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Title: Characterisation of O-glycans from HOC544 and the challenges in targeting pre-defined complex carbohydrate conformations of mucin-type glycoproteins
Author: Afrough, Babak
ISNI:       0000 0004 2671 401X
Awarding Body: University of Westminster
Current Institution: University of Westminster
Date of Award: 2008
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The role of glycosylation in the development, regulation, and progression of disease is the focus of considerable research. Glycosylation is a complex posttranslational modification which creates linear or multi-antennary chains of heterogeneous glycans on proteins or lipids. Glycosylation is a tissue specific and highly regulated process modulated by genomic loci encoding specific glycosyltransferases. In light of tissue-specific glycosylation and aberrant glycosylation in a number of disease areas, targeting glycans could potentially lead to selective therapeutic strategies directed at glycoconjugates originating from pathological tissue. However for such a strategy to work, we need to better understand the shape and overall topology of glycan presentation by the glycoprotein which modulates tissue specificity in the extracellular milieu.. Monoclonal antibodies with specificity towards glycan conformations possessing defined biological activity offer promising prospects for this purpose. With this point in view, two naIve .single chain fragment variable (scFv) phage libraries were investigated as potential sources of carbohydrate binding reagents. The defined glycan conformations of the blood group H active mucin-like glycoprotein from human ovarian cyst fluid (HOGS«) were used as target for scFv isolation. A number of different tools were exploited to understand better the nature of HOGS« and its glycans. These include lectins, antibodies, high performance liquid chromatography (HPLG) and mass spectrometry (MS). The purity of HOGS« glycoprotein was assessed by 80S-PAGE and monosaccharide composition determined by HPLG. The sugar linkages and glycan structures were explored by various MS strategies. A solid phase assay approach including inhibition experiments using a panel of 21 plant lectins was used to (1) optimise selection conditions in isolating glycan-specific scFv from the Tomlinson I and J phagemid libraries and (2) to characterise accessible sugar conformations of HOGS«. The Tomlinson I and J libraries were subsequently expanded and screened by selection against HOG544 for glycan specific scFv. SOS-PAGE analysis confirmed the absence of protein impurities and evidence of glycoprotein. HPLG monosaccharide composition analysis identified L-fucose, 0galactose, N-acetyl-:-O-glucosamine and N-acetyl-O-galactosamine. Linkage analysis of partially methylated alditol acetates of depolymerised glycans predominantly showed terminal-fucose, 2-linked galactose and 3-linked galactose amongst other linkages illustrating linkage heterogeneity. Structural analysis and sequencing of permethylated glycans indicates masses correlating to composite .conformations ranging from a trisaccharide to a potential dodecasaccharide and revealed information on branching and core architecture. A substantially modified solid phase assay demonstrated (1) bovine serum albumin is not an optimal blocking agent for detection of glycan on Nunclon Delta surfaces and (2) binding of the lectins Ulex europaeus agglutinin I, Jacalin, Soybean agglutinin, Wheat germ agglutinin and Ricinus communis agglutinin I to HOC544. Inhibition experiments confirmed lectin binding through carbohydrate recognition. An attempt to select for carbohydrate specific reagents using HOC544 as target for the Tomlinson I and J libraries did not produce any functional scFv capable of binding HOC544. The findings of these experiments show substantial heterogeneity in composition, linkage, sequence and overall masses of the oligosaccharides. Despite this complexity in glycan presentation by the glycoprotein, only 5 out of 21 lectins and type-2 H antigen-specific monoclonal antibody were found to bind the glycans of this naturally occurring mucin-like glycoprotein. Na'ive phage libraries may not be the best source for isolating carbohydrate-specific reagent despite their numerous advantages. Understanding the relationship between glycan structure(s) and how it mediates specificity as one entity in recognition events should provide in silico design of carbohydrate binding proteins with a superior approach for developing reagents in comparison to screening complex biological libraries.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available