Title:
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Characterisation of O-glycans from HOC544 and the
challenges in targeting pre-defined complex
carbohydrate conformations of mucin-type
glycoproteins
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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.
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