Mass spectrometric investigation of biomedically important glycosylation

Abstract

Glycobiology is the comprehensive study of the structure, biosynthesis, function and evolution of saccharides which are also named sugars or glycans. Glycosylation is a type of modification in which sugars are added to another molecule, such as a protein molecule or a ceramide. Abnormal glycosylation is frequently associated with diseases such as cancer and immune responses. Defining glycan structures is therefore important for understanding glycan function in health and disease. In addition, identification of glycan populations can provide essential information for further research on glycoproteins and glycolipids. In this thesis, glycomic experimental approaches were employed to characterize the structures and populations of glycans of glycoconjugates from HeLa cells, normal human dermal fibroblast (NHDF) cells, myoblasts, myotubes and trophoblasts. These approaches include sample preparation methodologies which were followed by the application of highly sensitive mass spectrometry, particularly MALDI-TOF MS, MALDI-TOF/TOF MS/MS and GC-MS. Ribosome inactivating proteins (RIPs) and lectins from elderberry are more toxic to HeLa cells than to NHDF cells. The difference in the cytotoxicity was hypothesized to be caused by the difference in the glycome patterns of HeLa and NHDF cells. To test the hypothesis, glycome patterns on both glycoproteins and glycolipids of HeLa and NHDF cells were investigated. Glycomic results have revealed that glycome patterns in HeLa cells and NHDF are different, and this gives a possible explanation for the difference observed in the cytotoxicity assay. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway which yields uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an essential substrate for protein glycosylation. N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. Mutations in the gene GFPT1 can result in “limb-girdle CMS with tubular aggregates” which is a subtype of congenital myasthenic syndromes (CMS). To investigate whether protein glycosylation at the neuromuscular junction might be involved in this impairment, the N-glycomes of myoblasts and myotubes derived from healthy controls and patients were investigated. My result showed that global glycosylation is not significantly impaired in the muscle cells from the CMS patients caused by GFPT1 mutations. The human fetoembryonic defense system hypothesis (hu-FEDS) is a hypothetical model depicting a way via which the human immune system is able to recognize foreign substances as "own species" as has been observed with maternal immune tolerance in pregnancy. The fundamental idea of this hypothesis is that glycoproteins existing in the reproductive system and exposed on gametes can either inhibit immune responses or prevent rejection of the foetus. This model has not been tested in human trophoblasts. My glycomic analyses of three trophoblast populations (CTB, STB and evCTB) revealed that functional glycan structures that are present on human gametes are also expressed on trophoblasts, and this provides further evidence for the hu-FEDS hypothesis.