The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the −2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the −2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native ‘QYNST’ sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity.

Summary The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens.

The reversible, mitochondrial membrane-associated transhydrogenase from the midgut of Manduca sexta (L.) (Lepidoptera: Sphingidae) catalyzes hydride-ion transfer between NADP(H) and NAD(H). The effects of ecdysone and 20-hydroxyecdysone were evaluated and compared to both the NADH-NADP+ and NADPH-NAD+ transhydrogenations. In the direction of NADPH-formation, the developmentally significant transhydrogenations occur as non-energy- or energy-linked reactions. The energy-linked activity couples with either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg++ -dependent ATPase. Upon the addition of ecdysone alone, all energy-linked reactions in the direction of NADPH formation exhibited a notable increase in activity level over the control reaction. The addition of 20-hydroxyecdysone yielded no significant increase in the activity of any of the transhydrogenations. Synergistic addition of both ecdysone and 20-hydroxyecdysone resulted in no significant effect on transhydrogenase activity. The results of this study make evident a relationship between the presence of ecdysone and 20-hydroxyecdysone on the overall activity of M. sexta midgut mitochondrial transhydrogenations. The potential mediation of the energy-linked mitochondrial transhydrogenations involved with NADPH synthesis through the developmental relationship of ecdysone and 20-hydroxyecdysone is considered.