Combined effects of glycan chain length and linkage type on the immunogenicity of glycoconjugate vaccines

The development and use of antibacterial glycoconjugate vaccines have significantly reduced the occurrence of potentially fatal childhood and adult diseases such as bacteremia, bacterial meningitis, and pneumonia. In these vaccines, the covalent linkage of bacterial glycans to carrier proteins augments the immunogenicity of saccharide antigens by triggering T cell-dependent B cell responses, leading to high-affinity antibodies and durable protection. Licensed glycoconjugate vaccines either contain long-chain bacterial polysaccharides, medium-sized oligosaccharides, or short synthetic glycans. Here, we discuss factors that affect the glycan chain length in vaccines and review the available literature discussing the impact of glycan chain length on vaccine efficacy. Furthermore, we evaluate the available clinical data on licensed glycoconjugate vaccine preparations with varying chain lengths against two bacterial pathogens, Haemophilus influenzae type b and Neisseria meningitidis group C, regarding a possible correlation of glycan chain length with their efficacy. We find that long-chain glycans cross-linked to carrier proteins and medium-sized oligosaccharides end-linked to carriers both achieve high immunogenicity and efficacy. However, end-linked glycoconjugates that contain long untethered stretches of native glycan chains may induce hyporesponsiveness by T cell-independent activation of B cells, while cross-linked medium-sized oligosaccharides may suffer from suboptimal saccharide epitope accessibility.

The Flexibility of Oligosaccharides Unveiled Through Residual Dipolar Coupling Analysis

The intrinsic flexibility of glycans complicates the study of their structures and dynamics, which are often important for their biological function. NMR has provided insights into the conformational, dynamic and recognition features of glycans, but suffers from severe chemical shift degeneracy. We employed labelled glycans to explore the conformational behaviour of a β(1-6)-Glc hexasaccharide model through residual dipolar couplings (RDCs). RDC delivered information on the relative orientation of specific residues along the glycan chain and provided experimental clues for the existence of certain geometries. The use of two different aligning media demonstrated the adaptability of flexible oligosaccharide structures to different environments.

Glycosylation decreases aggregation and immunogenicity of adalimumab fab secreted from Pichia pastoris

Glycoengineering of therapeutic proteins has been applied to improve the clinical efficacy of several therapeutics. Here, we examined the effect of glycosylation on the properties of the Fab of the therapeutic antibody, adalimumab. An N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis (H: L178N Fab), and the H: L178N Fab was produced in Pichia pastoris. Expressed mutant Fab contained long and short glycan chains (L-glyco Fab and S-glyco Fab, respectively). Under the condition of aggregation of Fab upon pH shift-induced stress, both of L-glyco Fab and S-glyco Fab were less prone to aggregation, with L-glyco Fab suppressing aggregation more effectively than the S-glyco Fab. Moreover, the comparison of the antigenicity of glycosylated and wild-type Fabs in mice revealed that glycosylation resulted in the suppression of antigenicity. Analysis of the pharmacokinetic behavior of the Fab, L-glyco Fab, and S-glyco Fab indicated that the half-lives of glycosylated Fabs in the rats were shorter than that of wild-type Fab, with L-glyco Fab having a shorter half-life than S-glyco Fab. Thus, we demonstrated that the glycan chain influences Fab aggregation and immunogenicity, and glycosylation reduces the elimination half-life in vivo.

Polysaccharide length affects mycobacterial cell shape and antibiotic susceptibility

Bacteria control the length of their polysaccharides, which can control cell viability, physiology, virulence, and immune evasion. Polysaccharide chain length affects immunomodulation, but its impact on bacterial physiology and antibiotic susceptibility was unclear. We probed the consequences of truncating the mycobacterial galactan, an essential linear polysaccharide of about 30 residues. Galactan covalently bridges cell envelope layers, with the outermost cell wall linkage point occurring at residue 12. Reducing galactan chain length by approximately half compromises fitness, alters cell morphology, and increases the potency of hydrophobic antibiotics. Systematic variation of the galactan chain length revealed that it determines periplasm size. Thus, glycan chain length can directly affect cellular physiology and antibiotic activity, and mycobacterial glycans, not proteins, regulate periplasm size.

Assessment of Structural Units Deletions in the Archaeal Oligosaccharyltransferase AglB

Oligosaccharyltransferases (OSTs) are enzymes that catalyze the transfer of a glycan chain to an acceptor protein. Their structure is composed by a transmembrane domain and a periplasmic / C-terminal domain, which can be divided into structural units. The Archaeoglobus fulgidus OST, AfAglB, has unique structural units with unknown functions. Here, we evaluate the stability role proposed for AfAglB units by employing molecular modelling and molecular dynamics simulations, to examine the effect of single and double deletions in the enzyme structure. Our results show a strong effect on the dynamics of the C-terminal domain for the mutated systems with increased fluctuations near the deleted areas. Conformational profile and stability are deeply affected, mainly in the double unit deletion, modifying the enzyme behavior and binding interfaces. Coordination at the catalytic site was not disrupted, indicating that the mutated enzymes could retain activity at some level. Hotspots of variation were identified and rationalized with previous data. Our data shows that structural units may provide stabilization interactions, contributing for integrity of the wild-type enzyme at high temperatures. By correlating our findings to structural units mutagenesis experimental data available, it was observed that structural units deletion can interfere with OSTs stability and dynamics but it is not directly related to catalysis. Instead, they may influence the OST structural integrity, and, potentially, thermostability. This work offers a basis for future experiments involving OSTs structural and functional characterization, as well as for protein engineering.

Squalamine and Aminosterol Mimics Inhibit the Peptidoglycan Glycosyltransferase Activity of PBP1b

Peptidoglycan (PG) is an essential polymer of the bacterial cell wall and a major antibacterial target. Its synthesis requires glycosyltransferase (GTase) and transpeptidase enzymes that, respectively, catalyze glycan chain elongation and their cross-linking to form the protective sacculus of the bacterial cell. The GTase domain of bifunctional penicillin-binding proteins (PBPs) of class A, such as Escherichia coli PBP1b, belong to the GTase 51 family. These enzymes play an essential role in PG synthesis, and their specific inhibition by moenomycin was shown to lead to bacterial cell death. In this work, we report that the aminosterol squalamine and mimic compounds present an unexpected mode of action consisting in the inhibition of the GTase activity of the model enzyme PBP1b. In addition, selected compounds were able to specifically displace the lipid II from the active site in a fluorescence anisotropy assay, suggesting that they act as competitive inhibitors.

Chemical synthesis of human syndecan-4 glycopeptide bearing O-, N-sulfation and multiple aspartic acids for probing impacts of the glycan chain and the core peptide on biological functions

Attaching heparan sulfate glycan on a peptide backbone can modulate biological functions of the glycan.

Use of a Glycan Library Reveals a New Model for Enteric Virus Oligosaccharide Binding and Virion Stabilization

ABSTRACT Enteric viruses infect the gastrointestinal tract, and bacteria can promote replication and transmission of several enteric viruses. Viruses can be inactivated by exposure to heat or bleach, but poliovirus, coxsackievirus B3, and reovirus can be stabilized by bacteria or bacterial polysaccharides, limiting inactivation and aiding transmission. We previously demonstrated that certain N-acetylglucosamine (GlcNAc)-containing polysaccharides can stabilize poliovirus. However, the detailed virus-glycan binding specificity and glycan chain length requirements, and thus the mechanism of virion stabilization, have been unclear. A previous limitation was our lack of defined-length glycans to probe mechanisms and consequences of virus-glycan interactions. Here, we generated a panel of polysaccharides and oligosaccharides to determine the properties required for binding and stabilization of poliovirus. Poliovirus virions are nonenveloped icosahedral 30-nm particles with 60 copies of each of four capsid proteins, VP1 to VP4. VP1 surrounds the 5-fold axis, and our past work indicates that this region likely contains the glycan binding site. We found that relatively short GlcNAc oligosaccharides, such as a six-unit GlcNAc oligomer, can bind poliovirus but fail to enhance virion stability. Virion stabilization required binding of long GlcNAc polymers of greater than 20 units. Our data suggest a model where GlcNAc polymers of greater than 20 units bind and bridge adjacent 5-fold axes, thus aiding capsid rigidity and stability. This study provides a deeper understanding of enteric virus-bacterial glycan interactions, which are important for virion environmental stability and transmission. IMPORTANCE Enteric viruses are transmitted through the fecal-oral route, but how enteric viruses survive in the environment is unclear. Previously, we found that bacterial polysaccharides enhance poliovirus stability against heat or bleach inactivation, but the specific molecular requirements have been unknown. Here, we showed that certain short-chain oligosaccharides can bind to poliovirus but do not increase virion stability. Long-chain polysaccharides bind and may bridge adjacent sites on the viral surface, thus increasing capsid rigidity and stability. This work defines the unique interactions of poliovirus and glycans, which provides insight into virion environmental stability and transmission.

Use of a glycan library reveals a new model for enteric virus oligosaccharide binding and virion stabilization

Enteric viruses infect the gastrointestinal tract and bacteria can promote replication and transmission of several enteric viruses. Viruses can be inactivated by exposure to heat or bleach, but poliovirus, coxsackievirus B3, and reovirus can be stabilized by bacteria or bacterial polysaccharides, limiting inactivation and aiding transmission. We previously demonstrated that certain N-acetylglucosamine (GlcNAc)-containing polysaccharides can stabilize poliovirus. However, the detailed virus-glycan binding specificity and glycan chain length requirements, and thus the mechanism of virion stabilization, has been unclear. A previous limitation was our lack of defined-length glycans to probe mechanisms and consequences of virus-glycan interactions. Here, we generated a panel of polysaccharides and oligosaccharides to determine the properties required for binding and stabilization of poliovirus. Poliovirus virions are non-enveloped icosahedral 30 nm particles with 60 copies of each of four capsid proteins, VP1-4. VP1 surrounds the fivefold axis and our past work indicates that this region likely contains the glycan binding site. We found that relatively short GlcNAc oligosaccharides, such as a six unit GlcNAc oligomer, can bind poliovirus but fail to enhance virion stability. Virion stabilization required binding of long GlcNAc polymers of greater than 20 units. Our data suggest a model where GlcNAc polymers greater than 20 units bind and bridge adjacent fivefold axes, thus aiding capsid rigidity and stability. This study provides a deeper understanding of enteric virus-bacterial glycan interactions, which is important for virion environmental stability and transmission.ImportanceEnteric viruses are transmitted through the fecal-oral route, but how enteric viruses survive in the environment is unclear. Previously, we found that bacterial polysaccharides enhance poliovirus stability against heat or bleach inactivation, but the specific molecular requirements have been unknown. Here we showed that certain short chain oligosaccharides can bind to poliovirus but do not increase virion stability. Long chain polysaccharides bind and may bridge adjacent sites on the viral surface, thus increasing capsid rigidity and stability. This work defines the unique interactions of poliovirus and glycans, which provides insight into virion environmental stability and transmission.

Bioinformatics analysis of diversity in bacterial glycan chain-termination chemistry and organization of carbohydrate-binding modules linked to ABC transporters

The structures of bacterial cell surface glycans are remarkably diverse. In spite of this diversity, the general strategies used for their assembly are limited. In one of the major processes, found in both Gram-positive and Gram-negative bacteria, the glycan is polymerized in the cytoplasm on a polyprenol lipid carrier and exported from the cytoplasm by an ATP-binding cassette (ABC) transporter. The ABC transporter actively participates in determining the chain length of the glycan substrate, which impacts functional properties of the glycoconjugate products. A subset of these systems employs an additional elaborate glycan capping strategy that dictates the size distribution of the products. The hallmarks of prototypical capped glycan systems are a chain-terminating enzyme possessing a coiled-coil molecular ruler and an ABC transporter possessing a carbohydrate-binding module, which recognizes the glycan cap. To date, detailed investigations are limited to a small number of prototypes, and here, we used our current understanding of these processes for a bioinformatics census of other examples in available genome sequences. This study not only revealed additional instances of existing terminators but also predicted new chemistries as well as systems that diverge from the established prototypes. These analyses enable some new functional hypotheses and offer a roadmap for future research.