Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays.

Galectins are ß-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His6-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His6-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6-3-fold increase in binding efficiency for HSYGal-3 (His6-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His6-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His6-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.

Methods of in vitro study of galectin-glycomaterial interaction.

Galectins are a family of carbohydrate-binding lectins modulating cell events such as cell proliferation, apoptosis, adhesion or migration by cross-linking the glycan structures of cell membranes and/or extracellular matrix components. In a diseased organism, galectins are upregulated and trigger the progression of diseases such as inflammation, cancerogenesis, fibrosis, cardiovascular and metabolic disorders. Targeting galectins with glycomaterials for the aims of diagnostics or therapy is, therefore, a focus of biotechnological and biomedicinal research, and already led to candidates for clinical trials. Testing and evaluation of galectin-glycomaterial interactions require informative and versatile analytical methods at several levels of knowledge, from basic inter-molecular interaction to complex cell-based assays. This review aims to classify and characterize a selection of the most promising methods to identify the prospective glycomaterials for translating galectin targeting from the molecular level to the level of tailored in vivo assays.

Enzymatic Synthesis of N-Acetyllactosamine (LacNAc) Type 1 Oligomers and Characterization as Multivalent Galectin Ligands.

Repeats of the disaccharide unit N-acetyllactosamine (LacNAc) occur as type 1 (Galß1, 3GlcNAc) and type 2 (Galß1, 4GlcNAc) glycosylation motifs on glycoproteins and glycolipids. The LacNAc motif acts as binding ligand for lectins and is involved in many biological recognition events. To the best of our knowledge, we present, for the first time, the synthesis of LacNAc type 1 oligomers using recombinant ß1,3-galactosyltransferase from Escherichia coli and ß1,3-N-acetylglucosaminyltranferase from Helicobacter pylori. Tetrasaccharide glycans presenting LacNAc type 1 repeats or LacNAc type 1 at the reducing or non-reducing end, respectively, were conjugated to bovine serum albumin as a protein scaffold by squarate linker chemistry. The resulting multivalent LacNAc type 1 presenting neo-glycoproteins were further studied for specific binding of the tumor-associated human galectin 3 (Gal-3) and its truncated counterpart Gal-3∆ in an enzyme-linked lectin assay (ELLA). We observed a significantly increased affinity of Gal-3∆ towards the multivalent neo-glycoprotein presenting LacNAc type 1 repeating units. This is the first evidence for differences in glycan selectivity of Gal-3∆ and Gal-3 and may be further utilized for tracing Gal-3∆ during tumor progression and therapy.