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Selective and Tunable Galectin Binding of Glycopolymers Synthesized by a Generalizable Conjugation Method.

ABSTRACT: Glycopolymers, conjugates of synthetic polymers with pendant carbohydrates, are becoming increasingly important to probe the role of carbohydrates in cellular processes and for applications like biosensors and drug delivery. A library of glycopolymers bearing different sugar moieties was synthesized by grafting amino-functionalized sugars to poly(acrylic acid) via DMTMM coupling. Primary amines were introduced at the anomeric (C-1) position to a number of unprotected mono-, di-, and trisaccharides using ammonium carbamate and conjugated to poly(acrylic acid) of different molecular weights, synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. This approach provides a simple and efficient route for the preparation of glycopolymers that differ only in the identity or degree of substitution of the sugar moiety on the polymer. The binding parameters (k_a, k_d, and K_D) of these new glycopolymers to galectins-1 and -3 were quantified using surface plasmon resonance. The galectins selectively bound only to lactose-containing polymers, and the binding affinity was dependent on the galectin type, degree of sugar substitution and the molecular weight of polymer chains. Binding to both galectin-1 and -3 increased with a higher degree of sugar substitution, and higher molecular weight of the polymer backbone, reaching K_D values on the order of 10^-11 M.

SUBMITTER: Zhou C

PROVIDER: S-EPMC8182684 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Site-selective conjugation generally requires both (i) molecular engineering of the protein of interest to introduce a conjugation site at a defined location and (ii) a site-specific conjugation technology. Three N-terminal interferon α2-a (IFN) variants with truncated histidine tags were prepared and conjugation was examined using a bis-alkylation reagent, PEG(10kDa)-mono-sulfone 3. A histidine tag comprised of two histidines separated by a glycine (His2-tag) underwent PEGylation. Two more IFN variants were then prepared with the His2-tag engineered at different locations in IFN. Another IFN variant was prepared with the His-tag introduced in an α-helix, and required three contiguous histidines to ensure that two histidine residues in the correct conformation would be available for conjugation. Since histidine is a natural amino acid, routine methods of site-directed mutagenesis were used to generate the IFN variants from E. coli in soluble form at titres comparable to native IFN. PEGylation conversions ranged from 28-39%. A single step purification process gave essentially the pure PEG-IFN variant (>97% by RP-HPLC) in high recovery with isolated yields ranging from 21-33%. The level of retained bioactivity was strongly dependent on the site of PEG conjugation. The highest biological activity of 74% was retained for the PEG10-106(HGHG)-IFN variant which is unprecedented for a PEGylated IFN. The His2-tag at 106(HGHG)-IFN is engineered at the flexible loop most distant from IFN interaction with its dimeric receptor. The biological activity for the PEG10-5(HGH)-IFN variant was determined to be 17% which is comparable to other PEGylated IFN conjugates achieved at or near the N-terminus that have been previously described. The lowest retained activity (10%) was reported for PEG10-120(HHH)-IFN which was prepared as a negative control targeting a IFN site thought to be involved in receptor binding. The presence of two histidines as a His2-tag to generate a site-selective target for bis-alkylating PEGylation is a feasible approach for achieving site-selective PEGylation. The use of a His2-tag to strategically engineer a conjugation site in a protein location can result in maximising the retention of the biological activity following protein modification.

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Selective and Tunable Galectin Binding of Glycopolymers Synthesized by a Generalizable Conjugation Method.

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