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In the biopharmaceutical industry, identification of glycans in a glycoprotein is a regulatory requirement and is a part of the characterization of the protein. Glycans are constructed of several monosaccharides linked together. N-linked glycans, which have been studied in this project, are attached to the nitrogen atom in asparagine. A method for separating N-linked glycans by high performance anion exchange chromatography had already been developed at the department. To develop a method for identification of the N-glycans by mass spectrometry, a desalting method on porous graphitic carbon (PGC) columns was used and optimized resulting in the eluents A (0,05% TFA in ACN:water 5:95 v/v) and B (0,05% TFA in ACN:water 50:50 v/v). Also the sample introduction on the mass spectrometer was optimized and resulted in a sensitive on-line liquid chromatography mass spectrometry (LC-MS) approach which gave mass spectrometric peaks with high signal to noise ratios and with high mass accuracy. The developed procedure was then successfully used on glycans cleaved from a glycoprotein separated by high performance anion exchange chromatography with pulsed amperometric detector.

Philosophiae Doctor - PhD / The HIV-1 envelope (Env) glycoprotein is the primary target of the humoral immune response and a critical vaccine candidate. However, Env is densely glycosylated and thereby substantially protected from neutralisation. Despite the importance of the HIV- 1 Env glycans, limited computational analyses have been employed to analyse these glycans. Here, the Env glycans of two HIV-1 wild-type subtype C isolates are examined, in detail, using computational approaches. These particular strains were used since in vitro data showed that the removal of a single glycan had a substantially different impact on the neutralisation sensitivity of the two strains. Molecular dynamics simulations, and the subsequent analyses, were carried out on the computationally determined, fully glycosylated, Env structures of these two wild-type strains and their N301A mutant counterparts. Detailed comparison of the molecular dynamics simulations demonstrated that unique glycan dynamics and conformations emerged and that, despite shared HXB2 reference sequence positions, the glycans adopted distinct conformations specific to each wild-type model. Furthermore, different changes in conformations were observed for each wild-type model compared to its N301A mutant counterpart and, interestingly, these N301A mutant model-specific glycan conformations were directly associated with the protein residues ultimately found to be exposed, which may explain the varied resistance to neutralising antibodies observed, in vitro, for the two N301A mutant strains.

Glycans and their conjugates form the largest and most diverse class of biological molecules found within nature. These glycosides are vital for numerous cellular functions including recognition events, protein stabilisation and energy storage, to name a few. Additionally, abnormalities within these structures are associated with a wide range of disease states. As a result, robust analytical techniques capable of in depth characterisation of carbohydrates and their binding partners are required. Currently, liquid chromatography coupled with tandem mass spectrometry (MS2) is the 'gold standard' for characterising these species. However there are inherent challenges for 'sequencing' carbohydrates given that most structures are diastereomeric. As a result MS alone is insufficient to fully elucidate all stereochemical and often regiochemical information and alternative analytical techniques have inherent issues meaning that they are not suitable for medium/high throughput analysis. To facilitate elucidation of these structures, ion mobility spectrometry (IMS) has been used in-line with MS2. IMS of mono- and di-saccharide product ions generate by collision-induced dissociation (CID) of various glycans and their conjugates enables unambiguous identification of the monomer and the regio-/stereo-chemistry of the glycosidic bond, independent of the precursor structure. Also, given the prominence of glycans in biological recognition events, high-throughput techniques capable of elucidating and characterising carbohydrate to glycan-binding protein (GBP) interactions are highly sought after. Historically, (micro)array strategies are employed to screen large numbers of biological interactions, with detection conventionally achieved with fluorescent tagging. The major disadvantage of this approach is the requirement of a labelling step to facilitate detection of glycan-GBP binding. MS offers the ability to unambiguously identify GBPs when combined with routine bottom-up proteomics strategies, namely on-chip proteolysis followed by mass fingerprinting and MS2 analysis and subsequent comparison to protein databases. It is anticipated that these methodologies developed throughout these studies, both for carbohydrate sequencing and the characterisation of glycan-binding proteins, will greatly add to the Glycomics toolbox.

Glycoengineering is a technology that could improve protein therapeutics. While protein glycosylation in general enhances solubility and stability, and reduces aggregation, immunogenicity and proteolysis, specific kinds of glycosylation may also be critical. For example, capping of glycans with N-acetylneuraminic acid (Neu5Ac) maximises circulatory half-life in humans. Moreover, some glycans directly participate in molecular recognition and other aspects of glycoprotein function. Glycoproteins produced by non-human mammalian cells carry glycans capped by N-glycolyl-neuraminic acid rather than Neu5Ac. Yet production in human cell lines is costly and slow, requires specialist facilities, produces low yields and is subject to additional regulations. Hence there is a case for glycoengineering alternative expression systems capable of rapid, low-cost, high-yield glycoprotein production. This report focuses on the glycoengineering of Pichia pastoris, a yeast, to produce recombinant human glycoprotein factor H (FH) bearing human-like glycans. FH is a potent down-regulator of the complement system. Mutations and SNPs in FH result in autoimmune diseases such as atypical haemolytic ureamic syndrome and age-related macular degeneration (AMD). Recombinant FH is an enticing therapeutic candidate for treating AMD, but high doses are required since FH is abundant (200-300 mg l-1) in normal human serum. Human FH (155 kDa), with eight sites of N-linked glycosylation and 40 disulphides, is a challenging target for recombinant production. Yet FH was previously expressed to 10s of milligrams in P. pastoris. In this study, methods were established to confirm that human plasma-derived (h)FH carries predominantly N-linked diantennary disialylated complex-type glycans, with monosialylated diantennary structures and triantennary structures in fucosylated and non-fucosylated forms, contributing to glycan heterogeneity. Functional comparison of native hFH, enzymatically desialylated (DeSia-) hFH and deglycosylated recombinant P. pastoris-produced (DeGly-r)FH showed that DeSia-hFH had the lowest affinity for complement protein C3b, its key target. Moreover, DeSia-hFH binds C3d, an opsonic C3b-breakdown product, whereas native hFH does not. DeSia-hFH had an improved ability to accelerate decay of the C3 convertase (an enzyme that cleaves C3 to C3b) compared to native hFH, but neither was as good as DeGly-rFH in this respect. In contrast, DeGly-rFH had reduced cofactor activity (for factor I-mediated degradation of C3b) compared to native hFH whereas DeSiahFH did not have reduced cofactor activity. These data suggest that sialylation of FH glycans may play a role in stabilising a conformation of circulating FH that is not fully effective, consistent with specificity for self-surfaces and resistance to bacterial hijack. Aiming eventually to produce human-like glycosylated FH in glycoengineered P. pastoris, the SuperMan 5 strain served as a starting point. While conventional strains of P. pastoris put hypermannosylated N-linked glycans on proteins, glycans on SuperMan 5-produced FH were shown to contain just five mannose (Man) residues. In further glycoengineering, and following unsuccessful efforts to use inABLE technology for this purpose, commercially available (GlycoSwitch) vectors were used to introduce genes encoding the glycosyltransferase enzymes N-acetylglucosamine (GlcNAc) transferase I (GnTI) and galactose (Gal) transferase. These catalysed the formation of a hybrid-type glycan containing an N-acetyllactosamine (Gal-β(1,4)-GlcNAc (LacNAc)) antennae on a five-mannose glycan. Then two more GlycoSwitch plasmids, containing genes encoding α-Mannosidase II (ManII) and GnTII, were introduced into P. pastoris to catalyse the formation of a second LacNAc antennae. MALDI-TOF analysis found the glycosylation of this strain to be heterogeneous, containing the humanised diantennary digalactosyl glycan as well as other endogenous yeast glycans. This strain was designated SuperGal. Large-scale expression of rFH with terminally galactosylated complex-type glycans (Gal-rFH) in SuperGal yielded 100s of milligrams of purified Gal-rFH. Yeast-type glycans were enzymatically removed from rFH and the remaining complex-type humanised glycans were sialylated with a recombinant bacterial α(2,6)-sialyltransferase from Photobacterium sp. expressed in E.coli. Purified sialylated (Sia-) and non-sialylated (Gal-) rFH expressed in SuperGal were functionally characterised in vitro using SPR-based assays. In C3b-binding assays Sia-rFH had lower affinity compared to Gal-rFH. Both bound with lower affinity than DeGly-rFH. A similar pattern of binding affinity was seen for C3d. In C3 convertase decay-acceleration assays, all rFH glycoforms performed equally well and had greater activity than hFH. Conversely, Sia-and Gal-rFH were shown to perform equally as well as hFH in CA assays, while all three versions outperformed DeGly-rFH. However, in vivo complement activity assay carried out in a FH-knockout mouse model showed that humanisation of the glycosylation of rFH did not significantly improve activity compared to DeGly-rFH. In addition, analysis of the circulatory half-life of rFH showed that humanisation did not improve half-life. Further engineering steps will be required to increase the complex-type glycan site occupancy on rFH with a view to improving circulatory half-life and efficacy. However, this study represents a significant step forward in developing a therapeutically useful source of rFH.

Arrhythmias are often caused by aberrant ion channel activity, resulting in remodeling of the cardiac action potential. Two K + currents, IKs and IKr, contribute to phase III repolarization of the human cardiac action potential. Human ether-a-go-go-related gene 1 (hERG1), a voltage-gated potassium channel, underlies IKr. Alterations in the repolarization phase of the action potential, and in particular IKr, can lead to arrhythmias, long or short QT syndrome, heart disease, and sudden cardiac death. HERG1A has two putative N-glycosylation sites located in the S5-S6 linker region, one of which is N-glycosylated. The aim of the first study was to determine whether and how N-linked glycosylation modifies hERG1A channel function. Voltage-dependent gating and kinetics of hERG1A were evaluated under conditions of full glycosylation, no sialylation, in the absence of complex N-glycans, and following the removal of the full N-glycosylation structure. The hERG1A steady state activation relationship was shifted linearly along the voltage axis by a depolarizing ~9 mV under each condition of reduced glycosylation. Steady state channel availability curves were shifted by a much greater depolarizing 20–30 mV under conditions of reduced glycosylation. There was no significant difference in steady state gating parameters among the less glycosylated channels, suggesting that channel sialic acids are responsible for most of the effect of N-glycans on hERG1A gating. A large rightward shift in hERG1A window current for the less glycosylated channels was caused by the observed depolarizing shifts in steady state activation and inactivation. The much larger shift in inactivation compared to activation leads to an increase in hERG1A window current. Together, these data suggest that there is an increase in the persistent hERG current that occurs at more depolarized potentials under conditions of reduced glycosylation. This would lead to increased hERG1A activity during the AP, effectively increasing the rate of repolarization, and reducing AP duration, as observed through in silico modeling of the ventricular AP. The data describe a novel mechanism by which hERG1A activity is modulated by physiological and pathological changes in hERG1A glycosylation, with increased channel sialylation causing a loss of hERG1A activity that would likely cause an extension of the ventricular AP. The second study was to evaluate possible changes in antibiotic drug block as a result of alterations to N-glycosylation. We determined that N-glycans play a protective role on the hERG1A channel. SMX, Erythromycin, and Penicillin G were assessed individually at three concentrations. The data showed increases in antibiotic block with decreases in N-glycans. In addition, alterations in the voltage-dependence of block with changes in N-glycans were observed. SMX block was voltage-independent at each drug concentration under conditions of reduced sialylation only. Overall, these data indicate a functional role for N-glycosylation in the modulation of hERG1A antibiotic block, suggesting that even small changes in channel N-glycosylation modulate hERG1A block, and thereby likely impact the rate of action potential repolarization. The data from these studies enhances our understanding of the role of N-glycosylation on hERG1A function and drug block, and how that role will impact the cardiac action potential and overall cardiac excitability.

Dendritic cells (DCs) are regulators of the immune system and express a class of pattern recognition receptors known as C-type lectin receptors (CLRs) to recognize and respond to carbohydrates (glycans). Dendritic cells are hypothesized to be key mediators in the immune response to implanted materials and ligation of CLRs has been shown to have diverse effects on DC phenotype ranging from tolerogenic to pro-inflammatory. Thus, designing future biomaterials and combination products that harness the potential of CLR ligation on DCs has great promise. Additionally, many of the proteins which adsorb to biomaterials when implanted are glycosylated and thus understanding this interaction would provide further insight into the host response to currently implanted materials. However, DC responses to glycans presented from non-phagocytosable surfaces has not been well characterized and optimal factors for DC phenotype modulation by surface presented glycans are unknown. Additionally, studies relating DC response to glycan structures from soluble and phagocytosable displays to that of non-phagocytosable display have not been performed. This is of critical importance to the field because of the extremely limited supply of complex glycan structures that are able to be obtained. Because of this limited supply of glycans the trend in glycomics has been toward creation of glycan microarrays to assess initial candidates of interest for further study. However, the assumption that cell response to these glycoconjugate microarrays is equivalent to soluble or phagocytosable conjugates has not been validated. Therefore, the purpose of this study was to 1) determine the optimal molecular contextual variables of glycoconjugate presentation from a non-phagocytosable surface, namely, charge, density, and glycan structure for modulating DC phenotype; and 2) determine if modality of glycoconjugate presentation, i.e. soluble, phagocytosable, or non-phagocytosable will modulate DC phenotype differentially. To determine the effect of the molecular contextual variables primary human immature DCs (iDCs) were exposed to a range of adsorbed glycoconjugates in a 384 well plate and their subsequent phenotype assessed via a novel in house produced high throughput (HTP) assay. Bovine serum albumin (BSA) was modified to have a range of glycan densities and isoelectric points to determine which of these were optimal for DC phenotype modulation. Next, several poly-mannose structures were presented to DCs to determine if DC response was structure specific. Finally, contextual variables were modeled in a multivariate general linear model to determine underlying trends in DC behavior and optimal factors for glycan presentation from non-phagocytosable surfaces. To determine the effect of the modality of glycoconjugate display on DCs, optimized glycoconjugates from 1) were adsorbed to the wells of a 384 flat well plate, delivered at varying soluble concentrations, or adsorbed to phagocytosable 1 µm beads and subsequent DC phenotype assessed via the HTP assay. The cell response to the glycoconjugates was then validated to be CLR mediated and the DC response to glycan modality was modeled in another general linear model. Results from these studies show that highly cationized high density glycoconjugates presented from non-phagocytosable flat well display modulate DC phenotype toward a pro-inflammatory phenotype to the greatest extent. Additionally, significant impacts on DC phenotype in response to adsorbed conjugates can be seen when grouping glycan structure by terminal glycan motif. Finally, DC response to glycoconjugates were found to be CLR mediated and that each modality of glycan display is significantly different, in terms of DC phenotype, from the others. These results provide indications for the future design of glycan microarray systems, biomaterials and combination products. Furthermore, this work indicates that different mechanisms are involved in binding and processing of surface bound versus soluble glycoconjugates. With further study these differences could be harnessed for use in the next generation of biomaterials.

Numerous toxins and pathogens gain entry into mammalian cells using cell surface glycans. The Iyer group at Georgia State University is working on the development of glycoconjugates for the accurate detection of infectious agents. In this thesis, I have focused on the development of glycans to detect influenza virus and norovirus. In the first section, I have focused on influenza viruses. A panel of synthetic glycans was synthesized as receptor mimics for the specific capture of influenza viruses. The synthetic glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. This glycan printed microarray was evaluated for its ability to capture three strains of influenza viruses. The analytical limit of detection is ~10 pfu/ml, (plaque forming units/milliliter) which is clinical relevant as 102 viral particles are typically required to cause infection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using the microarray and determined the feasibility of this system to determine antiviral resistance for different strains. In addition to optical detection, I developed an electrochemical assay to rapidly detect influenza viruses. Here, we utilized an unique property of influenza viral surface enzyme, Neuraminidase (NA), which cleaves terminal N-Acetyl Neuraminic acid (sialic acid) from cell surfaces and proteins. We designed an electrochemical assay that uses glucose bearing sialic acid substrates. Glucose is released when exposed to viral NA or intact viruses. The released glucose can be detected using repurposed glucose meters. Thus, personal glucose meters that were designed to assist diabetics and prediabetics monitor blood glucose can potentially be used to detect pathogens. Using this approach, we have detected 19 unique strains of influenza viruses. We also demonstrated drug susceptibility using this assay. The limit of detection of this assay is 102 pfu/sample, which is clinically relevant. The results were validated plaque assays and polymerase chain reaction (PCR). In the second part of this thesis, I focused on norovirus detection. I developed a focused glycan microarray that comprised of a library of histo blood group antigens (HBGAs). The HBGAs were attached to a carrier protein and printed onto activated glass slides. A panel of norovirus virus like particles (VLPs) and strains that included different genogroups was exposed to the microarray. We found that different VLPs and strains give rise to unique binding patterns. When the binding pattern of VLPs for a particular strain were compared to the corresponding intact virus, the binding patterns didn't match well, presumably because the virus does not recognize the same antibody as the VLPs. Unfortunately, antibodies for the virus cannot be generated because the virus cannot be grown in a laboratory setting. Indeed, all norovirus samples are obtained from human challenge studies. I also used surface plasmon resonance (SPR) studies in an effort to determine the binding affinities. Divalent biotinylated H type glycans were synthesized and their binding affinities with different VLPs and viral strains were determined. Initial studies suggest that the binding affinities are strain specific. These results demonstrate that glycans can be used to capture and isolate norovirus, although more research is required to develop glycan based norovirus detection kits.