title: Structural and Functional Investigation of Galectin-8 and Galectin-9: A Pathway to Develop Novel Cancer Drugs
Creators: name: Eslampanah Seyedi, Hadieh A
Publisher: name: Griffith University
Year: 2021
Subjects: subject: galectin-8
subject: galectin-9
subject: carbohydrate recognition domains

description: Galectins, or galactose binding proteins, are widely distributed in nature from sponges to humans and are defined by highly conserved carbohydrate recognition domains (CRDs). Up to now, 15 mammalian galectins have been identified, 12 of which are in humans. Galectins are found in both intracellular and extracellular environments and participate in vital biological processes such as cell growth, cell adhesion and signal transduction. The multiple functions of galectins, in particular as mediators of cell-cell and cell-matrix interactions, have been correlated to several physiological and pathological diseases containing cancer metastasis, angiogenesis, inflammation, immune response, bacterial and viral infections. This study aims to investigate two human galectins, galectin-8 and galectin-9, which can simultaneously bind two different carbohydrates through two distinct CRDs and are therefore called "tandem repeat" type galectins. The two CRDs in galectin-8 and galectin-9 are associated by a peptide linker of variable length showing high flexibility and protease susceptibility that make these proteins challenging for in vitro studies. To probe the molecular basis of galectin-8 and galectin-9 function in glycan ligand recognition, the work enclosed within this dissertation presents the recombinant expression of galectin-8 and galectin-9 in their native forms as the full-length protein isoforms called galectin-8M (medium isoform), galectin-8L (long isoform), and galectin-9M (medium isoform) as well as single N- and C-CRDs named galectin-8N, galectin-8C, galectin-9N and galectin-9C. Characterisation of the expressed proteins revealed for the first time different dimerisation states for two galectin-8 isoforms: galectin-8L’s high tendency to self-associate, while galectin-8M is found primarily in the monomeric form. For individual CRDs, galectin-8C indicated the disulfide-dependent dimeric form and galectin-8N partially formed tetrameric structure. The biochemical and biophysical assessments also revealed that galectin-9M forms a dimeric in addition to monomeric and partially turn to either trimeric or tetrameric structure. This very distinct tendency to form oligomers among recombinant galectins could affect the proteins’ capacity to bind glycans as well as to engage in cross-networking on the cell surface. For this purpose, a N-acetylneuraminic acid derivative with the biological relevant α-configuration (Methyl α-glycoside of Neu5Ac, Neu5Acα2Me) was analysed in complex with recombinant galectins. Structural and binding analytical investigations using various biophysical and computational methods revealed that Neu5Acα2Me binds to galectin-8N in a distinct conformation compared to 3'-Sialyllactose (3'SL), whilst maintaining essential Arg59-carboxylic acid interaction. The position of OMe-aglycon moiety of Neu5Acα2Me differed significantly compared to 3’SL that opens a new pathway for structure-guided inhibitor design. The thesis further showed for the first time that galectin-8L’s self-association predominantly occurs via N-CRD with picomolar affinity. The dimerisation of galectin-8L was found to completely block the binding to Neu5Acα2Me. This suggests that the protein self-association occurs in close proximity to the Neu5Acα2Me binding site of the N-CRD domain. Galectin-9M binding to Neu5Acα2Me occurred via its C-CRD domain and at much lower affinity compared to galectin-8M’s N-CRD binding to Neu5Acα2Me. In addition, it was found that Neu5Acα2Me has the ability to inhibit human erythrocyte agglutination in the presence of either galectin-8M or galectin-8N. This result is of significant importance for designing lectin-based inhibitors in human’s cancer use. Overall, the outcomes of this research thesis have contributed to achieve an in-depth understanding of galectin-8 and galectin-9 proteins, both in the form of full-length isoforms and single CRD domains. Furthermore, this thesis provides novel insights of galectins’ ability to oligomerise, influencing binding capacity, and engage in cross-network interactions. Structural and functional studies contributed to a comprehensive view of the binding of Neu5Acα2Me to galectins. Remarkably, the particular ability of galectin-8 protein in interaction with Neu5Acα2Me uncovered an important binding event and enable researchers to move towards a specific inhibitor design from neuraminic acid-based molecule targeting galectin-8 in cancer disease.