Multivalent scaffolds induce galectin-3 aggregation into nanoparticles

Abstract

Galectin-3 meditates cell surface glycoprotein clustering, cross linking, and lattice formation. In cancer biology, galectin-3 has been reported to play a role in aggregation processes that lead to tumor embolization and survival. Here, we show that lactose-functionalized dendrimers interact with galectin-3 in a multivalent fashion to form aggregates. The glycodendrimer-galectin aggregates were characterized by dynamic light scattering and fluorescence microscopy methodologies and were found to be discrete particles that increased in size as the dendrimer generation was increased. These results show that nucleated aggregation of galectin-3 can be regulated by the nucleating polymer and provide insights that improve the general understanding of the binding and function of sugar-binding proteins.

Keywords: dendrimers; galectin-3; glycodendrimers; multivalency; multivalent glycosylation; protein aggregation.

Scheme 1

Synthesis of isothiocyanato-functionalized lactoside 1.

Scheme 2

Synthesis of carbohydrate-functionalized PAMAM dendrimers. (Values for m and for x equivalents added are given in Supporting Information File 1.)

Figure 1

Effective diameter of galectin-3/glycodendrimer aggregates (DLS). Final concentration of galectin-3 31 μM; final concentration of glycodendrimers 0.14, 3.3, 11.5 μM for 220:1, 9:1, 3:1, respectively. 2 (blue), 3 (black), 4 (red) and 5 (green). Aggregate size was below the detection limit for 2 at 0.14 μM.

Figure 2

Schematic representation of galectin-3/glycodendrimer aggregates at varying stoichiometries.

Figure 3

Fluorescence microscopy images of labelled particles. Microbead standards at similar exposure times are shown in (a)–(d); (a) 190 nm, inset is a 4× enlargement of selected area, (b) 520 nm, inset is a 4× enlargement of selected area, (c) 1020 nm and (d) 1900 nm. Aggregates formed after ca. 60 min incubation of Alexa 488 labelled galectin-3 (A488gal-3) with lactose-functionalized dendrimers are shown in (e)–(h); (e) A488gal-3 and 2; (f) A488gal-3 and 3; (g) A488gal-3 and 4; (h) A488gal-3 and 5. Exposure times of lectin–glycodendrimer aggregates are provided in Table S6, Supporting Information File 1.

Figure 4

Aggregate diameter distribution of fluorescence microscopy images; 31 μM galectin-3 and 0.14 μM (a) 2, (b) 3, (c) 4 and (d) 5.