We report here the development of chemoenzymatic methods for the large-scale synthesis of cancer-associated antigens globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5 (SSEA4) by using overexpressed glycosyltransferases coupled with effective regeneration of sugar nucleotides, including UDP-Gal, UDP-GalNAc, GDP-Fuc, and CMP-Neu5Ac. The enzymes used in the synthesis were first identified from different species through comparative studies and then overexpressed in E. coli and isolated for synthesis. These methods provide multigram quantities of products in high yield with only two or three purification steps and are suitable for the evaluation and development of cancer vaccines and therapeutics.
A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120, thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.
The structural diversity of glycoproteins often comes from post-translational glycosylation with heterogeneous N-glycans. Understanding the complexity of glycans related to various biochemical processes demands a well-defined synthetic sugar library. We report herein a unified convergent strategy for the rapid production of bi-, tri-, and tetra-antennary complex type N-glycans with and without terminal N-acetylneuraminic acid residues connected via the α-2,6 or α-2,3 linkages. Moreover, using sialyltransferases to install sialic acid can minimize synthetic steps through the use of shared intermediates to simplify the complicated procedures associated with conventional sialic acid chemistry. Furthermore, these synthetic complex oligosaccharides were compiled to create a glycan array for the profiling of HIV-1 broadly neutralizing antibodies PG9 and PG16 that were isolated from HIV infected donors. From the study of antibody PG16, we identified potential natural and unnatural glycan ligands, which may facilitate the design of carbohydrate-based immunogens and hasten the HIV vaccine development.
In
this study, we report the fabrication of aluminum oxide-coated
glass (ACG) slides for the preparation of glycan microarrays. Pure
aluminum (Al, 300 nm) was coated on glass slides via electron-beam
vapor deposition polymerization (VDP),
followed by anodization to form a thin layer (50–65 nm) of
aluminum oxide (Al-oxide) on the surface. The ACG slides prepared
this way provide a smooth surface for arraying sugars covalently via
phosphonate formation with controlled density and spatial distance.
To evaluate this array system, a mannose derivative of α-5-pentylphosphonic
acid was used as a model for the optimization of covalent arraying
based on the fluorescence response of the surface mannose interacting
with concanavalin A (ConA) tagged with the fluorescence probe A488.
The ACG slide was characterized using scanning electron microscopy,
atomic force microscopy (AFM), and ellipsometry, and the sugar loading
capacity, uniformity, and structural conformation were also characterized
using AFM, a GenePix scanner, and a confocal microscope. This study
has demonstrated that the glycan array prepared from the ACG slide
is more homogeneous with better spatial control compared with the
commonly used glycan array prepared from the N-hydroxysuccinimide-activated
glass slide.
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