CHEMOENZYMATIC SYNTHESIS OF HOMOGENEOUS GLYCOPEPTIDES AND GLYCOPROTEINS FOR FUNCTIONAL STUDIES

Functional and structural studies on glycoproteins are hampered by lack of homogeneous structures due to the inherent structural heterogeneity in the pendant glycans which makes separation by routine chromatographic techniques extremely difficult. To address this problem, we used a chemoenzymatic method to make homogeneous glycopeptides and glycoproteins, which relies on the glycosynthase-based transglycosylation of a GlcNAc-peptide/protein using synthetic glycan oxazoline as the enzyme substrate. The thesis research presented here consists of three projects. First, we synthesized selectively fluorinated homogeneous Man3GlcNAc2-RNase glycoproteins for binding studies to the concanavalin A (con A) lectin. From SPR analysis, we observed that fluorine substitution at the C6-hydroxyl of the α-1,6-branched mannose residue of the Man3GlcNAc2 negated lectin binding suggesting the importance of that hydroxyl group in binding interactions. We found that the fluorinated glycan oxazoline corresponding to the Man3GlcNAc core could serve as an excellent substrate for the Endo-A enzyme during transglycosylation and a structural modeling analysis of the Endo-A enzyme binding pocket suggested favorable hydrophobic interactions between a suitably located tyrosine and the fluorine. This suggest that the selectively fluorinated glycoproteins can be used to probe specific protein-carbohydrate interactions. Secondly, we synthesized homogeneous HIV-1 V3 glycopeptides for characterizing the epitope of a recently discovered, potent anti-HIV-1 broadly neutralizing antibody (bnAb) PGT121 and related antibody 1074. SPR binding analysis indicated that antibody PGT121 recognizes complex type N-glycan while the PGT121-like antibody 10-1074 binds to high-mannose type N-glycan (Man9GlcNAc2) in the context of the V3 domain. Interestingly, these antibodies did not bind to free N-glycans or non-glycosylated V3 polypeptides under the same conditions. This study has facilitated the characterization of the glycan specificity of the two anti-HIV antibodies. Finally, we designed and synthesized a novel multifunctional BALT tag for separation of N-glycans for functional glycomics applications. It consists of an N-methylhydroxylamine group for attachment to reducing ends of glycans with preservation of ring structure, free primary amine for immobilization to microarrays, tyrosine group providing a chromophore and hydrophobicity during chromatographic separation and biotin for binding to streptavidin. The tag was successfully used for separation and recovery of individual N-glycans from a mixture of four N-glycans released from the glycoprotein bovine fetuin

CHEMOENZYMATIC SYNTHESIS OF HOMOGENEOUS GLYCOPEPTIDES AND GLYCOPROTEINS FOR FUNCTIONAL STUDIES

Functional and structural studies on glycoproteins are hampered by lack of homogeneous structures due to the inherent structural heterogeneity in the pendant glycans which makes separation by routine chromatographic techniques extremely difficult. To address this problem, we used a chemoenzymatic method to make homogeneous glycopeptides and glycoproteins, which relies on the glycosynthase-based transglycosylation of a GlcNAc-peptide/protein using synthetic glycan oxazoline as the enzyme substrate. The thesis research presented here consists of three projects. First, we synthesized selectively fluorinated homogeneous Man3GlcNAc2-RNase glycoproteins for binding studies to the concanavalin A (con A) lectin. From SPR analysis, we observed that fluorine substitution at the C6-hydroxyl of the α-1,6-branched mannose residue of the Man3GlcNAc2 negated lectin binding suggesting the importance of that hydroxyl group in binding interactions. We found that the fluorinated glycan oxazoline corresponding to the Man3GlcNAc core could serve as an excellent substrate for the Endo-A enzyme during transglycosylation and a structural modeling analysis of the Endo-A enzyme binding pocket suggested favorable hydrophobic interactions between a suitably located tyrosine and the fluorine. This suggest that the selectively fluorinated glycoproteins can be used to probe specific protein-carbohydrate interactions. Secondly, we synthesized homogeneous HIV-1 V3 glycopeptides for characterizing the epitope of a recently discovered, potent anti-HIV-1 broadly neutralizing antibody (bnAb) PGT121 and related antibody 1074. SPR binding analysis indicated that antibody PGT121 recognizes complex type N-glycan while the PGT121-like antibody 10-1074 binds to high-mannose type N-glycan (Man9GlcNAc2) in the context of the V3 domain. Interestingly, these antibodies did not bind to free N-glycans or non-glycosylated V3 polypeptides under the same conditions. This study has facilitated the characterization of the glycan specificity of the two anti-HIV antibodies. Finally, we designed and synthesized a novel multifunctional BALT tag for separation of N-glycans for functional glycomics applications. It consists of an N-methylhydroxylamine group for attachment to reducing ends of glycans with preservation of ring structure, free primary amine for immobilization to microarrays, tyrosine group providing a chromophore and hydrophobicity during chromatographic separation and biotin for binding to streptavidin. The tag was successfully used for separation and recovery of individual N-glycans from a mixture of four N-glycans released from the glycoprotein bovine fetuin

The Odd “RB” Phage—Identification of Arabinosylation as a New Epigenetic Modification of DNA in T4-Like Phage RB69

In bacteriophages related to T4, hydroxymethylcytosine (hmC) is incorporated into the genomic DNA during DNA replication and is then further modified to glucosyl-hmC by phage-encoded glucosyltransferases. Previous studies have shown that RB69 shares a core set of genes with T4 and relatives. However, unlike the other “RB” phages, RB69 is unable to recombine its DNA with T4 or with the other “RB” isolates. In addition, despite having homologs to the T4 enzymes used to synthesize hmC, RB69 has no identified homolog to known glucosyltransferase genes. In this study we sought to understand the basis for RB69’s behavior using high-pH anion exchange chromatography (HPAEC) and mass spectrometry. Our analyses identified a novel phage epigenetic DNA sugar modification in RB69 DNA, which we have designated arabinosyl-hmC (ara-hmC). We sought a putative glucosyltranserase responsible for this novel modification and determined that RB69 also has a novel transferase gene, ORF003c, that is likely responsible for the arabinosyl-specific modification. We propose that ara-hmC was responsible for RB69 being unable to participate in genetic exchange with other hmC-containing T-even phages, and for its described incipient speciation. The RB69 ara-hmC also likely protects its DNA from some anti-phage type-IV restriction endonucleases. Several T4-related phages, such as E. coli phage JS09 and Shigella phage Shf125875 have homologs to RB69 ORF003c, suggesting the ara-hmC modification may be relatively common in T4-related phages, highlighting the importance of further work to understand the role of this modification and the biochemical pathway responsible for its production

Glycosylation States on Intact Proteins Determined by NMR Spectroscopy

Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B

Systematic Synthesis and Binding Study of HIV V3 Glycopeptides Reveal the Fine Epitopes of Several Broadly Neutralizing Antibodies

A class of new glycan-reactive broadly neutralizing antibodies represented by PGT121, 10–1074, and PGT128 has recently been discovered that targets specific N-glycans and the peptide region around the V3 domain. However, the glycan specificity and fine epitopes of these bNAbs remain to be further defined. We report here a systematic chemoenzymatic synthesis of homogeneous V3 glycopeptides derived from the HIV-1 JR-FL strain carrying defined N-glycans at N332, N301, and N295 sites. Antibody binding studies revealed that both the nature and site of glycosylation in the context of the V3 domain were critical for high-affinity binding. It was found that antibody PGT128 exhibited specificity for high-mannose N-glycan with glycosylation site promiscuity, PGT121 showed binding specificity for glycopeptide carrying a sialylated N-glycan at N301 site, and 10–1074 was specific for glycopeptide carrying a high-mannose N-glycan at N332 site. The synthesis and binding studies permit a detailed assessment of the glycan specificity and the requirement of peptide in the context of antibody–antigen recognition. The identified glycopeptides can be used as potential templates for HIV vaccine design

Multivalent Antigen Presentation Enhances the Immunogenicity of a Synthetic Three-Component HIV‑1 V3 Glycopeptide Vaccine

HIV-1 envelope glycoproteins gp120 and gp41 are presented on the virus surface as a trimer of heterodimer and are the targets of broadly neutralizing antibodies (bNAbs). We describe here the synthesis and preliminary immunological evaluation of a three-component trivalent HIV-1 V3 glycopeptide immunogen aiming to raise glycopeptide epitope-specific antibodies. Click chemistry confers efficient synthesis of the lipopeptide–glycopeptide conjugate that carries three copies of HIV-1 JR-FL gp120 V3 glycopeptide with a high-mannose glycan at the N332 glycosylation site. We found that the multivalent presentation substantially enhanced the immunogenicity of the V3 glycopeptide. The antisera induced by the three-component multivalent glycopeptide immunogen exhibited stronger binding to heterologous HIV-1 gp120s and the trimeric gp140s than that from the monovalent glycopeptide immunogen. The antisera generated from this preliminary rabbit immunization did not show virus neutralization activity, probably due to the lack of somatic maturation. The ability to elicit substantial glycopeptide epitope-specific antibodies by the three-component trivalent glycopeptide immunogen suggests that it could serve as a valuable vaccine component in combination with other vaccine candidates for further immunization studies