Top-Down Chemoenzymatic Approach to Synthesizing Diverse High-Mannose N-Glycans and Related Neoglycoproteins for Carbohydrate Microarray Analysis

Abstract

High-mannose-type N-glycans are an important component of neutralizing epitopes on HIV-1 envelope glycoprotein gp120. They also serve as signals for protein folding, trafficking, and degradation in protein quality control. A number of lectins and antibodies recognize high-mannose-type N-glycans, and glycan array technology has provided an avenue to probe these oligomannose-specific proteins. We describe in this paper a top-down chemoenzymatic approach to synthesize a library of high-mannose N-glycans and related neoglycoproteins for glycan microarray analysis. The method involves the sequential enzymatic trimming of two readily available natural N-glycans, the ManGlcNAcAsn prepared from soybean flour and the sialoglycopeptide (SGP) isolated from chicken egg yolks, coupled with chromatographic separation to obtain a collection of a full range of natural high-mannose N-glycans. The Asn-linked N-glycans were conjugated to bovine serum albumin (BSA) to provide neoglycoproteins containing the oligomannose moieties. The glycoepitopes displayed were characterized using an array of glycan-binding proteins, including the broadly virus-neutralizing agents, glycan-specific antibody 2G12, Galanthus nivalis lectin (GNA), and Narcissus pseudonarcissus lectin (NPA).

Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides

“…The material was finally purified by reverse-phase HPLC to afford homogeneous Man 9 GlcNAc 2 Asn (20 mg) as a white powder after lyophilization, which was characterized by compositional analysis, HPLC, and electron spray ionization mass spectrometry (ESI-MS) 71 Supplementary Fig. 7) 72 . Specially, the mannosidase (10 μg) was added to a solution of Man 9 GlcNAc 2 Asn (10 mg, 0.005 mmol) in a buffer (PBS, 100 mM, pH 7.4, 1 mL).…”

Recent Progress in Chemo-Enzymatic Methods for the Synthesis of N-Glycans

“…This structure is considered the key intermediate of N-glycans in both the in vivo biosynthesis pathway and in vitro synthesis strategies, which can be elongated and elaborated by various GTs, and is thus called a core pentasaccharide (boxed structure in Figure 3A). Moreover, Man 3 GlcNAc 2 can be prepared from chemical synthesis or obtained from natural source digestion (Seeberger et al, 1996;Li et al, 2018b;Toonstra et al, 2018;Pistorio et al, 2019). Recently, the successful expression and purification of the ER ManTs Alg1 and Alg2 allow the reconstitution of the lipid-linked oligosaccharide (LLO) biosynthesis pathway up to the core pentasaccharide in vitro (Li et al, 2017b).…”

“…Two natural sources of N-glycans, i.e., Asn-linked Man 9 GlcNAc 2 from soybean flour and SGP from chicken egg yolks, were also successfully treated by sequential enzymatic digestion to produce a library of N-glycans. These N-glycans could be conjugated to bovine serum albumin (BSA) to give neoglycoprotein microarrays for the comprehensive analysis of critical virus-neutralizing epitopes (Toonstra et al, 2018).…”

“…For instance, Fmoc labeled high-mannose type N-glycan Man 9 GlcNAc 2 Asn-Fmoc, which can be obtained from the Fmoc modified fractional precipitate of soybean flour, could be digested by the α-1,2-mannosidase to produce several high-mannose type N-glycan intermediates. Since Man 9 GlcNAc 2 -Asn-Fmoc contains four terminal Man-α-1,2-Man linkages, it can generate Man 5−8 GlcNAc 2 -Asn-Fmoc by the treatment with an α-1,2-mannosidase from the human gut bacterial symbiont Bacteroides thetaiotaomicron under controlled conditions (Toonstra et al, 2018). On the other hand, sialylated bi-antennary complex type N-glycan (SCT), which is available at large scale from sialoglycopeptide (SGP) isolated from the chicken egg yolk, can be further trimmed by sequentially adding sialidase, galactosidase, and N-acetylglucosaminidase to give various N-glycan structures.…”

High-Throughput Methods in the Discovery and Study of Biomaterials and Materiobiology

“… 515 Typically, these microarrays display a variety of saccharide chains immobilized on epoxy-activated glass slides using contact printing at high humidity, and require a postprinting blocking step to remove any exposed hydroxyl groups that could promote protein deposition and cell adhesion during the out-put assays. 505 Covalently bound glycan microarrays have also been described, where maleimide-activated surfaces are used to bind a thiol-terminated linker on the carbohydrate chain. 516 The most challenging feature of these arrays has been the source glycans, with large libraries being assembled on the basis of their biological source and potential binding-specificity.…”