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 water) or HCl (20%, in water) to the stirring solution, while observing the pH with a pH-meter, and adjust the pH to 8.0.</li>
		<li>Add the volume of water needed to achieve a final volume of 100 mL. Filter the solution using a 0.22 &#181;m sterile filter system. 
		NOTE: 100 mL Tris-HCl buffer will contain 150 mM NaCl, 10 mM Tris-HCl, and 5 mM EDTA (pH 8.0). The solution can be stored at 4 &#176;C for several weeks.</li>
	</ol>
	</li>
	<li>Preparation of aprotinin solution
	<ol>
		<li>Dissolve 10 mg aprotinin in 1 mL water (1.54 mM). Aliquot the aprotinin solution into 10 x 1.5 mL-plastic tubes (100 &#181;L each). Store at -20 &#176;C for several weeks.</li>
	</ol>
	</li>
	<li>Preparation of leupeptin solution
	<ol>
		<li>Dissolve 10 mg leupeptin in 2 mL water (10 mM). Aliquot the leupeptin solution 10 x 1.5 mL-plastic tubes. Store at -20 &#176;C for several weeks.</li>
	</ol>
	</li>
	<li>Preparation of phenylmethylsulfonyl fluoride (PMSF) solution
	<ol>
		<li>Dissolve 34.8 mg PMSF in 2 mL ethanol (100 mM). Aliquot the PMSF solution in 10 x 1.5 mL-plastic tubes. Store at -20 &#176;C for several weeks.</li>
	</ol>
	</li>
	<li>Preparation of 1,2-diamino-4,5-methyldioxybenzol-dihydrochloride (DMB) solution
	<ol>
		<li>Combine 15.62 mg DMB, 352 &#181;L &#946;-mercaptoethanol, and 48 &#181;L sodium bisulfite-solution (39%), and add 9.6 mL water (6.9 mM DMB, 500 mM &#946;-mercaptoethanol, and 0.19% sodium bisulfite). Aliquot the DMB solution in 40 x 1.5 mL-plastic tubes (250 &#181;L each). Store protected from light at -20 &#176;C for several weeks.</li>
	</ol>
	</li>
	<li>Preparation of 1 M trifluoroacetic acid (TFA) solution
	<ol>
		<li>Add 770.3 &#181;L of 100% TFA to 9.23 mL water in a 15 mL plastic tube (1 M). Store at 4 &#176;C for several weeks.</li>
	</ol>
	</li>
	<li>Preparation of 120 mM TFA solution
	<ol>
		<li>Add 92.4 &#181;L TFA (100%) to 9.91 mL water (120 mM) in a 15 mL plastic tube. Store at 4 &#176;C for several weeks.</li>
	</ol>
	</li>
	<li>Preparation of 400 mM sodium hydroxide (NaOH) solution
	<ol>
		<li>Dissolve 160 mg NaOH in 10 mL water (400 mM) in a 15 mL plastic tube. Store at 4 &#176;C for several weeks.</li>
	</ol>
	</li>
	<li>Preparation of Western blot lysis buffer
	<ol>
		<li>Dissolve 5.84 g NaCl, 0.1 g Tris(hydroxymethyl)-aminomethan (Tris), 0.1 g CaCl2, 0.95 g MgCl2, and 1 g Triton-X100 in 100 mL water and adjust the pH to 7.8. Store at 4 &#176;C. Add 100 &#181;L of each protease inhibitor (aprotinin, leupeptin, and PMSF) before using the lysis buffer.</li>
	</ol>
	</li>
	<li>Preparation of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer
	<ol>
		<li>Dissolve 0.3 g Tris, 1.5 g glycine, and 0.1 g SDS in 100 mL water and adjust the pH to 7.3. Store at RT.</li>
	</ol>
	</li>
	<li>Preparation of Western blot buffer
	<ol>
		<li>Dissolve 0.3 g Tris, 1.1 g glycine in 90 mL water and add 10 mL ethanol. Store at RT.</li>
	</ol>
	</li>
	<li>Preparation of blocking solution
	<ol>
		<li>Dissolve 1 g of fat free milk power in 25 mL phosphate buffered saline (PBS). Always prepare fresh.</li>
	</ol>
	</li>
</ol>
2. Synthesis of ManNProp and Related N-acyl-modified Mannosamines
<ol>
	<li>Dissolve 431.2 mg mannosamine hydrochloride in 10 mL 3 mM sodium methoxide solution in a 50 mL glass bottle with a stir bar.</li>
	<li>Cool the mixture on ice to 0 &#176;C. To the stirring solution, slowly add dropwise 210 &#181;L propionyl chloride (2.4 mmol), or 248 &#181;L butanoyl chloride (2.4 mmol) to synthesize ManNProp or ManNBut, respectively. Incubate the stirring mixture at 0 &#176;C for 4 h.</li>
	<li>Transfer the solution into a 50 mL plastic tube. Poke 4 - 8 holes into the lid of the plastic tube with a thin needle. Rapidly freeze the solution using liquid nitrogen and subsequently lyophilize (freeze dry) it for 48 h or until it has completely dried.</li>
	<li>Mix 350 g silica gel 60 with 750 mL ethyl-acetate/methanol/water (15:2:1) (this is a suspension). Fill a glass column (35 mm diameter and 70 cm length) with the silica gel 60 suspension and wash the prepared column with an additional 100 mL ethyl-acetate/methanol/water (15:2:1) before use.</li>
	<li>Dissolve the dried products from step 2.3 (5 g dried product) in 10 mL ethyl-acetate/methanol/water (15:2:1) and load them using a pipette onto the silica gel column. Collect 4 mL fractions after 500 mL (for ManNProp). NOTE: ManNBut will elute from the column approximately after 700 mL.</li>
	<li>Transfer the eluted fractions into 15 mL plastic tubes. Poke 3 - 6 holes into the lid of the plastic tube with a thin needle. Rapid freeze the solution using liquid nitrogen and subsequently lyophilize it until it has completely dried. 
	NOTE: The lyophilized products can be stored for several months at RT.</li>
	<li>Verify the purity of the products by mass spectrometry (see section 9). 
	NOTE: Alternatively, purity can also be verified by 1H Nuclear Magnetic Resonance (NMR).</li>
</ol>
3. Cell Culture
<ol>
	<li>Culture Kelly neuroblastoma cells in Roswell Park Memorial Institute (RPMI) medium containing 10% fetal bovine serum, 100 U penicillin, 100 mg streptomycin, 2 mM L-glutamine containing 5 mM ManNAc, 5 mM ManNProp, or 5 mM ManNBut, at 37 &#176;C and 5% CO2. 
	NOTE: Cells cultured in medium without ManNAc or its analogues are used as a control.</li>
	<li>Prior to application of the mannosamines, detach the Kelly neuroblastoma cells by incubating them for 10 min with trypsin/EDTA (0.25%, 0.02%, in PBS) and count using a Neubauer-chamber or cell counter. Seed the cells at defined numbers based on the size of the plate/dish.
	<ol>
		<li>To measure sialic acid monosaccharides, seed 1 x 105 cells in 500 &#181;L medium into a 48-well tissue culture plate. For the analysis of polysialic acids, seed 1 x 106 cells in 3 mL medium onto a 6 cm diameter cell culture dish. Incubate at 37 &#176;C and 5% CO2. Replace the medium every 24 h. 
		NOTE: The effect of MGE increases with the total time of treatment. For high metabolic efficiency treat the cells for 5 - 7 days.</li>
	</ol>
	</li>
	<li>After treatment, decant the medium. Wash the plates/dishes with PBS. Detach the cells by incubating them for 10 min with trypsin/EDTA (0.25%, 0.02%, in PBS). Neutralize the trypsin by adding the same volume of cell culture medium to the detached cells. Transfer the detached cells into 15 mL plastic tubes. Centrifuge the samples for 3 min at 500 x g and discard the supernatant.
	<ol>
		<li>Add 5 mL PBS and centrifuge cells (see step 3.3). Repeat the washing procedure two more times. 
		NOTE: The washed cell pellet without supernatant can be stored at -20 &#176;C for several days. If the expression of polysialic acid is to be elucidated continue to section 10.</li>
	</ol>
	</li>
</ol>
4. Cell Lysis for HPLC Analysis
<ol>
	<li>Preparation of HPLC lysis buffer
	<ol>
		<li>Combine 5 mL Tris-HCl buffer, 5 &#181;L aprotinin solution, 20 &#181;L leupeptin solution, and 50 &#181;L PMSF solution. 
		NOTE: 5 mL HPLC Lysis buffer will contain 150 mM NaCl, 10 mM Tris-HCl, 5 mM EDTA, 1.54 &#181;M aprotinin, 40 &#181;M Leupeptin, and 1 mM PMSF (pH 8.0). This buffer should always be prepared fresh prior to cell lysis.</li>
	</ol>
	</li>
	<li>Re-suspend the collected cell pellets (step 3.3) each in 500 &#181;L ice-cold HPLC lysis-buffer, and ultra-sonicate the samples with an ultra-sonic processor needle three times for a period of 30 s at medium-high amplitudes. Cool the samples on ice for at least 1 min in between the cycles.</li>
</ol>
5. Separation of the Membrane Fraction
<ol>
	<li>Centrifuge the lysed cell samples for 2 h at 20,000 x g and 4 &#176;C.Separate the supernatant (approximately 480 &#181;L), representing cytosolic proteins, in 1.5 mL plastic tubes. Determine the protein concentration of these fractions using, e.g., the Bradford or the bicinchoninic acid (BCA) assay. 
	NOTE: The protein concentration (approximately 1 mg/mL) of the cytosolic fractions can later be related to the measured amounts of the respected sialic acid species. The pellet represents the membrane fraction, which is used in step 6.1.</li>
</ol>
6. Acidic Hydrolysis
NOTE: Oligo- and polysaccharides in the cell membranes are hydrolyzed to monosaccharides. Further, possible O-modifications in the monosaccharides are hydrolyzed, as well. This is necessary for quantitative HPLC analysis, because the overwhelming majority of sialic acids in the membrane fractions are naturally incorporated in glycoconjugates and might possibly bear O-acetyl or O-lactolyl modifications.
<ol>
	<li>Add 150 &#181;L 1 M TFA-solution to the separated membrane fractions (pellet from section 5). Incubate the samples for 4 h at 80 &#176;C with shaking at 200 - 600 rpm.</li>
	<li>Centrifuge the samples for approximately 30 min at 20,000 x g and 21 &#176;C using 0.5 mL filter tubes with 3 kDa exclusion membranes, until the upper phase volume is less than 20 &#181;L. 
	NOTE: This step is important to dispose higher molecular debris.</li>
	<li>Transfer the lower phase containing smaller molecules, including the sialic acid species, into 2 mL plastic tubes. Poke 2 - 4 holes into the caps of the plastic tubes with a thin needle. Rapid freeze the samples using liquid nitrogen and then lyophilize them overnight. 
	NOTE: The dried samples can be stored for several days at RT. Replace a cap without holes for longer storage.</li>
</ol>
7. Fluorescent Labeling
<ol>
	<li>Re-suspend the dried samples in 10 &#181;L 120 mM TFA solution and add 50 &#181;L DMB solution. Transfer samples into dark 1.5 mL tubes to protect them from ultraviolet light.</li>
	<li>For standards, dissolve 1 &#181;L Neu5Ac (60 ng/mL, in water) or 1 &#181;L Neu5Gc (60 ng/mL, in water) in 10 &#181;L 120 mM TFA solution in dark 1.5 mL tubes. Add 50 &#181;L DMB solution.</li>
	<li>Incubate the cell membrane samples and the standards for 1.5 h at 56 &#176;C protected from light. After incubation, add 4 &#181;L 400 mM NaOH solution to each sample to stop the labeling reaction.</li>
</ol>
8. HPLC Analysis
NOTE: Labeled samples are analyzed on a HPLC system equipped with a C18 RP column (110 &#197;, 3 &#181;m particle size, 4.6 x 150 mm), a fluorescence detector, and a fraction collector. The solvents used are methanol, acetonitrile, and water. Make sure to degas all solvents prior to the measurements, if the HPLC system lacks an internal degasser.
<ol>
	<li>Set the temperature of the column to 40 &#176;C and configure the fluorescence detector with 373 nm for excitation and 448 nm for emission, respectively. Inject 10 &#181;L sample volume and separate the probes for 50 min at 0.5 mL/min flow rate with methanol/acetonitrile/water (6:8:86) as the eluent. For mass spectrometry analysis, collect the peaks of interest. 
	NOTE: Neu5Gc is expected to eluate after 6-8 min, Neu5Ac after 9 -&#160;12 min, Neu5Prop after 17 - 23 min, and Neu5But after 38 - 44 min. The fractionated samples can be stored for several h at 4 &#176;C protected from light.</li>
	<li>After measuring each sample, wash the column for 7.5 min at 1.0 mL/min flow rate (&#8776; 1.5 column volumes) with methanol/acetonitrile/water (6:25:69) and re-equilibrate the system for 7.5 min at 1.0 mL/min flow rate with methanol/acetonitrile/water (6:8:86).</li>
	<li>For quantitative analysis, calculate the area under the curve of the sialic acid peaks of interest using the operating software of the respective HPLC-system22. 
	NOTE: Obtained data can be related to the Neu5Ac or the Neu5Gc standard and to the measured protein concentrations in the cytosolic fractions (see section 5.1).</li>
</ol>
9. Mass Spectrometry Analysis of Sialic Acid Monosaccharides
NOTE: HPLC retention peaks of interest can be further analyzed by liquid chromatography (LC) electrospray-ionization mass spectrometry (ESI-MS), in order to verify the unnatural sialic acid species.
<ol>
	<li>For mass spectrometry analysis, inject 20 &#181;L of collected sample into a LC/Mass Selective Detector (MSD) system with 79.9% methanol, 20% isopropyl alcohol, and 0.1% formic acid as eluent, 0.5 mL/min flow rate, 4 kV capillary voltage, and 350 &#176;C capillary temperature22,23.</li>
	<li>In the evaluation software of the respective LC/MSD system, select the peak of interest in the total ion chromatogram. View the mass spectrum of the resolved peak. Display the positive mass/charge ratio between 300 and 700. 
	NOTE: DMB labeling of sialic acids leads to an increase in the molecular mass of 116.2 g/mol. The DMB-labeled sialic acid species have the following molecular masses: DMB-Neu5Ac = 424.2 g/mol, DMB-Neu5Gc = 441.8 g/mol, DMB-Neu5Prop = 436.2 g/mol, DMB-Neu5But = 453.2 g/mol. Common adducts are acetonitrile, sodium, or isopropyl alcohol.</li>
</ol>
10. Western Blot Analysis of Polysialic Acid in Kelly Neuroblastoma Cells
<ol>
	<li>Preparation of cell lysates
	<ol>
		<li>Add 1 mL Western blot lysis buffer to the cell pellets from step 3.3 and vortex. Incubate for 30 min on ice. Vortex every 5 min.Centrifuge the samples for 1.5 h at 20,000 x g and 4 &#176;C. Collect the supernatant containing the proteins from the lysed cells.</li>
		<li>Determine the protein concentration of the protein fractions, e.g., the Bradford or the BCA assay. Dilute the samples to a protein concentration of 1.5 &#181;g/&#181;L in lysis buffer.</li>
		<li>Prepare the samples for the SDS-PAGE by adding 10 &#181;L Laemmli sample buffer to 90 &#181;L protein fraction (1.5 &#181;g/&#181;L) and boil the sample for 5 min24.</li>
	</ol>
	</li>
	<li>Immunoblotting
	<ol>
		<li>Use 8% SDS-acrylamide gels with 20 - 30 &#181;L pockets and 0.75 or 1 mm thickness. Run the gel at 25 mA for 2 h at RT.</li>
		<li>Carefully remove the glass cover from the gel. Cut out and discard the upper part of the gel containing the loading pockets. Place the gel on a nitrocellulose membrane (0.2 &#181;m), previously soaked in Western blot buffer. Transfer the proteins to nitrocellulose according to the recommendation of the system (e.g., 250 mA for 1 h at 4 &#176;C).</li>
		<li>Remove the nitrocellulose membrane from the blotting system. Stain the blot with Ponceau red to visualize the proteins. Transfer the nitrocellulose membrane into a plastic chamber and add 10 mL blocking solution. Incubate for 1 h at RT or overnight at 4 &#176;C.</li>
		<li>Decant the blocking solution and add monoclonal anti-polySia 735 antibody (1 &#181;g/mL) in PBS. Incubate the membrane for 1 h at RT or overnight at 4 &#176;C. After incubation, wash the membrane at least three times for 5 min with PBS.</li>
		<li>Add polyclonal anti-mouse IgG secondary antibody (1 &#181;g/mL) coupled to horseradish peroxidase (HRP) or a fluorescent label in PBS. Incubate the membrane for 1 h at RT. After incubation, wash the membrane at least three times for 5 min with PBS.</li>
		<li>Transfer the membrane onto a plate of an appropriate imager. Detect the signal according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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 &amp;Aring;, 3 &amp;micro;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>Agilent</td><td></td><td></td></tr><tr><td>ESI-MSD-System</td><td>Agilent</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 ...

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

Research

JoVE Journal

Bioengineering

12.6K Views.  LMU München. 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.

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

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

Behavior

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.

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.

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 ...

Chemistry

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having multifarious bioactivities. Although chemical O-glycosylation is a commonly beneficial strategy to synthesize disaccharide nucleosides, the preparation of substrates such as glycosyl donors and acceptors requires&#160;tedious protecting&#160;group manipulations and a purification at each synthetic step. Meanwhile, several research groups have reported that boronic and borinic esters serve as a protecting or activating group of carbohydrate derivatives to achieve the regio- and/or stereoselective acylation, alkylation, silylation, and glycosylation. In this article, we demonstrate the procedure for the regioselective O-glycosylation of unprotected ribonucleosides utilizing boronic acid. The esterification of 2&#39;,3&#39;-diol of ribonucleosides with boronic acid makes the temporary protection of diol, and, following O-glycosylation with a glycosyl donor in the presence of p-toluenesulfenyl chloride and silver triflate, permits the regioselective reaction of the 5&#39;-hydroxyl group to afford the disaccharide nucleosides. This method could be applied to various nucleosides, such as guanosine, adenosine, cytidine, uridine, 5-metyluridine, and 5-fluorouridine. This article and the accompanying video represent&#160;useful (visual) information for the O-glycosylation of unprotected nucleosides and their analogs for the synthesis of not only disaccharide nucleosides, but also a variety of biologically relevant derivatives.

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2&#39;,3&#39;-diol moieties utilizing a cyclic boronic ester. This method applies to several unprotected nucleosides such as adenosine, guanosine, cytidine, uridine, 5-methyluridine, and 5-fluorouridine to give corresponding disaccharide nucleosides.

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having ...

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

Biochemistry

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Immunology and Infection

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

De-Sialylated Monocyte-Derived-DCs from Isolated PBMCs Using Sialidase Treatment

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

Author Spotlight: Dendritic Cells Maturation Using Sialidases-Based Enzymatic Treatment of the Cell Surface 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Biology

Expansion Microscopy for Visualizing Intracellular Sialylated Glycans via MOE

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes within cells through click and bioorthogonal chemistry. Metabolic oligosaccharide engineering (MOE) has attracted considerable interest due to the essential role of glycosylation in numerous biological processes that involve molecular recognition and its impact on pathologies ranging from cancer to genetic disorders to viral and bacterial infections.
Although MOE is better known for the detection of cell surface glycoconjugates, it is also a very important methodology for the study of intracellular glycans in physiological and pathological contexts. Such studies greatly benefit from high spatial resolution. However, super-resolution microscopy is not readily available in most laboratories and poses challenges for daily implementation. Expansion microscopy is a recent alternative that enhances the resolution of microscopy by physically enlarging biological specimens labeled with fluorescent markers. By embedding the sample in a swellable gel and causing it to expand isotropically through chemical treatment, subcellular structures can be visualized with enhanced precision and resolution without the need for super-resolution techniques.
In this work, we illustrate the capacity of expansion microscopy to visualize intracellular sialylated glycans through the combined use of MOE and click chemistry. Specifically, we propose a procedure for bioorthogonal labeling and expansion microscopy that employs a reporter targeting sialylation, which may be associated with immunofluorescence for co-localization studies. This protocol enables localization studies of sialoconjugate biosynthesis, intracellular trafficking, and recycling.

Here, we propose a simple protocol combining metabolic oligosaccharide engineering, click chemistry, and expansion microscopy that allows bioimaging of intracellular sialylated N-glycoproteins with improved resolution using routine microscopy equipment.

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes ...

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

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