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Synthesis and Immunological Study of N-Glycan-Bacteriophage Qβ Conjugates Reveal Dominant Antibody Responses to the Conserved Chitobiose Core.

ABSTRACT: N-Glycosylation plays an important role in many biological recognition processes. However, very few N-glycan-specific antibodies are available for functional studies and potentially for therapeutic development. In this study, we sought to synthesize bacteriophage Qβ conjugates with representative N-glycans and investigate their immunogenicity for raising N-glycan-specific antibodies. An array of Qβ glycoconjugates bearing five different human N-glycans and two different chemical linkers were synthesized, and the immunization of the N-glycan-Qβ conjugates was performed in mice. We found that the N-glycan-Qβ conjugates raised significant IgG antibodies that recognize N-glycans, but, surprisingly, most of the glycan-dependent antibodies were directed to the shared chitobiose core and were nonspecific for respective N-glycan structures. The linker chemistry was found to affect antibody specificity with adipic acid-linked N-glycan-Qβ immunogens raising antibodies capable of recognizing both the N-acetylglucosamine (GlcNAc) moieties of the chitobiose core. In contrast, antibodies raised by N-glycan-Qβ immunogens with a triazole linker preferentially recognized the innermost N-acetylglucosamine moiety at the reducing end. We also found that sialylation of the N-glycans significantly suppressed the immune response. Furthermore, the N-glycan-Qβ immunogens with an adipic acid linker elicited higher glycan-specific antibody titers than the N-glycan-triazole-Qβ immunogens. These findings delineate several challenges in eliciting mammalian N-glycan-specific antibodies through the conventional glycoconjugate vaccine design and immunization.

SUBMITTER: Donahue TC

PROVIDER: S-EPMC9308761 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Synthesis and Immunological Study of N-Glycan-Bacteriophage Qβ Conjugates Reveal Dominant Antibody Responses to the Conserved Chitobiose Core.

Donahue Thomas C TC Zong Guanghui G O'Brien Nicholas A NA Ou Chong C Gildersleeve Jeffrey C JC Wang Lai-Xi LX

Bioconjugate chemistry 20220610 7

N-Glycosylation plays an important role in many biological recognition processes. However, very few N-glycan-specific antibodies are available for functional studies and potentially for therapeutic development. In this study, we sought to synthesize bacteriophage Qβ conjugates with representative N-glycans and investigate their immunogenicity for raising N-glycan-specific antibodies. An array of Qβ glycoconjugates bearing five different human N-glycans and two different chemical linkers were syn ...[more]

PMID: 35687881

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Project description:UnlabelledChanges in protein function and other biological properties, such as RNA structure, are crucial for adaptation of organisms to novel or inhibitory environments. To investigate how mutations that do not alter amino acid sequence may be positively selected, we performed a thermal adaptation experiment using the single-stranded RNA bacteriophage Qβ in which the culture temperature was increased from 37.2°C to 41.2°C and finally to an inhibitory temperature of 43.6°C in a stepwise manner in three independent lines. Whole-genome analysis revealed 31 mutations, including 14 mutations that did not result in amino acid sequence alterations, in this thermal adaptation. Eight of the 31 mutations were observed in all three lines. Reconstruction and fitness analyses of Qβ strains containing only mutations observed in all three lines indicated that five mutations that did not result in amino acid sequence changes but increased the amplification ratio appeared in the course of adaptation to growth at 41.2°C. Moreover, these mutations provided a suitable genetic background for subsequent mutations, altering the fitness contribution from deleterious to beneficial. These results clearly showed that mutations that do not alter the amino acid sequence play important roles in adaptation of this single-stranded RNA virus to elevated temperature.ImportanceRecent studies using whole-genome analysis technology suggested the importance of mutations that do not alter the amino acid sequence for adaptation of organisms to novel environmental conditions. It is necessary to investigate how these mutations may be positively selected and to determine to what degree such mutations that do not alter amino acid sequences contribute to adaptive evolution. Here, we report the roles of these silent mutations in thermal adaptation of RNA bacteriophage Qβ based on experimental evolution during which Qβ showed adaptation to growth at an inhibitory temperature. Intriguingly, four synonymous mutations and one mutation in the untranslated region that spread widely in the Qβ population during the adaptation process at moderately high temperature provided a suitable genetic background to alter the fitness contribution of subsequent mutations from deleterious to beneficial at a higher temperature.

Dataset Information

Synthesis and Immunological Study of N-Glycan-Bacteriophage Qβ Conjugates Reveal Dominant Antibody Responses to the Conserved Chitobiose Core.

Publications

Synthesis and Immunological Study of N-Glycan-Bacteriophage Qβ Conjugates Reveal Dominant Antibody Responses to the Conserved Chitobiose Core.

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