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>
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			<td style="width:164px;">http://www.graphpad.com/guides/prism/6/user-guide/</td>
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			<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>
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		<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>
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		<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>
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		<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 ...

The overall goal of this methodology, is to develop a modular chemo-enzymatic approach to the synthesis of N-glycans by constructing a new glycan microarray on a aluminum oxide coated glass slide for detecting heteroglycan binding of HIV antibodies. This methods can help answer key questions in glycobiology for identification of cell virus specific glycans for much development. The major advantage of this method is to detect weak binding and identify a mixture of sugars that bind to a protein such as, antibody from HIV patient.The implications of this technique extend to a diagnosis of influenza infections because sialic catabolist express on human airways epithelial cells and issues are common while the symptoms. Therefore, glycoarray will be an ideal tool to determine specificity of wild subtypes. Though this method can provide inside into the glycan specificities of HIV antibodies it can also be applied to other systems such as, diagnosis of various cancers based on the binding pattern of human serum antibodies toward human specific serum antigens printed on the surface.To begin this procedure, add 2-5 micromoles of previously prepared glycan and a five milliliter round bottom flask. Add a magnetic stir bar to the flask and cap it with a rubber septum. Add 400 microliters of freshly dried DMF and 10-25 micromoles of the phosphonic acid linker to the flask.Then, stir the reaction mixture at 800 rpm for five hours. Once the reaction is complete, evaporate the solvent using a rotary evaporator at five to 10 millibar vacuum pressure and 40 degrees Celsius. Following this, dissolve the dried reaction mixture and 0.5 milliliter of water and load on top of polyacrylamide gel bed.Elute the products using water and collect one to two milliliter fractions. To check the collected fractions by TLC, spot each fraction on a TLC plate and dip the plate in 0.25 molars Cerium Ammonium Molybdate. Heat the stained plate with a hot plate to visualize the product.After combining the product containing fractions and freezing the solution biofolize to obtain the desired product as a white powder. Next, dissolve the dried product in two milliliters of water and add Palladium Hydroxide. Attach a hydrogen containing balloon to the flask and stir the reaction mixture at 800 rpm for 15 hours under hydrogen atmosphere.Once the reaction is complete, filter the mixture through a Celite Bed and wash with two milliliters of methanol and two milliliters of distilled deionized water. Then, evaporate the solvent using a rotary evaporator at 300 millibar vacuum pressure and 40 degree Celsius. Following this, dissolve the crud mixture in approximately one milliliter of water and load it on top of a polyacrylamide gel bed.Elute the product with water and collect one to two milliliter fractions. After combining the product containing fractions and freezing the solution biofolize to obtain the desired product as a white powder. Once the product is dry, characterize it by enimar and mass spectroscopy using Deuterium Oxide as the solvent.For surface anodization, set a temperature controlled incubator to four degree Celsius. Then transfer a previously prepared 0.3 molar aqueous oxalic acid solution to a 500 milliliter beaker containing a magnetic stir bar. Place a 10 centimeter long platinum rod as a cathode into the solution.Keep stirring the oxalic acid at 300 rpm throughout the anodization process. In the lab tracer software, click on the setup button and then click on function choice Sweep Voltage. Set the start and stop voltage to 25.8 volts.The number of points to 100, the compliance to one, and the sweep delay to 1200 milliseconds. Then, click okay. Clamp an aluminum coated glass slide facing toward the cathode.Click on the run test button and observe the current measurement. After surface anodization, wash the glass slide thoroughly with double distilled water and purge dry with nitrogen gas. Store the glass slide in a 30%relative humidity chamber until further use.For fabrication, prepare 100 microliters solutions of all monovalent glycans in ethanol glycol at 10 millimolar concentration. Dilute the glycan solutions with printing buffer to make 100 micromolar solutions. For the Heteroligand Study, add five microliters of MAN-5 Glycan to five microliters of each glycan solution.Transfer the prepared glycans into the 384 well microplate for microarray printing step. After inserting a microplate on the printer array tech, print microarrays by robotic pin by depositing 0.6 nanoliters of the glycan solutions onto each aluminum coated glass slide. Next prepare 70 microliters of primary antibody PG-9 and PTSB buffer containing 3%BSA.Then prepare 120 microliters of secondary fluorescent tag antibody Donkey and Anti-human Immunoglobulin G and PBST buffer containing 3%BSA in the dark. Mix PG9 and the secondary fluorescent tag antibody in a 1:1 ratio. Then incubate the premixed antibodies for 30 minutes at four degrees Celsius.Following incubation, load an aluminum coated glass slide into the slide incubation chamber, which is divided into 16 wells. Transfer 100 microliters of the premixed antibodies to the glycan array. After incubating at four degrees Celsius for 16 hours remove the premixed antibodies from the glycan array with a pipette and then remove the slide incubation chamber.Wash the glass slide in PBST buffer and deionized water. Then spin dry the glass slide at 2000 times gravity. Next, open the microarray image analysis software.Insert the glass dried slide into the array scanner with the arrayed features facing down. Click on the settings menu. Set the image resolution to five micrometers per pixel.And set the wavelength at 635 nanometers with PMT 450 and power to 100%Click on the scan button to start imaging the slide. Finally click the file icon to save the scan image in TIF format for image analysis. In this study, a wide array of N-Glycans were synthesized using a modular chemo enzymatic strategy.To demonstrate the effectiveness of the strategy a biantennary isometric structure was synthesized by chemoenzymatic methods. Enzymatic galactosylation for disaccharide 1 followed by fucosylation of trisaccharide 2 afforded the desired tetrasaccharide 3. Per satylation and reducing end modifications of building block two and three, afforded to desire molecule four and five.Stereoselective three O-glycosalyation of module four of trisaccharide six provided hexasaccharide seven. De-protection and reaction before module five yielded dedecasaccharide nine. Global detection afforded glycan 10 which was characterized by enimar and mass spectroscopy.Glycans containing a Pentylamine tail at the reducing end were modified with phosphonic acid linkers and attached to the aluminum glass coated surface through phospinide chemistry. HIV-1 broadly neutralizing antibody PG9 was screened for it's glycan specificity using homo and heteroglycan arrays to demonstarte for the first time it's ineteraction with adjacent heteroglycans in the V1/V2 loop of HIV1 PG120 surface. Once mastered, this technique can be done in 24 hours from glycan immobilization to beta analysis if it was performed properly.While attempting this procedure, it is important to remember to use freshly anodized aluminum coated glass slide for Glycan 10. For laying this procedure are the med techs slides. Determination of the body constant can be performed in order to quantify the binding interactions between carbohydrate and protein of interest.After the development of this technique, it paved the way for researchers to better understand the role of carbohydrate in infectious disease in Kenya and help develop new therapies against diseases. Don't forget that working with toxic agents can be extremely hazardess and precautions such as wearing of the clothes, glasses and masks should always be taken when preforming experiments.

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