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 glycandependent 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 Nacetylglucosamine 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.

Synthetic Site-Specific Antibody–Ligand Conjugates Promote Asialoglycoprotein Receptor-Mediated Degradation of Extracellular Human PCSK9

“…To enable the chemoenzymatic synthesis of ASGPR-specific LYTACs and explore the substrate scope of the ASGPR receptor, several natural N -glycan structures were first prepared from the sialoglycopeptide (SGP) isolated from chicken egg yolks. Thus, the asialo-biantennary complex-type N -glycan-Asn (G2-Asn, 1 ) was prepared by protease digestion of SGP followed by sialidase digestion to remove terminal α­(2,6)-sialic acid, as described in our previous reports. , The asialylated N -glycan (G2-Asn) was then reacted with 1.5 mol equiv NHS-PEG 5 -DBCO to prepare G2-Asn-DBCO ( 2 ) and install a chemical handle at the Asn N-terminus for click conjugation (Scheme ). The final product was purified by reversed-phase preparative HPLC to give 2 .…”

“…Thus, the asialo-biantennary complex-type N-glycan-Asn (G2-Asn, 1) was prepared by protease digestion of SGP followed by sialidase digestion to remove terminal α(2,6)-sialic acid, as described in our previous reports. 42,43 The asialylated N-glycan (G2-Asn) was then reacted with 1.5 mol equiv NHS-PEG 5 -DBCO to prepare G2-Asn-DBCO (2) and install a chemical handle at the Asn N-terminus for click conjugation (Scheme 1). The final product was purified by reversed-phase preparative HPLC to give 2.…”

Catanionic Vesicles as a Facile Scaffold to Display Natural N-Glycan Ligands for Probing Multivalent Carbohydrate–Lectin Interactions

“…For this purpose, we chose to use the asparagine-containing natural N-glycans as the starting materials and take advantage of the free amino group in the asparagine residue to introduce a lipid chain. We selected four typical N-glycans as model sugars, including the sialylated bi-antennary complex-type glycan (S2G2-Asn), the asialylated bi-antennary Asn-glycan (S0G2), and the two high-mannose type N-glycans (Man9-Asn and Man5-Asn), which we have previously prepared by digestion of the chicken egg yolk sialoglycopeptide (SGP) and soybean agglutinin with pronase, respectively, followed by size-exclusion chromatographic purification. , Glycolipids bearing lipids with different lengths were synthesized to investigate if a longer lipid chain is required to incorporate the large natural N-glycans in the vesicles. Thus, reaction of the asialo-complex-type N-glycan ( 1 ) with the N-hydroxysuccinimide (NHS)-activated ester of dodecanoic acid gave the corresponding N-glycolipid ( 5 ) containing a C12 lipid chain in excellent yield.…”

Chemoenzymatic Synthesis of Genetically-Encoded Multivalent Liquid N-glycan Arrays

“…LiGA constructs-glycosylated bacteriophages or "glycophages"-like polymers, liposomes, hollow bacteriophage Qβ capsids 20,33 or protein cages 70 have an optimal 'soluble' format suitable for cell-based assays and in vivo assays but differ from these more traditional formats, as they can be genetically encoded. Other cell-based glycoarrays 50,53,71 can potentially give rise to similar, DNA-encoded multivalent constructs displayed in a natural milieu.…”

“…Furthermore, with the exception of BSA-conjugate arrays 19,29,30 , it is challenging to systematically probe the effect of density using solid-phase arrays. The synthesis of homogeneous multivalent displays of glycans on polymers, dendrimers, liposomes, and other carriers offers a higher level of control of density [31][32][33][34][35][36] . Many such displays have been used to study cellular responses 20,33−38 .…”