Influence of distal glycan mimics on direct electron transfer performance for bilirubin oxidase bioelectrocatalysts.

Bilirubin oxidase (BOD) is a bioelectrocatalyst that reduces dioxygen (O2) to water and is capable of direct electron transfer (DET)-type bioelectrocatalysis via its electrode-active site (T1 Cu). BOD from Myrothecium verrucaria (mBOD) has been widely studied and has strong DET activity. mBOD contains two N-linked glycans (N-glycans) with N472 and N482 binding sites distal to T1 Cu. We previously reported that different N-glycan compositions affect the enzymatic orientation on the electrode by using recombinant BOD expressed in Pichia pastoris and the deglycosylation method. However, the individual function of the two N-glycans and the effects of N-glycan composition (size, structure, and non-reducing termini) on DET-type reactions are still unclear. In this study, we utilize maleimide-functionalized polyethylene glycol (MAL-PEG) as an N-glycan mimic to evaluate the aforementioned effects. Site-specific enzyme-PEG crosslinking was carried out by specific binding of maleimide to Cys residues. Recombinant BOD expressed in Escherichia coli (eBOD), which does not have a glycosylation system, was used as a benchmark to evaluate the effect. Site-directed mutagenesis of Asn residue (N472 or N482) into Cys residue is utilized to realize site-specific glycan mimic modification to the original binding site.

Combining CuAAC reaction enables sialylated Bi- and triantennary pseudo mannose N-glycans for investigating Siglec-7 interactions.

Naturally occurring N-glycans display much diversity in modifications, linkages, and peripheral presentation of the oligosaccharide chain. Despite continued advancements in oligosaccharide synthesis, synthetic access to these natural glycans remains challenging. Biologically relevant complex N-glycan mimetics with various natural and unnatural modifications are an alternate way for investigating glycan-protein interactions. Further supporting this pattern, we report here a new class of sialylated bi- and triantennary pseudo mannose N-glycans reproducing orientation of the underlying glycan chain and branching patterns and replacing the two inner mannopyranosyl units with 1,2,3-triazole rings. Such mimetics are straightforwardly generated by implementing multiple intermolecular Cu(I)-catalyzed azide-alkyne cycloaddition between chemoenzymatically synthesized azido sialosides and rationally designed C-3 and C-6 di-O- or C-2, C-3, and C-6 tri-O-alkynylated mannoside. Human recombinant Siglec-7-Fc fusion protein recognizes almost all sialylated pseudo mannose N-glycans in the microarray. However, a differential Sia-binding pattern was also observed. Given the library size, comparison of pairwise mannose N-glycan combinations showed that biantennary linear α(2,3)α(2,8)- and α(2,6)α(2,8)- or branched α(2,3)α(2,6)-, and triantennary branched α(2,3)α(2,6)-sialyl pseudo N-glycans possess similar binding capabilities and affinity to recombinant Siglec-7-Fc. While the full range of topological mannose arms remain elusive, the bi- and triantennary mimics are simpler structures for interrogating Siglec interactions.