Catanionic Vesicles as a Facile Scaffold to Display Natural N-Glycan Ligands for Probing Multivalent Carbohydrate-Lectin Interactions

Abstract

Multivalent interactions are a key characteristic of protein-carbohydrate recognition. Phospholipid-based liposomes have been explored as a popular platform for multivalent presentation of glycans, but this platform has been plagued by the instability of typical liposomal formulations in biological media. We report here the exploitation of catanionic vesicles as a stable lipid-based nanoparticle scaffold for displaying large natural N-glycans as multivalent ligands. Hydrophobic insertion of lipidated N-glycans into the catanionic vesicle bilayer was optimized to allow for high-density display of structurally diverse N-glycans on the outer membrane leaflet. In an enzyme-linked competitive lectin-binding assay, the N-glycan-coated vesicles demonstrated a clear clustering glycoside effect, with significantly enhanced affinity for the corresponding lectins including Sambucus nigra agglutinin (SNA), concanavalin A (ConA), and human galectin-3, in comparison with their respective natural N-glycan ligands. Our results showed that relatively low density of high-mannose and sialylated complex type N-glycans gave the maximal clustering effect for binding to ConA and SNA, respectively, while relatively high-density display of the asialylated complex type N-glycan provided maximal clustering effects for binding to human galectin 3. Moreover, we also observed a macromolecular crowding effect on the binding of ConA to high-mannose N-glycans when catanionic vesicles bearing mixed high-mannose and complex-type N-glycans were used. The N-glycan-coated catanionic vesicles are stable and easy to formulate with varied density of ligands, which could serve as a feasible vehicle for drug delivery and as potent inhibitors for intervening protein-carbohydrate interactions implicated in disease.

Figure 1.

(A) Vesicle elution profile from the Sepharose G-50 column, peak observed between 13 and 19 mL elution volume by DLS at 633 nm. (B) Glycolipid incorporation for C12- and C16-functionalized S0G2-GlcNAc₂-Asn at varying concentrations of N-glycolipid (0.2, 0.45, and 0.85 mM). (C) Glycolipid incorporation of S2G2-GlcNAc₂-Asn-C16 at 0.2, 0.8, and 1.6 mM N-glycolipid. (D) Glycolipid incorporation of Man9-GlcNAc₂-Asn-C16 at 0.2, 0.6, and 1.6 mM N-glycolipid. (D) Glycolipid incorporation of Man5-GlcNAc₂-Asn-C16 at 0.2, 0.8, and 1.6 mM N-glycolipid. All measurements of glycolipid incorporation were done in triplicate.

Figure 2.

Endo-CC glycan release experiments on N-glycan-coated vesicles to confirm N-glycolipid incorporation (A) MALDI-TOF MS spectra for S0G2-GlcNAc₁, calculated, M = 1437.5 Da; found, m/z 1460.7 [M + Na]⁺, (B) S2G2-GlcNAc₁, calculated, M = 2019.7 Da; found, m/z 2040.8 [M + Na − 2H]⁻, (C) Man9-GlcNAc₁, calculated, M = 1679.6 Da; found, m/z 1702.6 [M + Na]⁺, and (D) Man₅-GlcNAc₁, calculated, M = 1031.3 Da; found, m/z 1054.9 [M + Na]⁺.

Figure 3.

(A) Design of microplate-based competitive inhibition assay, N-glycan-coated vesicles inhibit lectin binding to synthetic N-glycan-BSA conjugate coating antigens. Detection is done by Streptavidin-HRP (Strep-HRP), or a mAb-HRP conjugate in the case of Galectin-3. Colored shapes represent different lectins. (B) Inhibition curve of ConA binding to Man9-BSA conjugates with Man9-gCVs as inhibitors. (C) Inhibition of SNA binding to S2G2-BSA conjugates with S2G2-gCVs. (D) Inhibition of Gal-3 binding to S0G2-BSA conjugates with S0G2-gCVs as the inhibitors. (E) Inhibition curve of ConA binding to Man5-BSA conjugates with Man5-gCVs as inhibitors. Insets in each graph provide the IC₅₀ and multivalent enhancement (β) for the corresponding inhibitors shown on the right side. The molar concentrations are defined as the concentration of the equivalent sugar ligands on the particle, instead of the molar concentration of the particles themselves.

Figure 4.

(A) Scheme for the preparation of heteromultivalent vesicles from glycolipids 7 and 9. (B) N-Glycolipid incorporation of heteromultivalent S2G2/Man5-gCV vesicles as quantified by HPLC-based assay.

Figure 5.

(A) Competitive inhibition of ConA binding to Man5-BSA conjugates by S2G2/Man5-C16 heteromultivalent N-gCVs. (B) Competitive inhibition of SNA binding to S2G2-BSA conjugates by S2G2/Man5-C16 heteromultivalent N-gCV and homomultivalent S2G2-C16 N-gCV.

Scheme 1.

Synthesis of N-Glycan Lipids Using Free Asparagine-Linked N-Glycans as the Starting Materials

Scheme 2.

Assembly of N-Glycan-Coated Catanionic Vesicles^{a a}Catanionic vesicles were formulated from sodium dodecylbenzene sulfonate (SDBS) and cetyltrimethylammonium tosylate (CTAT) detergents in water and allowed to equilibrate. Preformed vesicles were mixed with N-glycolipids 5−9 at room temperature for 16 h to obtain N-glycan-coated vesicles