Name: metabolic glycoengineering of sialic acid using n-acyl-modified mannosamines
Uploaded: 2017-11-25T14:00:00
Description: Watch this Scientific Journal Video about Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines at JoVE.com

We thank L. D. Nguyen for proofreading the manuscript and for fruitful discussions. Furthermore, we thank J. Dernedde and H. G. Nguyen for helping us preparing the video shoot. Most scenes of the video were shot in the laboratories of R. Tauber. We also thank the Max Planck Institute for the colloids and interfaces, and for giving us free access to their mass spectrometry facility. RH was supported by the DFG (ProMoAge).

1. Preparation of Buffers and Reagents
<ol>
	<li>Preparation of 3 mM sodium methoxide solution
	<ol>
		<li>Dissolve 8.1 mg sodium methoxide in 50 mL methanol (3 mM) in a 100 mL glass bottle with a stir bar. Store at room temperature (RT) for several weeks.</li>
	</ol>
	</li>
	<li>Preparation of Tris-HCl buffer
	<ol>
		<li>Combine 8.766 g NaCl, 157 mg Tris-HCl, and 146 mg EDTA in a 100 mL glass bottle with a stir bar and dissolve in 80 mL water.</li>
		<li>Add sodium hydroxide (1 M, in ...

If the chemically synthesized ManNAc derivatives, ManNProp and ManNBut are analyzed via mass spectrometry, only the correct mass peak for both specimens should be identified. Therefore, the products can be assumed to have a purity of over 99%. Small amounts of Neu5Gc, which is normally not found in human cells29, are detected in the membrane fractions of the lysed cells. This most likely occurs through a salvage pathway that recruits Neu5Gc from fetal bovine serum sialoglycoconjugates in the media...

Sialic acid is a monosaccharide that can typically be found at the non-reducing termini of glycoconjugates, such as N- and O-glycans or glycolipids. Among all monosaccharides, sialic acid has some unique chemical characteristics. It has a 9 C-atom backbone, a carboxylic group in the C-1 position, that is deprotonated and thereby negatively charged under physiological conditions, and an amino function in the C-5 position. Although over 50 naturally occurring variants of sialic acid have been characterized to date1, the predominant form of sialic acid found in humans is N-acetylneuraminic acid (Neu5Ac). Other mammals also express higher amounts of N-glycolylneuraminic acid (Neu5Gc)2,3.
Due to its exposed position in glycoconjugates, sialic acid is involved in a plethora of receptor-ligand interactions, e.g., the hemagglutinin dependent binding of the influenza virus to host cells4. A sialic acid epitope with important biological functions, especially during embryogenesis and in the nervous system, is polysialic acid. Polysialic acid is a polymer of up to 200 alpha 2,8-linked sialic acids. The major protein carrier of polysialic acid is the neural cell adhesion molecule (NCAM). Polysialic acid expression modulates the adhesive property of NCAM in that polysialic acid expression decreases the adhesion and increases plasticity with the nervous system5.
Changes in the expression of (poly)sialic acid will ultimately affect a multitude of different biological interactions. This can be used to study known sialic acid dependent processes on a molecular level, to uncover novel glycoconjugate interactions, or explore possible therapeutic approaches. There are different methods available by which the expression of sialic acid on the cell surface can be modulated, for example treatment with sialic acid specific glycosidases (sialidases), inhibition of enzymes involved in the sialic acid biosynthesis6,7,8, or knocking down or changing the expression of the key enzyme of sialic acid biosynthesis9.
Another versatile method to modulate sialic acid expression is MGE (also known as metabolic oligosaccharide engineering, MOE). Herein, cells, tissues, or even animals are treated with non-natural derivatives of ManNAc that bear C2-amino modifications. Being precursor molecules for sialic acid, after cellular uptake, these ManNAc analogs are unidirectional metabolized to non-natural sialic acids and can be expressed on sialylated glycoconjugates. Cells treated with ManNAc derivatives carrying aliphatic C2-modifications, such as ManNProp or ManNBut, do incorporate N-propionylneuraminic acid (Neu5Prop) or N-butanoylneuraminic acid (Neu5But) in their glycoconjugates10,11. By using functional groups introduced to the C2-position of ManNAc, the occurring non-natural sialic acids can be coupled, e.g., via the Staudinger ligation or the azide alkine cycloaddition, with fluorescent dyes and therefore visualized on the cell surface12.
The expression of these non-natural sialic acids has intriguing effects on many biological processes, including pathogen infections, the adhesion and migration of tumor cells, general cell adhesion, as well as vascularization and differentiation (for review see: Wratil et al.13). Interestingly, MGE with N-acyl modified mannosamines can also be used to interfere with the expression of polysialic acid. Polysialic acid is generated by two different polysialyltransferases (ST8SiaII and ST8SiaIV). It has been demonstrated, that polysialyltransferase ST8SiaII is inhibited by unnatural sialic acid precursors, such as ManNProp or ManNBut14,15. In addition, it has been demonstrated in human neuroblastoma cells that ManNProp or ManNBut application also reduces sialylation in total15.
MGE with N-acyl modified mannosamines is an easy to apply method that has been successfully used, not only in mammalian and bacteria cell culture but also in entire animals of different species, such as Caenorhabditis elegans16, zebrafish17, or mice18,19,20,21. Especially ManNAc derivatives bearing aliphatic modifications, including ManNProp and ManNBut, are negligibly cytotoxic, even at millimolar concentrations in cell culture medium or blood plasma. Furthermore, they are relatively easy to synthesize.
Here, we provide details on how to use MGE with N-acetyl modified mannosamines. First, the chemical synthesis of two of the most widely used ManNAc derivatives in this field, ManNProp and ManNBut, is explained. Next, we show how MGE can be applied in an in vivo experiment. As an example, the neuroblastoma cell line Kelly was chosen to demonstrate decreased expression of the polysialic epitope by Western blot after treatment with the ManNAc derivatives. The non-natural sialic acids on the cell surface were quantified by HPLC and further analyzed via mass spectrometry.

<table>
	<tbody>
		<tr>
			<td>Cells</td>
			<td>Sigma-Aldrich</td>
			<td>92110411</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>75 ml tissue culture flasks</td>
			<td>Greiner</td>
			<td>690175</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>PAA</td>
			<td>A15-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsine</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Roth</td>
			<td>X986.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Serva</td>
			<td>37190.03</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Roth</td>
			<td>2326.2</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE equipment (tanks, glassware etc., machine</td>
			<td>VWR</td>
			<td>SDS Gel/Blot</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Roth</td>
			<td>3019.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein ladder</td>
			<td>Fisher Scientific</td>
			<td>267620</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>GE Healthcare</td>
			<td>10600002</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau red</td>
			<td>Roth</td>
			<td>5938.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Milk powder</td>
			<td>Roth</td>
			<td>T145.3</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL</td>
			<td>Millipore</td>
			<td>WBLUF 0500</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 ml Centrifugal Filter Unit with 3 kDa membrane</td>
			<td>Merck-Millipore</td>
			<td>UFC500324</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Sigma-Aldrich (Corning)</td>
			<td>CLS430791-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Sigma-Aldrich</td>
			<td>34965-1L</td>
			<td>HPLC gradient grade</td>
		</tr>
		<tr>
			<td>4,5-Methylenedioxy-1,2-phenylenediamine dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>D4784-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td>48 well, flat bottom tissue culture plate</td>
			<td>Sigma-Aldrich (Corning)</td>
			<td>CLS3548-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Sigma-Aldrich (Corning)</td>
			<td>CLS430829-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34967-1L</td>
			<td>HPLC gradient grade</td>
		</tr>
		<tr>
			<td>Aprotinin from bovine lung</td>
			<td>Sigma-Aldrich</td>
			<td>A1153-10MG</td>
			<td>lyophilized powder, 3-8 TIU/mg solid</td>
		</tr>
		<tr>
			<td>Butyryl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>109614-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 RP column</td>
			<td>Phenomenex</td>
			<td>00F-4435-E0</td>
			<td>110 &#197;, 3 &#181;m particle size, 4.6 x 150 mm</td>
		</tr>
		<tr>
			<td>D-Mannosamine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>M4670-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco`s Phosphate Buffered Salt Solution</td>
			<td>PAN Biotech</td>
			<td>P04-36500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>E9884-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma-Aldrich</td>
			<td>56302-50ML-GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>H1758-100ML</td>
			<td>36.5-38.0%, in water</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma-Aldrich</td>
			<td>L2884-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl-Roth</td>
			<td>T169.2</td>
			<td>HPLC gradient grade</td>
		</tr>
		<tr>
			<td>N-Acetyl-D-mannosamine</td>
			<td>Sigma-Aldrich</td>
			<td>A8176-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylneuraminic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A0812-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Glycolylneuraminic acid</td>
			<td>Sigma-Aldrich</td>
			<td>G9793-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>P7626-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionyl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P51559-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes, 1.5 mL, amber (light protection)</td>
			<td>Eppendorf</td>
			<td>30120191</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes, 1.5 mL, colorless</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bisulfite solution</td>
			<td>Sigma-Aldrich</td>
			<td>13438-1L-R-D</td>
			<td>40%, in water</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>746398-500G-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>795429-500G-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>319511-500ML</td>
			<td>1.0 M, in water</td>
		</tr>
		<tr>
			<td>Sodium methoxide</td>
			<td>Sigma-Aldrich</td>
			<td>164992-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508-100ML-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>T5941-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25 %/EDTA 0.02 % in PBS</td>
			<td>PAN Biotech</td>
			<td>P10-019100</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Carl-Roth</td>
			<td>T905.1</td>
			<td>HPLC gradient grade</td>
		</tr>
		<tr>
			<td>Silica Gel 60</td>
			<td>Carl-Roth</td>
			<td>9779.1</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC</td>
			<td>Shimadzu</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

metabolic glycoengineering of sialic acid using n-acyl-modified mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

HPLC chromatograms of the fluorescent labeled Neu5Ac and the Neu5Gc standards are depicted in Figure 2. Using the herein described method, DMB-labeled Neu5Gc typically elutes between 7 - 9 min elution time, and DMB-Neu5Ac between 10 - 12 min. Several smaller peaks in the chromatogram usually appear between 2 - 6 min. These peaks represent unreacted DMB and reaction intermediates25.
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Watch this Scientific Journal Video about Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines at JoVE.com

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

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