Receptor glycosylation is critical in receptor-ligand interactions in immune cells, but the exact role of glycosylation in receptor activation upon ligand binding has not been elucidated. In neuronal cells, we have shown that when neurotrophic factors bind their respective Trk tyrosine kinase receptors, receptor activation and subsequent neurotrophin-mediated signaling is dependent upon the induction and activity of an endogenous sialidase enzyme. In this thesis, we report that toll-like receptor (TLR) activation upon ligand binding is similarly dependent on the induction of a cellular sialidase, which we have identified as Neu1 sialidase, which specifically targets and hydrolyses alpha-2,3-linked sialic acid residues on the receptor. Blocking Neu1 sialidase activity with specific inhibitor Tamiflu detrimentally impacts ligand-induced TLR4/MyD88 interaction, NFkappaB activation and TLR-mediated effector responses like nitric oxide and pro-inflammatory cytokine production. Diminished cytokine production is also seen in vivo in Neu1-deficient mice. We propose a mechanism for the induction of Neu1 sialidase, upon ligand binding to TLR, that involves the activation of heterotrimeric G-alpha protein-dependent G-protein coupled receptor (GPCR) signaling to activate a matrix metalloproteinase (MMP) enzyme, likely MMP-9. It is suggested that MMP-(9) targets the cell surface elastin receptor complex of Neu1/protective protein cathepsinA/elastin binding protein (EBP), which potentially catalytically activates Neu1. In addition, we report an association between Neu1 and TLR2, TLR3 and TLR4 on the plasma membrane that has not previously been described. The idea that the multiple functionality and diversity of TLRs and TLR-mediated signaling may be an immunologic paradigm capable of explaining all human disease is provocative but plausible. Certainly, the structural integrity of TLRs, their ligand interactions and activation are essential for immunological protection. Thus, understanding the molecular mechanism of Neu1 sialidase regulation of TLR activation will provide important opportunities for disease control through TLR manipulation. The future directions of this research will also open a new area of glycobiology research (the glycomics of innate immune responses) and will widen the scope for the development of novel therapeutic drugs to combat infections and inflammatory diseases. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2009-01-26 12:33:32.743

N-glycosylation is a metabolic process found in all three domains of life. It is the attachment of a polysaccharide glycan to asparagine (Asn) residues within the amino acid motif, Asn-Xaa-Ser/Thr. In the archaeon, Methanococcus maripaludis, a tetrasaccharide glycan was isolated from purified flagella and its structure determined by mass spectrometry analysis. The linking sugar to the protein is surprisingly, N-acetylgalactosamine (β-GalNAc), with the next proximal sugar a derivative of N-acetylglucosamine (β-GlcNAc), being named β-GlcNAc3Ac, and the third sugar a derivative of N-acetylmannosamine (β-ManNAc), with an attached threonine residue on the C6 carbon (β-ManNAc3NAm). The terminal sugar is an unusual diglycoside of aldulose ((5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose). Previous genetic analyses identified the glycosyltransferases (GTs) responsible for the transfer of the second and third sugars of the glycan, as well as the oligosaccharyltransferase (OST) which attaches the glycan to protein. Left unidentified were the first and fourth GTs, the flippase as well as any genes involved in glycan sugar biosynthesis and modification. In this work, genes suspected to be involved in the biosynthesis of N-linked sugars, as well as those that might encode the missing GTs and flippase were targeted for in-frame deletion. Mutants with a deleted annotated GT gene (MMP1088) had a small decrease in flagellin molecular weight as determined by immunoblotting. Mass spectrometry (MS) analysis confirmed that the N-linked glycan was missing the terminal sugar as well as the threonine found on the third sugar of wildtype cells. Mutants with a deleted gene annotated to be involved in acetamidino synthesis (a functional group that is present on the third sugar), also had a decrease in flagellin molecular weight. MS analysis determined that the N-linked glycan was missing the acetamidino group on the third sugar as well as its attached threonine, along with the terminal sugar. Both mutants were able to assemble functional flagella but had impaired motility compared to wildtype cells in mini-swarm agar. Deletions were also constructed in four other GT genes considered candidates in assembly of the linking sugar. However, none of these mutants had the expected decrease in flagellin molecular weight. With the work done in this study, the glycosyl transferase that attaches the last sugar of the M. maripaludis N-linked assembly pathway has been identified as well as a gene involved in the biosynthesis and modification of the glycan sugars. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-07-07 15:45:19.052

The archaeal flagellum is a rotating prokaryotic motility apparatus used for swimming motility and adhesion; however, it is more closely related to the bacterial type IV pilus system than its bacterial namesake. Methanococcus maripaludis is a highly flagellated, obligately anaerobic methanogen and is used as the archaeal model system during this study. The identified structural genes of the archaeal flagella are transcribed by a single fla operon; however, the interactions between the majority of the Fla proteins has yet to be elucidated. In this work, several techniques were attempted to determine the protein-protein interactions between Fla proteins, including membrane fractionation experiments and in vitro dimerization assays. Evidence from these experiments suggests that two proteins, FlaC and FlaE, have the ability to self-associate. The M. maripaludis flagella system is also used as a model for the study of the N-linked glycosylation pathway in the domain, due to the presence of a tetrasaccharide N-linked to flagellin monomers. Previous work has identified several of the processes involved in the assembly of this glycan, including glycosyltransferases, the oligosaccharide transferase and several of the key components involved in the biosynthesis of the sugar residue precursors. However, many of the enzymes responsible for biochemical modifications to the sugar residues remain to be determined. The operon structure of the genes between mmp1080 and mmp1095 was experimentally confirmed using RT-PCR, and each of the operons contains at least one gene involved in the biosynthesis of the N-linked glycan. In-frame deletions of genes in this region were characterized for effects on the N-linked glycan. Evidence suggests that Mmp1082 and Mmp1083 are acting in conjunction with Mmp1081 in the addition of an acetamidino functional group to the third sugar residue. Mmp1085 was determined to be a methyltransferase responsiblefor the methylation of the terminal sugar residue. Additionally, Mmp1087 and Mmp1094 were identified as potentially having an effect on the glycan. Though this work, the breadth of knowledge in regards to both the archaeal flagella and the N-linked glycosylation process in the domain has been increased. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-01-28 11:50:05.542

No description available.

No description available.

Therapeutic monoclonal antibodies (MAb) are produced as secreted complex glycoproteins from mammalian cell systems and represent one of the most important classes of therapeutic medicines for the treatment of a variety of human diseases. Their benefit in health care and high economic impact provide the driving force for the development of improved production levels with the focus of optimizing clinical efficacy. One important issue is the optimization of monoclonal antibody production. A frequent approach used to address this challenge is the engineering of mammalian cell lines to increase antibody production levels through genetic manipulation. Valuable information can then be obtained by monitoring the effects of genetic changes on the biochemistry of the cell associated with MAb production. Global protein expression profiling of mammalian cells used for the production of biopharmaceuticals may reveal key biochemical characteristics associated with MAb-producing cell lines. A better understanding of these characteristics can in turn lead to more rational strategies for cell line and process development. The proposed research relates to a larger NSERC Strategic Network (MAbNet) Grant to develop and establish a novel platform for the large-scale manufacture of specific glycoforms of therapeutic monoclonal antibodies. The efficacy of these recombinant MAbs will be enhanced by the control of their glycosylation profiles. The work presented in this thesis will assist MAbNet in meeting their objectives. Specifically, we use 2D-Differential In-Gel Electrophoresis (2D-DIGE) to quantify protein expression differences between EG2-hFc1-producing Chinese Hamster Ovary cells (CHO-1A7) with its parental cell line (CHO-BRI). Here, we identified 34 unique differentially expressed proteins associated with EG2-hFc1 production that relate to various biological processes including protein processing, carbohydrate metabolism, amino acid metabolism, energy metabolism, apoptosis, and cell proliferation pathways. The majority of identified significant protein expression changes and their associated metabolic processes seem to prioritize energy production in CHO-1A7 cells. Due to the metabolic load of recombinant antibody production, the CHO-1A7 cell line attempts to meet the energy requirements needed for recombinant protein biosynthesis while maintaining cell viability and efficient protein folding mechanisms. A 2-D proteome reference map was also constructed for the CHO-BRI host cell line containing 131 identified protein spots. The map provides information that will further expand our understanding of this particular cell line. It will be a useful tool for studies investigating physiological responses and protein expression patterns of CHO-BRI to genetic and environmental perturbations. The set of identified differentially expressed proteins provides data on the downstream changes in protein expression due to genetic manipulation, and furthermore can provide targets for cell-line specific optimization of antibody production. The work described in this thesis furthers our understanding of antibody production in a specific CHO cell line.

Class I heavy chain (HC) must assemble with β-microglobulin (β2m) and acquire optimal peptide in order to be presented to cytotoxic T cells (CTLs). Calnexin is involved in the initial folding of class I HC and subsequent assembly with β2m. Incorporation of "empty" or suboptimally loaded class I molecules into the multimolecular loading complex is essential for them to acquire optimal peptides. The loading complex consists of several cofactors: TAP, tapasin, ERp57 and calreticulin. The precise role of calnexin and calreticulin in the regulated assembly and peptide loading and the significance of their physical interaction with other cofactors of the loading as well as preloading complex still remains unclear. Using mouse fibroblasts that lack calreticulin, I have studied the role of calreticulin in the assembly and loading of H2-K^b and H2-D^b expressed in these cells. MHC class I molecules in calreticulin-deficient cells are able to assemble with β2m normally, but their subsequent loading with optimal, stabilising peptides is defective despite their ability to interact with the TAP complex. The "empty" or suboptimally loaded class I molecules exit the ER rapidly. Reflecting the loading defect, presentation of endogenously processed antigens by class I molecules in calreticulin-deficient cells is impaired. I have used a human calnexin-deficient cell line CEM.NK^R to study assembly of class I in the absence of calnexin. The results demonstrate that contrary to current understanding, calnexin has an important role in class I HC assembly with 32- microglobulin. The role of heavy chain glycosylation in class I biogenesis is still controversial. My findings suggest asparagine (N)-linked glycosylation of human class I heavy chain at position 86 is optimal and any deviations from "normal" glycosylation results in poor loading with peptides and some defect in the assembly with β2m. Despite affecting the loading function, glycosylation did not have significant effect on presentation of a high affinity binding epitope to HLA-A*0201 specific CTLs. Finally, I show that co-operation from all domains of calreticulin is essential in order to generate a fully functional calreticulin. Interestingly, proline-rich (P) -domain of calreticulin downregulated expression of a number of cellular proteins including MHC class I HC, despite restoring the cytosolic calcium levels in calreticulindeficient cells. The effect of P-domain on class I expression was at the level of transcription.

Glycosylation is a fundamental post-translational modification of proteins that occurs in all eukaryotes, but the biological significance of specific glycosylation structures is still largely unknown. We have chosen to study this process by cloning and characterizing the genes involved in the N-linked glycosylation pathway in Drosophila melanogaster and analysing mutants of these genes. Since this biochemical pathway for the production of N-linked glycans is considerably conserved, studying it in Drosophila, a useful organism for genetic research, should yield results applicable to other eukaryotes. This thesis describes the attempt to identify a Drosophila homologue of rat ER mannosidase and how this investigation resulted in the identification of a Drosophila cDNA with considerable homology to calnexin, an enzyme involved in the folding of N-glycosylated proteins. It also describes the cloning and characterization of the genomic sequence for the putative Golgi mannosidase II (GmII)* in Drosophila. The expression and control of expression of GmII have been investigated further, and have suggested expression of this gene throughout Drosophila development. Analysis of the GmII promoter region has shown expression from a TATA-less promoter contained within 288bp of sequence 5' to the start of transcription.

This thesis is concerned with structural aspects of the recognition and effector functions of antibody molecules. The recognition process is investigated in the dinitrophenyl (DNP) binding mouse IgA produced by the myeloma MOPC 315. The studies on effector functions utilize a DNP binding mouse hybridoma IgG2a to examine the role of N-glycosylation in IgG. The combining site of protein 315. The involvement of tyrosyl residues in the combining site of protein 315 was examined by preparing specifically nitrated NO₂-Tyr-33_H and NO₂-Tyr-34_L derivatives of the Fv fragment of this protein. The ionizations of tnese derivatives were studied in the presence and absence of various DNP-ligands. Perturbations to the nitrotyrosine ionizations were found to be caused by the side chains of certain of these ligands, allowing an indication of the distance of these tyrosines from the bound hapten. On examination of the compatibility of these data with the model of the combining site of protein 315 proposed by Dower <en>et al. (1977) (Biochem. J. 165, 207-225) it was found that while the location of Tyr-33_H is consistent with this model, the position of Tyr-34_L is not. A remodelled combining site using the modified ring-current treatment of Perkins and Dwek (1980) (Biochemistry 19, 245-258) is presented. This allows a better rationalization of the nitration data and of previous experimental observations on protein 315. The role of the conserved C 2 domain oligosaccharide of IgG. This was examined by a functional comparison of native IgG with an aglycosylated IgG preparation. Aglycosylation was acheived by cell culture of the hybridoma cells in the presence of the glycosylation inhibitor tunicamycin. The conditions for preparation and purification of this aglycosyl IgG are described. Aglycosylated IgG is found to be correctly assembled as an H₂L₂ unit. It retains the antigen binding and Staphylococcal protein A binding abilities of the native glycosylated molecule. Using an assay system designed specifically to overcome certain problems in comparing Clq binding to different preparations of IgG it was found that the aglycosylated preparation showed only slightly reduced affinity for Clq. In addition the aglycosylated IgG is able to activate bound Cl. The above results are consistent with the structure of the Fc region being only minimally altered in the absence of oligosaccharide. The structural integrity of the aglycosylated molecule may be compromised however, as its ability to bind to monocyte Fc receptor is significantly reduced. In addition the aglycosylated molecule becomes much more susceptible to proteolytic digestion. A computational model-building analysis of the quaternary structure of Fc allows an explanation of at least some of the effects of aglycosylation in terms of reduced conformational stability of the C_H2 domains.

This thesis describes approaches to the transformation of simple carbohydrates into a polyhydroxylated pyrrolidine and the formation of its glucosides. Chapter one describes the synthesis of the naturally occurring pyrrolidine 2,5-dideoxy-2,5-imino-D-mannitol. Synthesised from di-O-isopropylidene-D-glucose, the key steps are the introduction of nitrogen at C-5 with retention of configuration. Then cyclisation of the nitrogen onto the C-2 position with inversion to form the pyrrolidine ring. Reduction of the aldehyde furnished the polyhydroxylated heterocycle in 3.4% yield over 16 steps. The synthetic compound matched the naturally occurring compound in all respects. Chapter two contains a review of commonly used glycosylation methods. It also describes the glycosylation of di-O-isopropylidene-α-D-glucose as a model system comparing the Koenig-Knorr method to the trichloroacetimidate method using several reaction conditions. Glycosylation of 2,5-dideoxy-2,5-imino-D-mannitol was carried out using the trichloroacetimidate method to synthese all four glucosides. Boron trifluoride etherate and trimethylsilyl trifluoromethanesulphonate were used as catalysts in dichloromethane, diethyl ether and acetonitrile under strictly anhydrous conditions. All four glucosides were prepared 1-O-(αβ-D-glucopyranosyl)-2,5-dideoxy-2,5-imino-D-mannitol and 3-O-(αβ-D-glucopyranosyl)-2,5-dideoxy-2,5-imino-D-mannitol. Biological screening carried out against a wide range of glycosidases and glycosyl transferases indicated that the glucosides showed little inhibition in comparison to 2,5-dideoxy-2,5-imino-D-mannitol. Chapter three describes the isolation and identification of 1-O-(β-D-glucopyranosyl)- 2,5-dideoxy-2,5-imino-D-mannitol from Nephthytis poisonii N.E.Br. a member of the Araceae family found in tropical Africa. Identification was made by comparison with the previously synthesised glucosides of 2,5-dideoxy-2,5-imino-Dmannitol. Investigations of Hyacinthoides non-scriptus (L.) chouard ex Rothm are also discussed. Chapter four describes the synthesis of a diazidolactone that could be used to form a 1,5 disubstituted tetrazole. This would have a second nitrogen functionality in the molecule allowing the possibility of the inclusion of the tetrazole into a peptide sequence. The synthesis was carried out from L-gulono-1,4-lactone. An azido group was introduced selectively at C-2, this unexpectedly occurred with retention of configuration. A second azide was then introduced at C-5, this occurring with the more commonly observed inversion of configuration to afford the 2,5-diazido-2,5-dideoxy-D-manno-1,4-lactone.