Arabinofuranoside–Antibody Interactions: A Case Study of Furanoside–Protein Binding

• Author / Creator

  Lak, Parnian

• Carbohydrate–protein interactions play key roles in a range of biological processes. Noticeable advancements have been made in understanding the features associated with the recognition of pyranosides (six-membered ring sugars) by proteins. In contrast, similar detailed studies about the interaction of furanosides (five-membered ring sugars) with proteins are scarce. Such interactions are increasingly recognized as important, particularly in host–pathogen recognition. In this thesis, we provide a detailed investigation of a furanoside–protein interaction system, by studying the recognition of a cell-wall arabinofuranoside oligosaccharide fragment of mycobacteria by antibodies. The first binding system addressed is the interaction of a hexaarabinofuranoside antigen (Ara6) of mycobacterial lipoarabinomannan with the CS-35 antibody. Our approach was centered on developing CS-35 single chain variable fragment (scFv) as a useful tool to engineer the binding site. This enabled us to create a library of its mutants. Structural and binding aspects of various fragments interacting with Ara6 were studied from different aspects by various methods such as surface plasmon resonance (SPR), saturation transfer difference NMR spectroscopy, and circular dichroism spectroscopy. Ultimately, we determined the specificity motifs of this binding system including key amino acids, the epitope of the furanoside ligand, and the affinities and kinetics of the binding. In addition to the molecular-level study of Ara6–CS-35 system, we also investigated Ara6-CS-40 and Ara6-906.4321 systems from several aspects. ScFv technology, SPR spectroscopy, STD NMR spectroscopy, CD spectroscopy, and homology modelling assisted us in developing a more detailed picture of these interactions. Finally, we compared the three systems in search for the common features in the recognition of Ara6 by these antibodies. Overall, this research serves to provide a more detailed understanding of the molecular recognition of furanosides by proteins.

• Subjects / Keywords

  • Antibody
  • Binding
  • Furanoside

• Graduation date

  Fall 2014

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3736M69T

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.