Name: chemo-enzymatic synthesis of n-glycans for array development and hiv antibody profiling
Uploaded: 2018-02-05T13:00:00
Description: Watch this Scientific Journal Video about Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling at JoVE.com

The authors thank the Thin Film Technology Division, Instrument Technology Research Center (ITRC) and National Applied Research Laboratories, Hsinchu Science Park, Taiwan. This work was supported by the National Science Council (grant no. MOST 105-0210-01-13-01) and Academia Sinica.

1. Preparation of D1/D2 Arm Modules22
<ol>
	<li>Preparation of intermediate 2
	<ol>
		<li>Weigh starting material 1 (shown in Figure 2, p-methoxyphenyl-O-2-acetamido-2-deoxy-&#946;-D-glucopyranosyl-(1&#8594;2)-&#945;-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.</...

<ol>
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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 antibodies23,24,25. As a part of our ongoing efforts to identify the glycan epi...

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 recognition1,2. 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 elements in synthesis of complex oligosaccharides. To solve this problem of complexity, a great amount of work to advance N-glycan synthesis was reported recently3,4. In spite of these robust approaches, finding an effective method for the preparation of a wide range of N-glycans (~20,000) remains a major challenge.
The rapid mutation rate of HIV-1 to achieve the extensive genetic diversity and its ability to escape from neutralizing antibody response, is among the greatest challenges to develop a safe and prophylactic vaccine against HIV-15,6,7. One effective tactic that HIV uses to avoid the host immune response is the post-translational glycosylation of envelope glycoprotein gp120 with a diverse N-linked glycans derived from the host glycosylation machinery8,9. A recent report regarding the precise analysis of recombinant monomeric HIV-1 gp120 glycosylation from human embryonic kidney (HEK) 293T cells suggests the occurrence of structural microheterogeneity with a characteristic cell-specific pattern10,11,12. Therefore, understanding the glycan specificities of HIV-1 bNAbs requires well characterized gp120 related N-glycan structures in a quantity sufficient for analysis.
The discovery of glycan microarray technology provided high throughput-based exploration of specificities of a diverse range of carbohydrate-binding proteins, viruses/bacterial adhesins, toxins, antibodies, and lectines13,14. The systematic glycans arrangement in an arrayed chip-based format could determine problematic low affinity protein-glycan interactions through multivalent presentation15,16,17,18. This chip-based glycan arrangement conveniently appears to effectively mimic cell-cell interfaces. To enrich the technology and overcome the uneven issue associated with conventional array formats, our group recently developed a glycan array on an aluminum oxide-coated glass (ACG) slide using phosphonic acid-ended glycans to enhance the signal intensity, homogeneity, and sensitivity19,20.
To improve the current understanding about glycan epitopes of newly isolated HIV-1 broadly neutralizing antibodies (bNAbs), we have developed a highly efficient modular strategy for the preparation of a broad array of N-linked glycans21,22 to be printed on an ACG slide (see Figure 1). Specificity profiling studies of HIV-1 bNAbs on an ACG array offered the unusual detection of hetero-glycan binding behavior of highly potent bNAb PG9 that was isolated from HIV infected individuals23,24,25.

<table width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetic acid</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">64197</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetonitrile</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">75058</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetic anhydride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">108247</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Anhydrous magnesium sulfate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7487889</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Boron trifluoride ethyl etherate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">109637</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Bovine serum albumin</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">9048468</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Bio-Gel P2 polyacrylamide</td>
			<td style="width:123px;">Bio-Rad</td>
			<td style="width:119px;">1504118</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Bis(cyclopentadienyl)hafnium(IV) dichloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">12116664</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">&#946;-1, 4 Galactosyl transferases from bovine milk</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">48279</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="width:168px;height:84px;">BioDot Cartesion technology with robotic pin SMP3 (Stealth Micro Spotting Pins)</td>
			<td style="width:123px;">Arrayit</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Cerium ammonium molybdate</td>
			<td style="width:123px;">TCI</td>
			<td style="width:119px;">C1794</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Cerium ammonium nitrate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">16774213</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Clean glass slide&#160;</td>
			<td style="width:123px;">Schott&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Cytidine-5&#8242;-monophospho-N-acetylneuraminic acid</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">3063716</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Deuterated chloroform</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">865496</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Donkey Anti-Human IgG (Alexa Fluor647 conjugated</td>
			<td style="width:123px;">Jackson Immuno Research, USA</td>
			<td style="width:119px;">709605098</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Dichloromethane</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">75092</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Diethylaminosulfur trifluoride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">38078090</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Dimethylformamide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">68122</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Ethyl acetate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">141786</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Ethylene glycol</td>
			<td style="width:123px;">Acros Organic</td>
			<td style="width:119px;">107211</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">FAST frame slide incubation chambers</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Guanosine 5&#39;-diphospho-b-L-fucose disodium salt&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">15839700</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lab tracer 2.0 software&#160;</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:164px;">Section 4 of the Protocol</td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">GenePix Pro 4300A reader (microarray image analysis)</td>
			<td style="width:123px;">moleculardevices</td>
			<td style="width:119px;"></td>
			<td style="width:164px;">www.moleculardevices.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">GraphPad Prism Software (Image processing )</td>
			<td style="width:123px;">GraphPad Software, Inc</td>
			<td style="width:119px;"></td>
			<td style="width:164px;">http://www.graphpad.com/guides/prism/6/user-guide/</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Lithium hydroxide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">1310652</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Manganese chloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7773015</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Methanol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">67561</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">N-butanol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">71363</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Oxalic acid</td>
			<td style="width:123px;">Acros Organic</td>
			<td style="width:119px;">144627</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Palladium hydroxide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">12135227</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Phosphate Buffered Saline</td>
			<td style="width:123px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">10010023</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Pyridine</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">110861</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">P-Toluene sulfonic acid monohydrate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">773476</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Silver triflate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">2923286</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium bicarbonate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">144558</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium chloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7647145</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Sodium hydrogen carbonate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">144558</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium methoxide&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">124414</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium sulfate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7757826</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Toluene&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">108883</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Tris buffer&#160;</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:119px;">N/A</td>
			<td style="width:164px;">Ultra-pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Tween-20</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:119px;">9005645</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Uridine diphosphate galactose (UDP-galactose)</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">137868521</td>
			<td style="width:164px;"></td>
		</tr>
	</tbody>
</table>

chemo-enzymatic synthesis of n-glycans for array development and hiv antibody profiling

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 &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-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.

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 &#945;-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 diversi...

Watch this Scientific Journal Video about Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling at JoVE.com

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

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.

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

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 ...

Research

JoVE Journal

Chemistry

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