The Development and Application of Approaches to Discover Siglec Ligands

• Author / Creator

  Schmidt, Edward N.

• Across all domains of life, the surface of all cells is coated in a dense array ofcarbohydrates appended to proteins and lipids, collectively referred to as glycans. The interactionbetween glycans and glycan-binding proteins, also known as lectins, are at the interface of nearlyevery biological interaction at the cellular level. Unlike other biological macromolecules (nucleicacids and proteins), glycan biosynthesis is not template driven and glycans can be branchedpolymers, resulting in heterogenous structures that are difficult to predict, and which are verystructurally complicated. Glycans terminating with the monosaccharide sialic acid are capable ofengaging with the Siglec lectin family. Siglecs facilitate many cellular functions, however, all Siglecfunctions are initiated by engaging with a glycan terminating with sialic acid. In humans, Siglecsare a family of fifteen cell surface receptors, which are generally found on immune cells. Theimmunomodulatory properties of Siglecs motivates a better understanding of their physiologicaland pathophysiological roles. Therefore, the first step in understanding the biology of a Siglecstarts with understanding the ligands of that Siglec. Nevertheless, the complexity and diversity ofglycans coupled with multiple Siglecs often being expressed by a single cell makes deconvolutingSiglec-glycan interactions challenging. Through the development and optimization of approachesthat enable the systematic description of Siglec ligands a better understanding of the roles ofSiglecs in health and disease can be established. Insights gained by describing Siglec ligandscan then be leveraged to investigate the function of the Siglec and potentially apply thesediscoveries to develop novel therapeutics towards the treatment of many pathologies such ascancer, bacterial/viral infection, autoimmune disease, neurodegenerative disease and manyothers.iiiIn Chapter 2, improvements with respect to versatility, activity, yield, and storage of apreviously established soluble version of a Siglec referred to as a Siglec-Fc-chimera were made.The versatility of this construct was demonstrated by its use in many different approaches fordescribing Siglec-ligands including a cell-based glycan array, bead assay, ELISA, and massspectrometry-based assay. Using these different approaches many insights were made withrespect to Siglec ligands. Two examples of new insights are: the relationship between cis andtrans binding of Siglecs on primary human immune cells and the glycan ligands of CD33. It isnoteworthy that the tools and approaches developed in this Chapter were extensively applied inthe later Chapters.In Chapter 3, a novel liposomal nanoparticle formulation was developed for optimalengagement of Siglecs by glycolipids. Using this formulation, the human and murine Siglec familywas screened against a panel of glycolipids. During this interrogation, it was revealed thatglycolipids are ligands for Siglec-6. Additionally, the glycolipid binding profiles between humanand murine Siglecs were compared and the appropriateness of murine models to study humansSiglec-glycolipid interactions was assessed.Building on the work from Chapter 3, the ability of Siglec-6 to bind glycolipids wassystematically dissected using a panel of synthetic glycolipids referred to as neoglycolipidscoupled with mutagenesis studies. These studies revealed that a solvent exposed tryptophanresidue is critical for Siglec-6 engagement of sialosides when presented from a liposome.Additionally, Siglec-6-liposome binding was probed on genuine human cells and tissuesdemonstrating that glycolipid bearing liposomes can target Siglec-6 in physiologically relevantcontexts. A possible biological role was ascribed to Siglec-6 when it was found that Siglec-6 canfacilitate the internalization of natural nanoparticles, known as extracellular vesicles, in aglycolipid-dependant manner. In summary, the work described above demonstrates howsystematic description of Siglec ligands leads to the development of probes which can be appliedto learn more about the biological roles Siglecs play in health and disease.

• Subjects / Keywords

  • Siglec
  • Glycan
  • Flow cytometry

• Graduation date

  Fall 2024

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-w7r3-vc68

• License

  This thesis is made available by the University of Alberta Library 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.