Design of Artificial Glycosidases: Metallopeptides that Remove H Antigen from Human Erythrocytes

Abstract

Catalysts that promote carbohydrate degradation have a wide range of potential applications, but the use of either enzyme glycosidases or small-molecule catalysts in biological systems raises significant challenges. Herein, we demonstrate a novel strategy for the design of synthetic agents that mimic natural glycosidases and address current problems for biological use. This strategy is illustrated by application to the development of potential blood substitutes for the rare Bombay blood type that is characterized by a deficiency of H2 antigen. Metallopeptides with 16 to 20 amino acids were constructed as artificial fucosidases that exhibit selective carbohydrate cleavage reactivity toward l-fucose over d-glucose. Selective fucose cleavage from the H2-antigen saccharide enables efficient removal of H2 antigen from erythrocytes and thereby accomplishes the conversion of regular human type-O blood into a potential blood substitute for the rare Bombay blood type.

Keywords: bioinorganic chemistry; blood antigens; carbohydrates; enzymes; peptides.

Figure 1

Cleavage of type 2 H-trisaccharide monitored by LC-ESI-MS. (a) Removal of fucose from the type 2 H-trisaccharide (Fucα1-2Galβ1-4GlcNAc) forms the disaccharide Galβ1-4GlcNAc. (b) and (c) A 25 μM solution of H-trisaccharide was incubated with 50 μM CuGGH-tOL-NH₂, 1 mM ascorbate and 1 mM H₂O₂ at 37°C for 0, 15, 30, 60, 90, and 120 min. Extracted ion chromatogram (EIC) at m/z 530.2 ± 0.1 (b) and m/z 384.1 ± 0.1 (c). (d) Time-dependant cleavage of 25 μM H-trisaccharide by 50 μM metallopeptides, 1 mM ascorbate and 1 mM H₂O₂ at 37°C.

Figure 2

Removal of H2-antigen from erythrocytes. a) Flow cytometric analysis of H2 antigen on the surface of human red blood cells (RBC). After erythrocytes were incubated with 20 μM CuGGH-tOL-NH₂ and 200 μM ascorbate for 4 h, immunofluorescent staining was performed with a FITC-labelled anti-H2 antibody. b) Relative amount of H2 antigen on erythrocytes, after incubation with or without peptide or ascorbate. Solutions containing 200 μM ascorbate and 20 μM CuGGH-tOL-NH₂ were used. c) Relative amount of H2 antigen on erythrocytes after incubation with various amount of CuGGH-tOL-NH₂. d) The half-maximal effective concentration (EC₅₀) for H2-antigen removal by artificial fucosidases in the presence of 200 μM ascorbate. e) and f) Inhibitory effect of saccharides. Human erythrocytes were incubated with 20 μM CuGGH-tOL-NH₂ (e), or CuGGH (f), with the indicated concentration of saccharides, and 200 μM ascorbate.