The Structure and Function of Glycoforms

Rudd, Pauline Mary (1995). The Structure and Function of Glycoforms. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000fb9f

Abstract

Glycoproteins generally consist of populations of glycosylated variants of a single protein (glycoforms). While the potential oligosaccharide processing pathways available to a protein are dictated by the cell in which it is expressed, the final glycosylation pattern reflects constraints imposed by the three dimensional structure of the individual protein. This thesis explores the extent to which a protein controls its own glycosylation by comparing the glycosylation patterns of soluble forms of leucocyte surface antigens (CD2, CD5d1, CD59, CD5/CD4d3,4 and CD48) expressed in Chinese Hamster Ovary cells (ch.2). In chapter 3 the Nand 0-linked and anchor glycans of human erythrocyte CD59 were characterised; the data suggested that the individual protein may influence the processing of glycosylphosphatidyl inositol anchor glycans. In chapter 4-7 consequences of variable glycosylation were explored by preparing defined sets of glycoforms for testing in functional assays. Individual glycoforms of RNase B reduced the susceptibility of the protein to proteases and modulated the activity of the enzyme (ch.4). IgG rheumatoid factor, from patients with rheumatoid arthritis, contained a specific subset of IgG glycoforms with Fc oligosaccharides terminating in GlcNAc (IgGO) and an extra-glycosylated heavy chain. Exposed GlcNAc residues on multiply presented IgGO bound serum mannose binding protein, suggesting a role for Fc sugars in recognition (ch.6). In chapter 7 a subset of highly active tissue plasminogen activator (tPA) glycoforms, type II dimers, was identified. In the fibrin stimulated activation of plasminogen, glycosylation of tPA site 184 (type I glycoforms) decreased kcat, while the Km was decreased by glycosylation at the variably occupied plasminogen site, Asn289. Chapter 8, which deals with technology, explores applications of HPLC and mass spectrometry to the analysis of oligosaccharides and in chapter 5 capillary electrophoresis was used to resolve transferrin glycoforms, suggesting a rapid diagnostic for carbohydrate deficient glycoprotein syndrome.