Genotype-dependent glycoproteoforms of histidine-rich glycoprotein illustrate the fine-tuning of the plasma proteome

Abstract

In the research chapters of this thesis, I focus on possible variants of glycoproteins and in particular HRG. By using primarily MS. I investigated the natural occurrence of sequence variants, and variants in glycosylation. In more details: In Chapter 2, I review how the recombinant production of glycoproteins in various host expression systems affects their glycosylation. These N-glycosylation profiles vary due to the different abundance and activity of the palette of glycotransferases present in the host cell lines. This review highlights the importance in choosing the right host system for recombinant protein production, especially when making biopharmaceuticals, including monoclonal antibodies. In Chapter 3, I explore potential relationships among HRG allelic variants in 44 human plasma HRG samples by using peptide-centric LC-MS/MS methods. Our proteomics data as well as genomics data retrieved from the 1000 Genome project show that several mutations of HRG are mutually exclusive, while other combinations have elevated frequencies of occurrence. The observed variability is related to ethnicity and global spread in population. In Chapter 4, I investigate in detail the glycosylation of endogenous HRG purified from human plasma and recombinant HRG samples expressed in human HEK293 cells. The latter were produced with selected combinations of allelic mutations. The work clearly shows that HRG N-glycosylation profiles vary between plasma and the HEK293 expression systems. We also uncover a novel O-glycosylation site, that could be pinpointed by using EThcD fragmentation. In addition, we show in this chapter that sialylation of HRG and the rs9898 mutation influence HRG cleavage by plasmin. Finally, in Chapter 5, I explore the relationships between HRG allelic variations, glycosylation, and inhibition of Factor XII autoactivation. Interestingly, HRG glycosylation (sialylation) can significantly protect FXII autoactivation from HRG inhibition, while mutation had minimal effects.