Biotinylated N-Acetyllactosamine- and N,N-Diacetyllactosamine-Based Oligosaccharides as Novel Ligands for Human Galectin-3

Abstract

Galectin inhibitor design is an emerging research field due to the involvement of galectins in cancer. Galectin-3, in particular, plays an important role in tumor progression. To generate inhibitors, modifications of the glycan structure can be introduced. Conjugation of hydrophobic compounds to saccharides has proven to be promising as increased binding of galectin-3 can be observed. In the present study, we report on neo-glycans carrying hydrophobic biotin as novel ligands for human galectin-3. We modified N-acetyllactosamine- and N,N-diacetyllactosamine-based tetrasaccharides at the C6-position of the terminal saccharide unit using selective enzymatic oxidation and subsequent chemical conjugation of biotinamidohexanoic acid hydrazide. These neo-glycans were much better bound by galectin-3 than the unmodified counterparts. High selectivity for galectin-3 over galectin-1 was also proven. We generated multivalent neo-glycoproteins by conjugation of neo-glycans to bovine serum albumin showing high affinity for galectin-3. Compared to non-biotinylated neo-glycoproteins, we achieved high binding levels of galectin-3 with a lesser amount of conjugated neo-glycans. Multivalent ligand presentation of neo-glycoproteins significantly increased the inhibitory potency towards galectin-3 binding to asialofetuin when compared to free monovalent glycans. Our findings show the positive impact of 6-biotinylation of tetrasaccharides on galectin-3 binding, which broadens the recent design approaches for producing high-affinity ligands.

Keywords: LacdiNAc; biotin; chemo-enzymatic synthesis; galectin-3; neo-glycoprotein.

Scheme 1

Two-step synthesis of 6-biotin tetrasaccharides 6 and 7.

Scheme 2

Neo-glycoprotein synthesis by reaction of BSA and squaric acid monoamide ester glycosides 11 and 12.

Figure 1

SDS-PAGE and streptavidin blot analysis of neo-glycoproteins. The BSA-based neo-glycoproteins were analyzed by SDS-PAGE with Coomassie staining (A) and by a streptavidin blot (B); M, Marker; C, control (BSA); 13a–f, 6-biotin LacNAc-LacNAc conjugated BSA; 14a–f, 6-biotin LacdiNAc-LacNAc conjugated BSA.

Figure 2

Influence of 6-biotinylation of tetrasaccharides LacNAc-LacNAc and LacdiNAc-LacNAc on galectin binding and inhibition. Binding curves (A) and maximal binding signals (B) of Gal-3 to immobilized glycans show differences between biotinylated and non-biotinylated ligands. In competitive inhibition assays, the inhibitory potency was not affected by C6-biotinylation of the tetrasaccharides (C). Comparison of Gal-3 and Gal-1 at a 1 µM protein concentration indicates high specificity of biotinylated tetrasaccharides towards Gal-3 (D).

Figure 3

Comparison of Gal-1 and Gal-3 binding to immobilized neo-glycoproteins. In the ELISA-type assay, binding of 1 µM Gal-1 ($■$) and Gal-3 ($■$) to neo-glycoproteins 13a–f and 14a–f and to asialofetuin (ASF) as the control was measured. Signals showed different binding signals of both galectins to BSA decorated with C6-biotinylated tetrasaccharides.

Figure 4

Binding of human Gal-3 to neo-glycoproteins presenting different numbers of glycans. In an ELISA-type assay, Gal-3 at different concentrations was incubated on immobilized 6-biotin LacNAc-LacNAc- (13a–f) (A) and 6-biotin LacdiNAc-LacNAc-conjugated (14a–f) BSA (B) (5 pmol per well). Binding signals were plotted against galectin concentration and fitted.

Figure 5

Apparent K_d values of Gal-3 for binding to immobilized neo-glycoproteins. The Gal-3 concentration (µM) at half-maximal binding (K_d) is shown for the different neo-glycoproteins 13a–f ($■$) and 14a–f ($■$), as well as ASF ($■$). K_d values decreased with increasing modification degree, indicating higher affinity of human Gal-3 (see Table S3 in the Supplementary Materials).

Figure 6

Inhibition of Gal-3 binding to ASF by BSA-based neo-glycoproteins. With increasing concentrations of 6-biotin LacNAc-LacNAc-conjugated (A) and 6-biotin LacdiNAc-LacNAc-conjugated BSA (B) as well as monovalent controls (6 and 7), Gal-3 binding signals on ASF decreased. The curves shifted to lower inhibitor concentrations with rising glycosylation density.

Figure 7

IC₅₀ values of neo-glycoproteins for Gal-3 inhibition. IC₅₀ values of selected neo-glycoproteins ($■$) and the corresponding free saccharides ($■$) are compared. Neo-glycoproteins showed higher inhibitory potential compared to monovalent glycans, as well as with increasing number of glycans per molecule. Calculated IC₅₀ values are depicted in Table 2.

Figure 8

K_D values resulting from binding of neo-glycoproteins on immobilized Gal-3 in SPR measurements. Gal-3 was immobilized on a sensor chip, and binding to neo-glycoproteins as well as ASF was monitored in flow (20 µL/min). Apparent K_D values were calculated for one concentration by fitting association and dissociation using the software Scrubber2. The numbering of the ligands is explained in Table 3. (* K_D was not detectable).