Design and characterization of galectin-3 fusion proteins and novel multivalent galectin-3 ligands

Aachen (2018) [Dissertation / PhD Thesis]

Page(s): 1 Online-Ressource (IV, 133 Seiten) : Illustrationen


Galectin-3 (Gal-3) is a key player in a variety of biological processes including cancer progression and immune response driven by specific binding and cross-linking of galactose based carbohydrate ligands. In tumor regions, Gal-3, composed of an N-terminal domain and a carbohydrate recognition domain, can be N-terminally cleaved by metalloproteinases. Truncation of Gal-3 plays an important role in regulation of affinity and self-association. In this work, we aimed for a detailed analysis of Gal-3 from two points of view: Firstly, binding of Gal-3 was analyzed after modification of the protein. Twelve Gal-3 constructs with N-terminal truncation (∆1-62 and ∆1-116) and fusions with SNAP-tag and/or yellow fluorescent protein (YFP) were designed and recombinantly produced. We investigated the influence of the truncation and the fusion partners on Gal-3 binding. Altered binding to asialofetuin (ASF) in ELISA-type and surface plasmon resonance (SPR) binding assays was observed. Highest affinity was proved for Gal-3(Δ1-62) and native Gal-3, respectively, whereas Gal-3(Δ1-116) shows only weak binding. Moreover, we demonstrate here for the first time that SNAP-tag and YFP fusions of Gal-3 and truncated Gal-3 modulated and improved binding affinity to ASF. Fusion of truncated Gal-3 with YFP reconstituted binding properties similar to native Gal-3. In combination with a SNAP-tag even improved binding characteristics were obtained. These results emphasize that the N-terminal domain is important for ligand binding. The self-association potential of Gal-3 seemed not be affected by the modifications leading us to the conclusion that Gal-3 interaction involves the C-terminus. Secondly, we analyzed Gal-3 binding from the ligand’s point of view. As Gal-3 is strongly upregulated in many tumor cells it is a potential target for anti-cancer therapy and cancer diagnosis. In search of high-affinity ligands for Gal-3, we established a method to efficiently synthesize multivalent neo-glycoproteins. By conjugating N-acetyllactosamine (LacNAc) based tetrasaacharides to albumin via the homobifunctional linker squaric acid diethyl ester we obtained glycan-protein conjugates with tunable multivalency with up to 29 binding sites. For the first, time the influence of defined glycan density of neo-glycoproteins on Gal-3 binding was investigated. We observed higher affinity with increasing glycosylation density and found multivalent effects at serum level concentrations of Gal-3. As modifications of glycans can lead to promising ligands, we synthesized novel biotin modified glycans and proved high affinity and selectivity for Gal-3 over galectin-1. After conjugation to albumin, biotinylated neo-glycoproteins achieved high binding levels of Gal-3 at lower glycosylation density compared to non-biotinylated neo-glycoproteins. The efficient and selective binding of our tailor-made neo-glycoproteins make them suitable candidates for targeting Gal-3 in cancer related biomedical research. The two points of view of this work complete the circle of Gal-3 binding to carbohydrate ligands paving the path to future applications in tumor therapy and diagnostics. On the one hand, it gives important insights into the binding characteristics of truncated Gal-3 and how galectins may be tailored by fusions and beneficially tuned for higher binding affinity. On the other hand, it broadens the recent design approaches for producing high-affinity ligands.



Böcker, Sophia


Elling, Lothar
Jahnen-Dechent, Wilhelm


  • REPORT NUMBER: RWTH-2018-223859