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. 2022 Dec 6;144(50):23134–23147. doi: 10.1021/jacs.2c10757

Figure 1.

Figure 1

Mannose receptor (MR, CD206) glycopolymer ligands. (a) Structure of CD206-binding multivalent glycans designed in this study. These glycopolymer ligands possess variable chain length (n = 32 and 187) and either mannose (Man) or SO4-3-galactose (SO4-3-Gal) CD206-binding sugar units. Glycopolymer contents in Man (x) and SO4-3-Gal (y) are systematically varied (0, 33, 66, and 100%), using galactose (Gal) as the remaining non-CD206-binding polymer repeating units. Glycopolymers containing only non-CD206-binding Gal repeating units, G32 and G187, were used in this study as negative control ligands. All glycopolymers are fluorescently labeled with 1% mol/mol of either 2,4,5,7,7′-pentafluorofluorescein (Oregon Green (OG), λex/em 488/530 nm) or Nile blue (λex/em 629/670 nm) to enable detection in in vitro and in vivo assays. Table S1 shows the full composition and characterization of the glycopolymers. (b) Schematic representation of the mannose receptor (MR, CD206). CR: Cysteine-rich domain which binds 3/4-O-sulfate galactose (SO4-3/4)-Gal) and N-(acetyl galactosamine) (SO4-3/4)-GalNAc); FNII: collagen-binding fibronectin type II domain; CTLD: C-type lectin-like domain which binds mannose (Man), fucose (Fuc), and N-(acetyl glucosamine) (GlcNAc). (c) Binding of glycopolymers to CTLD4-7 and CR CD206 fragments as assessed by ELISA assay. Glycopolymers were first immobilized, and then for (i) lectin ELISA tests, they were treated with CTLD4-7-Fc or CR-Fc CD206 Fc-chimeras, while for (ii) inhibition ELISA tests, CTLD4-7-Fc and CR-Fc fragments were first co-incubated with 25 mM 2′-azidoethyl-O-α-D-mannopyranoside or 3-O-sulfo-2′-azidoethyl-O-β-D-galactopyranoside monovalent ligands and then added to the immobilized glycopolymers. In both sets of experiments, CD206 fragments were detected with anti-human IgG Fc-specific, alkaline phosphatase conjugates. G32 = (G100%)32, G187 = (G100%)187, M187 = (M100%)187, and S187 = (S100%)187. Results are from three independent experiments performed in quadruplicate. Data represent mean ± s.d. CSA: chondroitin sulfate A.