Glycosylation is a vital modification for many macro-biomolecules to obtain structural heterogeneity and functional diversity. In the immune system, nearly all engaged molecules are glycosylated, and their glycosylation status directly correlates with their functions. Therefore, structural studies of glycosylation are important and fundamental to allow understanding of the basic mechanisms underlying the immune system. In this thesis, comprehensive mass spectrometric based glycomic and glycoproteomic strategies were applied to investigate the glycosylation of the following immunologically important glycoproteins and animal models: pregnancy specific glycoprotein 1 (PSG1), canine melanoma, fragment crystallisable (Fc) fusion protein and cryoglobulins.
Glycomic and glycoproteomic studies of native PSG1 showed that four of the seven N-glycosylation sites on this protein were occupied by glycans, which were dominated by mono- and bi-antennary complex structures with sialylation and partial core-fucosylation. N-glycosylation of recombinant N-terminal domain of PSG1 from Chinese Hamster Ovary (CHO) systems differs from the native materials for universal core-fucosylation and extensive poly-LacNAc elongation. N-glycosylation of the new Expi-CHO system was characterised as abundant truncated multi-antennary complex structures.
Analysis of the glycoprotein and glycolipid glycans expressed by stage III canine melanoma cells indicates that the glycosylation of this animal model is highly consistent with that of human melanoma with an exception of species-specific expression of Gal-α-Gal epitopes. These results support the idea that canine melanoma should be an excellent animal model for the pre-clinical studies of melanoma.
Glycomic studies of the Fc fusion protein with engineered additional N-glycosylation sites indicated an improvement of sialylation and branching on N-glycans in a CHO cell system. Identical fusion proteins expressed in a HEK293 cell system exhibited more diverse epitopes and considerable antennary truncation.
Finally, analysis of the N-glycosylation of cryoglobulin indicated that glycosylation might not play a vital role in the formation of the cryoprecipitate of mixed-type cryoglobulins.