Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

Summary

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

Abstract

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise α-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial β-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man₅GlcNAc₂ (Man₅) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Introduction

N-glycans on glycoproteins are covalently linked to the asparagine (Asn) residue of the consensus Asn-Xxx-Ser/Thr sequon, which affect several biological processes such as protein conformation, antigenicity, solubility, and lectin recognition^1,2. The chemical synthesis of N-linked oligosaccharides represents a significant synthetic challenge because of their huge structural micro heterogeneity and highly branched architecture. Careful selection of protecting groups to tune reactivity of building blocks, achieving selectivity at anomeric centers, and proper use of promoter/activator(s) are key....

Protocol

1. Preparation of D1/D2 Arm Modules²²

1. Preparation of intermediate 2
   1. Weigh starting material 1 (shown in Figure 2, p-methoxyphenyl-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranoside (100 mg, 0.204 mmol)) into a 15 mL tube and dissolve in Tris buffer (25 mM, pH 7.5) containing manganese dichloride (MnCl₂, 10 mM) to achieve a final glycan concentration of 5 mM.
   2. Add 2 equivalents of uridine diphosphate galactose (UDP-Gal).
   3. Add 150 units of enzyme β-1, 4 galactosyl tr....

Results

A modular chemo-enzymatic strategy for the synthesis of a wide array of N-glycans is presented in Figure 1. The strategy is based on the fact that diversity can be created at beginning by chemo-enzymatic synthesis of the three important modules, followed by the α-specific mannosylation at the 3-O and/or 6-O position of the mannose residue of the common core trisaccharide of N-glycans. Considering the structural diversity of.......

Discussion

A class of HIV-1 bNAbs including PG9, PG16, and PGTs 128, 141 - 145 were reported to be highly potent in neutralizing 70 - 80% of circulating HIV-1 isolates. The epitopes of these bNAbs are highly conserved among the variants of the entire HIV-1 group M, therefore they may guide the effective immunogen design for an HIV vaccine to elicit neutralizing antibodies^23,24,25. As a part of our ongoing efforts to identify the glycan epi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic acid	Sigma Aldrich	64197
Acetonitrile	Sigma Aldrich	75058
Acetic anhydride	Sigma Aldrich	108247
Anhydrous magnesium sulfate	Sigma Aldrich	7487889
Boron trifluoride ethyl etherate	Sigma Aldrich	109637
Bovine serum albumin	Sigma Aldrich	9048468
Bio-Gel P2 polyacrylamide	Bio-Rad	1504118
Bis(cyclopentadienyl)hafnium(IV) dichloride	Sigma Aldrich	12116664
β-1, 4 Galactosyl transferases from bovine milk	Sigma Aldrich	48279
BioDot Cartesion technology with robotic pin SMP3 (Stealth Micro Spotting Pins)	Arrayit
Cerium ammonium molybdate	TCI	C1794
Cerium ammonium nitrate	Sigma Aldrich	16774213
Clean glass slide	Schott
Cytidine-5′-monophospho-N-acetylneuraminic acid	Sigma Aldrich	3063716
Deuterated chloroform	Sigma Aldrich	865496
Donkey Anti-Human IgG (Alexa Fluor647 conjugated	Jackson Immuno Research, USA	709605098
Dichloromethane	Sigma Aldrich	75092
Diethylaminosulfur trifluoride	Sigma Aldrich	38078090
Dimethylformamide	Sigma Aldrich	68122
Ethyl acetate	Sigma Aldrich	141786
Ethylene glycol	Acros Organic	107211
FAST frame slide incubation chambers	Sigma Aldrich
Guanosine 5'-diphospho-b-L-fucose disodium salt	Sigma Aldrich	15839700
Lab tracer 2.0 software			Section 4 of the Protocol
GenePix Pro 4300A reader (microarray image analysis)	moleculardevices		www.moleculardevices.com
GraphPad Prism Software (Image processing )	GraphPad Software, Inc		http://www.graphpad.com/guides/prism/6/user-guide/
Lithium hydroxide	Sigma Aldrich	1310652
Manganese chloride	Sigma Aldrich	7773015
Methanol	Sigma Aldrich	67561
N-butanol	Sigma Aldrich	71363
Oxalic acid	Acros Organic	144627
Palladium hydroxide	Sigma Aldrich	12135227
Phosphate Buffered Saline	Thermo Fisher Scientific	10010023
Pyridine	Sigma Aldrich	110861
P-Toluene sulfonic acid monohydrate	Sigma Aldrich	773476
Silver triflate	Sigma Aldrich	2923286
Sodium bicarbonate	Sigma Aldrich	144558
Sodium chloride	Sigma Aldrich	7647145
Sodium hydrogen carbonate	Sigma Aldrich	144558
Sodium methoxide	Sigma Aldrich	124414
Sodium sulfate	Sigma Aldrich	7757826
Toluene	Sigma Aldrich	108883
Tris buffer	Amresco	N/A	Ultra-pure grade
Tween-20	Amresco	9005645
Uridine diphosphate galactose (UDP-galactose)	Sigma Aldrich	137868521