylation analysis of further Env immunogens – a glycan-depleted trimer and a flexibly-linked, uncleaved trimer (both based on BG505 SOSIP.664) – provides important insights into the robustness of the HIV-1 glycan shield and the Env maturation pathway. Overall, this thesis reveals how structural constraints shape Env glycosylation and the network of bNAb-targeted glycans that should be preserved on recombinant vaccine candidates.

Immunological function declines with age, increasing the susceptibility and severity of infections in the elderly. For example, those who are 65 years of age and older make up around 15% of the U.S. population but represents nearly 90% of the deaths associated with influenza. This age-associated reduction in immunity has been attributed to the dysfunction of both the innate and adaptive immune system. Indeed, functional capacity of T cells is severely diminished and is exacerbated with age. Our lab has revealed that N-linked glycans play a critical role in controlling T cell immunity in mice and humans. Furthermore, irregularity in N-glycosylation as a mechanism for dysfunction of T cells in the elderly has not been examined as of yet. Here we report N-glycan branching in T cells increases with age in females, leading to T cell hypo-activity and increased susceptibility to infection. Reducing N-glycan branching rejuvenates T cell activation, proliferation and pro-inflammatory TH17 over anti-inflammatory Treg differentiation in aged female T cells. Susceptibility of aged female mice to Salmonella typhimurium invasion/dissemination was reduced by lowering N-glycan branching. A critical metabolic precursor of N-glycosylation and branching is N-acetylglucosamine (GlcNAc), a common amino sugar that is part of the regular human diet. GlcNAc is endogenous to human serum, increases with age and correlates with N-glycan branching in female > male human T cells. Interleukin-7 (IL-7) signaling synergistically regulates N-glycan branching with environmental GlcNAc. Both adoptive transfer of aged female T cells into young mice and negatively regulating IL-7 reversed aged-dependent increase in N-glycan branching. These results suggest that age-dependent increases of N-glycan branching, via increased supply of GlcNAc and IL-7 signaling, impairs T cell immunity in the elderly females, providing a novel mechanism to supplement current treatments for age-related diseases.

Eukaryotic cells employ vast and intricate molecular mechanisms to carry out biological functions that help them thrive. One such mechanism is the dynamic movement and reorganizing of the plasma membrane in order to facilitate inter- and intra-cellular events via receptor and/or ligand engagement. The plasma membrane is surrounded by a very dense layer of sugars, or glycans, which extend from glycolipids and glycoproteins, and can interact with galectins, a family of sugar-binding proteins. These interactions form molecular networks called galectin-glycoprotein lattices that function to control the localization, clustering, and retention of glycoproteins to globally regulate cell activation, growth, arrest, and differentiation.Glycoproteins are built in the endoplasmic reticulum and the Golgi apparatus, in which proteins are folded and then extensively modified with glycans before transport to the cell surface. In the Golgi, the N-acetylglucosaminyl transferases (Mgat1, 2, 4, and 5) mediate the branching of Asparagine (N)-linked glycans in an “assembly line” manner to progressively increase the production of ligands for galectins. More N-glycan branching increases galectin avidity to maintain lattice integrity, cellular homeostasis, and appropriate functional outcomes. Less N-glycan branching reduces galectin avidity, weakens the lattice, and promotes cellular dysfunction and disease states. We have shown the galectin-glycoprotein lattice and the N-glycan branching pathway are critical regulators of T cells. An intact lattice on T cells promotes basal TCR signaling and appropriate activation states. Furthermore, the lattice inhibits pro-inflammatory TH1/TH17 differentiation, and promotes humoral/immunomodulatory TH2 and anti-inflammatory iTREG differentiation. When the lattice is weakened due to reduced N-glycan branching, T cells become hyperactive and more pro-inflammatory, exacerbating autoimmunity (i.e., EAE and MS). How the galectin-glycoprotein lattice and N-glycan branching regulates B cells has not been shown before. We provide evidence N-glycan branching promotes B cell generation, differentially regulates CD19/BCR and TLR surface expression and signaling, and affects antigen presentation to CD4+ T cells to influence TH1, TH17, and iTREG differentiation. Furthermore, reduced N-glycan branching in B cells resulted in more severe EAE progression. These studies have begun to delineate how the galectin-glycoprotein lattice regulates B cell homeostasis, and may have implications in B cell targeting therapies.

Oral squamous cell carcinoma (OSCC) accounts for majority of head and neck cancers and ranks as the sixth most common cancer in the world. OSCC belongs to the most understudied cancers and little is known about molecular mechanisms underlying its etiology and progression to metastasis. A hallmark of cancer is the enhanced posttranslational modification of cell surface proteins with complex N-glycans. Our studies have shown that induced protein N-glycosylation via activation of the core N-glycosylation-regulating gene, DPAGT1, is associated with reduced E-cadherin adhesion, as well as deregulation of several oncogenic signaling pathways, including Wnt/β-catenin and Hippo. Modest increases in DPAGT1 expression are associated with dramatic amplification of Wnt/β-catenin activity and increased expression and nuclear localization of the Hippo pathway effectors TAZ /YAP. The goal of this study was to align the expression and localization of DPAGT1, complex N-glycans, β-catenin, and TAZ/YAP with the progression of oral cancer in vivo from dysplasia to OSCC. Human oral tissues from different stages of OSCC pathogenesis were characterized for DPAGT1/β-catenin/α-catenin/YAP/TAZ expression and localization and correlated with cell surface expression of complex N-glycans by PHA lectin staining and with expression of primitive cell surface markers, CD44, CD24 and CD29. Results showed that high DPAGT1 expression and nuclear TAZ became increasingly associated with disorganized E-cadherin junctions as oral epithelium progressed from mild to severe dysplasia to OSCC. This correlated with increasing expression of cell surface complex N-glycans and CD44. These studies suggest that DPAGT1/β-catenin/TAZ and high PHA staining represent novel signatures for OSCC pathogenesis.

Plant secretory peroxidases are important commercial enzymes and play a central role in plant stress responses. The focus of this work is a unique peroxidase from the leaves of a cold tolerant palm, Trachycarpus fortunei, (i.e., windmill palm tree). Like other palm tree peroxidases, windmill palm tree peroxidase is stable at high temperatures and in the presence of denaturants. It is distinct from other palm tree peroxidases in its substrate specificity. The amino acid sequence and glycosylation had not been determined. Because glycosylation is known to play a critical role in plant peroxidase stability and activity, this knowledge is essential for structure activity studies, selection of an expression system for enzyme production, and engineering the enzyme. Glycosylation is a complex modification that is difficult to study; furthermore, there is a lack of analytical tools for characterizing plant glycoproteins. The complete amino acid sequence was determined using cDNA sequencing and biological mass spectrometry. The mature amino acid sequence is 306 residues in length. The presence of a C-terminal signal peptide predicts vacuolar targeting of the enzyme. Native windmill palm tree peroxidase was analyzed at the glycopeptide level to give a qualitative and quantitative assessment of glycosylation at each site. Windmill palm tree peroxidase has 13 sites for N-linked glycosylation, 2 of which are unique. Each site is at least partially occupied by a glycan. Major glycans are paucimannosidic, which supports the assignment of windmill palm tree peroxidase as a vacuolar peroxidase. To carry-out this work, a workflow for analyzing the glycopeptide mass spectrometry data was developed. Included in the workflow are novel tools for glycan database construction, pGlycoFilter, and validation of glycopeptide assignment, gPSMvalidator. New analytical methods are needed for the emerging field of plant glycoproteomics. The novel methods developed in this dissertation will be useful for the study of other important plant glycoproteins. This knowledge can be used to study the roles of glycosylation in this exceptionally stable and unique palm peroxidase.

Rapidly proliferating cells such as T cells undergo the Warburg effect [1-3], a long-standing and poorly understood phenomenon that increases glucose uptake yet paradoxically reduces energy from glucose via a switch from oxidative phosphorylation to aerobic glycolysis. This has been rationalized as increased requirements for glucose-derived metabolites in biomass generation and/or limiting oxidative stress [4-7]. Asn (N)-linked protein glycosylation requires glucose and multiple glucose-derived sugars [8], yet surprisingly this pathway has not been investigated as a target of the Warburg effect. Here we report that the Warburg effect drives T cell growth and differentiation by controlling glucose flux to Golgi N-glycosylation. Branched N-glycans regulate the concentration and signaling of multiple surface receptors and transporters simultaneously to coordinate cellular and disease phenotypes [8-20]. Branching depends on UDP-GlcNAc biosynthesis via the hexosamine pathway [10, 11, 21, 22], a competitor of glycolysis for fructose-6-phosphate. In glycolytic T cells, the hexosamine pathway is starved of fructose-6-phosphate by glycolysis, limiting de novo UDP-GlcNAc biosynthesis and N-glycan branching dependent IL-2 receptor-α (CD25) surface retention. Restoring branching or UDP-GlcNAc levels via salvage of N-acetylglucosamine does not alter the glycolytic state yet inhibits T cell growth and switches cell fate from pro-inflammatory TH17 to anti-inflammatory induced T regulatory (iTreg) cells by restoring CD25 surface expression. Forcing oxidative phosphorylation inhibits growth and drives iTreg over TH17 differentiation by raising branching, demonstrating that glycolysis is not required for growth/differentiation control beyond its regulation of branching. Thus, the Warburg effect primarily targets N-glycosylation via altered glucose flux to control T cell growth and differentiation.

Essential biological systems are tightly regulated and their dysregulation is often associated with disease states. Recently, plasma membrane dynamics that globally control cell growth and differentiation have been shown to be regulated by the interaction of galectins with branched N-glycans at the cell surface. Galectins bind N-acetyllactosamine units (LacNAc: Gal beta;1,4GlcNAc) in branched N-glycans attached to surface glycoproteins, forming a molecular lattice that controls receptor clustering/surface-retention/signaling. LacNAc density and galectin avidity for N-glycans increase proportional to the number of LacNAc branches initiated via the sequential action of the medial Golgi branching enzymes Mgat1, 2, 4, and 5. Mgat5 deficiency marginally reduces LacNAc content by limiting N-glycans to three branches, yet results in T-cell hyperactivity and autoimmunity in mice. Despite its importance, little is known about endogenous mechanisms that regulate N-glycosylation and the galectin-glycoprotein lattice. Here we show that multiple MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and Vitamin D3, including genetic variants in interleukin-7 receptor-alpha (IL7RA*C), interleukin-2 receptor-alpha (IL2RA*T), MGAT1 (IVAVT-T) and CTLA-4 (Thr17Ala). Down-regulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IVAVT-T) and Vitamin D3, optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway, namely N-glycosylation. We also report a startling homeostatic mechanism that maintains cell surface LacNAc content when branching is severely disrupted. Based on the model of galectin-glycoprotein lattice, restricting N-glycans to a single branch via Mgat2 deficiency or mannosidase II inhibition is expected to result in a dramaticly hyperactive state, when compared to Mgat5 deficiency. However, we find that a marked increase in poly-LacNAc extension maintains LacNAc content and galectin binding to N-glycans, thereby preventing further increases in T cell activity and autoimmunity; phenotypes reversed by targeted deficiency of the poly-LacNAc extension enzyme B3GnT2 or complete blockade of branching. Homeostatic maintenance of LacNAc content in N-glycans appears to be a general mechanism, being present in epithelial, mesenchymal and stem cells. These data define Golgi proofreading and LacNAc content, rather than unique N-glycan structures, as critical regulators of the galectin lattice, cell homeostasis and autoimmunity.

The porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen of swine and is known to cause abortion and infertility in pregnant sows and respiratory distress in piglets. PRRSV contains a major glycoprotein (GP5) and three minor glycoproteins (GP2a, GP3, and GP4) on the virion envelope, all of which are required for infectious virus production. To study their interactions amongst each other and with a cellular receptor for PRRSV, CD163, I cloned each of the viral glycoproteins and CD163 in various expression vectors. My studies have shown that while the GP2a, GP3, and GP4 are co-translationally glycosylated, the GP5 is post-translationally glycosylated. By using co-immunoprecipitation (co-IP) assays, strong interaction was demonstrated between GP4 and GP5 proteins, although weak interactions among the other envelope glycoproteins were also detected. Further, GP4 was found to mediate interactions leading to formation of multiprotein glycoprotein complex. My results also show that GP2a and GP4 proteins are the only two GPs that specifically interact with the CD163 molecule and that glycosylation of these GPs is required for efficient interaction. Based on these studies, I have developed an interactome map of the viral GPs and CD163 and have proposed a model of the viral glycoprotein complex and its interaction with CD163. Studies reported here also show that glycan addition at residue 184 (N184) of GP2a, and residues N42, N50, and N131 of GP3 is essential for recovery of infectious virus. Although single site glycosylation mutants of GP4 had no effect on infectious virus production, introduction of double mutations was lethal. The loss of glycan moieties of GP2a, GP3, and GP4 proteins had no effect on host neutralizing antibody production. Overall, I conclude that the PRRSV glycoproteins are co-translationally and post-translationally glycosylated, the GP4 protein is central to mediating interglycoprotein interactions, and along with GP2a, serves as the viral attachment protein that is responsible for interactions with the viral receptor, CD163. Further, glycosylation of GP2a, GP3, and GP4 proteins is required for infectious virus production, efficient interaction with CD163, but does not play any role in neutralizing antibody response in infected animals.