Publicação

Bio-orthogonal site-selective labelling of carbohydrates and proteins

Detalhes bibliográficos
Resumo:	Carbohydrates and proteins represent two large groups of biomolecules which are tremendously important for biological processes in health and disease state. Although protein-structures are encoded in the genome, cellular glycan structures are template independent and can only be addressed in an indirect manner. The development of metabolic oligosaccharide engineering (MOE) gave rise to new methods to study carbohydrate structures in the context of different disease settings and in different organisms. While in many cases mannose derivatives are used to study the sialic acid structures in cancer cells, this work presents the results on the metabolic incorporation of galactose derivatives into cell membrane glycans of human hepatic cells. Three unnatural galactose derivatives containing terminal alkene groups in C2 or C6 position were synthesized and their reaction rates in inverse electron demand Diels Alder reactions (iEDDA) were evaluated, by using a high-throughput screening method in 96-well plates. It was shown that none of the developed galactose derivatives exhibit any cell toxic effect in HepG2 or Huh7 cell lines. Furthermore, all monosaccharides could be successfully incorporated in cell membrane glycan structures of both cell lines and the localization on the cell membrane was confirmed by co-localization with a plasma membrane dye. After developing this incorporation and labeling strategy of unnatural galactose derivatives in the cell membrane of human hepatic cells, the change in incorporation during an infection of these cells with Plasmodium berghei sporozoites was investigated. By using different techniques, such as confocal microscopy, flow cytometry and imaging flow cytometry, only a small trend for an increased uptake of the unnatural galactose derivative in P. berghei infected cells was observed. To explain this result, the pathway for the diffusion of the unnatural galactose derivative was determined. The application of specific and non-specific inhibitors for the glucose transporter GLUT1 revealed that this transporter is involved in the delivery of galactose derivatives into cultured cells. The enhanced translocation of this transporter to the surface of infected hepatic cells explains the observed tendency for an increased incorporation of the unnatural galactose derivative in these cells. Apart from cell studies, MOE was applied for the first time to study a possible transfer of galactose monosaccharides from the mosquito host to the parasite. Biosynthetic pathways for glycan assembly in the parasite are poorly understood. Suggestions on the participation of the mosquito host in some of these pathways, led to the idea to apply MOE in this situation. It was possible to show an uptake of the presented galactose derivatives by the mosquito but only reduced transfer to the parasite seems to occur. In addition to the development of monofunctional galactose derivatives, also a bifunctional derivative containing two orthogonal reporter groups was synthesized. However, so far it was not possible to achieve a metabolic incorporation or labeling of this derivative on cell membrane glycans. After developing cellular tools to study carbohydrate structures, a site-selective method for protein modification was generated, to be used for the development of new glycoconjugate vaccine candidates. By introducing selectively two dehydroalanine residues in place of the disulfide bond C186-C201 of the immunogenic protein CRM197, a new chemical moiety for the conjugation of carbohydrate antigens was obtained. It was shown that these moieties can be used for the selective introduction of polysaccharide antigens from group B Streptococcus (GBS) or Streptococcus pneumoniae. Both types of glycoconjugates could be synthesized and first trials on the purification methods were undertaken. This concept will be developed further for future vaccine candidates. Finally, a synthetic method was developed which could facilitate the synthesis of defined antigenic oligosaccharide structures. This method uses the thiophilic promoter O-mesitylenesulfonylhydroxylamine (MSH) for the activation of thioglycoside donors. It was demonstrated that different thioglycoside donors are activated with different kinetics, depending on the presented protecting groups or the anomeric leaving group. Apart from applying the developed activation method for the synthesis of several glycosylation products, the sequential activation of S-alkyl before S-phenyl anomeric groups was shown during the synthesis of a model trisaccharide. Overall, bio-orthogonal methods were developed and applied for the investigation of carbohydrate structures in the context of malaria disease, and for the site-selective modification of protein carriers during the development of glycoconjugate vaccine candidates.
Autores principais:	Kitowski, Annabel Katharina
Assunto:	Metabolic oligosaccharide engineering Bio-orthogonal labeling Glycoconjugate vaccine candidates Site-selective protein modification Teses de doutoramento - 2019
Ano:	2019
País:	Portugal
Tipo de documento:	tese de doutoramento
Tipo de acesso:	acesso aberto
Instituição associada:	Universidade de Lisboa
Idioma:	inglês
Origem:	Repositório da Universidade de Lisboa