Method Article

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

DOI:

10.3791/55746

⸱

November 25th, 2017

 , 
1Max von Pettenkofer-Institut & Genzentrum, Virologie, Nationales Referenzzentrum für Retroviren, Medizinische Fakultät, LMU München, 2Institut für Laboratoriumsmedizin, klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, 3Institut für Physiologische Chemie, Martin-Luther-Universität Halle-Wittenberg

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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 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.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

1. Preparation of Buffers and Reagents

  1. Preparation of 3 mM sodium methoxide solution
    1. 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.
  2. Preparation of Tris-HCl buffer
    1. 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.
    2. 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.
    3. Add the volume of water needed to achieve a final volume of 100 mL. Filter the solution using a 0.22 µm 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 °C for several weeks.
  3. Preparation of aprotinin solution
    1. Dissolve 10 mg aprotinin in 1 mL water (1.54 mM). Aliquot the aprotinin solution into 10 x 1.5 mL-plastic tubes (100 µL each). Store at -20 °C for several weeks.
  4. Preparation of leupeptin solution
    1. Dissolve 10 mg leupeptin in 2 mL water (10 mM). Aliquot the leupeptin solution 10 x 1.5 mL-plastic tubes. Store at -20 °C for several weeks.
  5. Preparation of phenylmethylsulfonyl fluoride (PMSF) solution
    1. 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 °C for several weeks.
  6. Preparation of 1,2-diamino-4,5-methyldioxybenzol-dihydrochloride (DMB) solution
    1. Combine 15.62 mg DMB, 352 µL β-mercaptoethanol, and 48 µL sodium bisulfite-solution (39%), and add 9.6 mL water (6.9 mM DMB, 500 mM β-mercaptoethanol, and 0.19% sodium bisulfite). Aliquot the DMB solution in 40 x 1.5 mL-plastic tubes (250 µL each). Store protected from light at -20 °C for several weeks.
  7. Preparation of 1 M trifluoroacetic acid (TFA) solution
    1. Add 770.3 µL of 100% TFA to 9.23 mL water in a 15 mL plastic tube (1 M). Store at 4 °C for several weeks.
  8. Preparation of 120 mM TFA solution
    1. Add 92.4 µL TFA (100%) to 9.91 mL water (120 mM) in a 15 mL plastic tube. Store at 4 °C for several weeks.
  9. Preparation of 400 mM sodium hydroxide (NaOH) solution
    1. Dissolve 160 mg NaOH in 10 mL water (400 mM) in a 15 mL plastic tube. Store at 4 °C for several weeks.
  10. Preparation of Western blot lysis buffer
    1. 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 °C. Add 100 µL of each protease inhibitor (aprotinin, leupeptin, and PMSF) before using the lysis buffer.
  11. Preparation of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer
    1. 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.
  12. Preparation of Western blot buffer
    1. Dissolve 0.3 g Tris, 1.1 g glycine in 90 mL water and add 10 mL ethanol. Store at RT.
  13. Preparation of blocking solution
    1. Dissolve 1 g of fat free milk power in 25 mL phosphate buffered saline (PBS). Always prepare fresh.

2. Synthesis of ManNProp and Related N-acyl-modified Mannosamines

  1. Dissolve 431.2 mg mannosamine hydrochloride in 10 mL 3 mM sodium methoxide solution in a 50 mL glass bottle with a stir bar.
  2. Cool the mixture on ice to 0 °C. To the stirring solution, slowly add dropwise 210 µL propionyl chloride (2.4 mmol), or 248 µL butanoyl chloride (2.4 mmol) to synthesize ManNProp or ManNBut, respectively. Incubate the stirring mixture at 0 °C for 4 h.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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).

3. Cell Culture

  1. 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 °C and 5% CO2.
    NOTE: Cells cultured in medium without ManNAc or its analogues are used as a control.
  2. 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.
    1. To measure sialic acid monosaccharides, seed 1 x 105 cells in 500 µL 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 °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.
  3. 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.
    1. 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 °C for several days. If the expression of polysialic acid is to be elucidated continue to section 10.

4. Cell Lysis for HPLC Analysis

  1. Preparation of HPLC lysis buffer
    1. Combine 5 mL Tris-HCl buffer, 5 µL aprotinin solution, 20 µL leupeptin solution, and 50 µL PMSF solution.
      NOTE: 5 mL HPLC Lysis buffer will contain 150 mM NaCl, 10 mM Tris-HCl, 5 mM EDTA, 1.54 µM aprotinin, 40 µM Leupeptin, and 1 mM PMSF (pH 8.0). This buffer should always be prepared fresh prior to cell lysis.
  2. Re-suspend the collected cell pellets (step 3.3) each in 500 µL 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.

5. Separation of the Membrane Fraction

  1. Centrifuge the lysed cell samples for 2 h at 20,000 x g and 4 °C.Separate the supernatant (approximately 480 µL), 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.

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.

  1. Add 150 µL 1 M TFA-solution to the separated membrane fractions (pellet from section 5). Incubate the samples for 4 h at 80 °C with shaking at 200 - 600 rpm.
  2. Centrifuge the samples for approximately 30 min at 20,000 x g and 21 °C using 0.5 mL filter tubes with 3 kDa exclusion membranes, until the upper phase volume is less than 20 µL.
    NOTE: This step is important to dispose higher molecular debris.
  3. 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.

7. Fluorescent Labeling

  1. Re-suspend the dried samples in 10 µL 120 mM TFA solution and add 50 µL DMB solution. Transfer samples into dark 1.5 mL tubes to protect them from ultraviolet light.
  2. For standards, dissolve 1 µL Neu5Ac (60 ng/mL, in water) or 1 µL Neu5Gc (60 ng/mL, in water) in 10 µL 120 mM TFA solution in dark 1.5 mL tubes. Add 50 µL DMB solution.
  3. Incubate the cell membrane samples and the standards for 1.5 h at 56 °C protected from light. After incubation, add 4 µL 400 mM NaOH solution to each sample to stop the labeling reaction.

8. HPLC Analysis

NOTE: Labeled samples are analyzed on a HPLC system equipped with a C18 RP column (110 Å, 3 µm 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.

  1. Set the temperature of the column to 40 °C and configure the fluorescence detector with 373 nm for excitation and 448 nm for emission, respectively. Inject 10 µL 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 - 12 min, Neu5Prop after 17 - 23 min, and Neu5But after 38 - 44 min. The fractionated samples can be stored for several h at 4 °C protected from light.
  2. After measuring each sample, wash the column for 7.5 min at 1.0 mL/min flow rate (≈ 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).
  3. 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).

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.

  1. For mass spectrometry analysis, inject 20 µL 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 °C capillary temperature22,23.
  2. 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.

10. Western Blot Analysis of Polysialic Acid in Kelly Neuroblastoma Cells

  1. Preparation of cell lysates
    1. 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 °C. Collect the supernatant containing the proteins from the lysed cells.
    2. 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 µg/µL in lysis buffer.
    3. Prepare the samples for the SDS-PAGE by adding 10 µL Laemmli sample buffer to 90 µL protein fraction (1.5 µg/µL) and boil the sample for 5 min24.
  2. Immunoblotting
    1. Use 8% SDS-acrylamide gels with 20 - 30 µL pockets and 0.75 or 1 mm thickness. Run the gel at 25 mA for 2 h at RT.
    2. 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 µm), 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 °C).
    3. 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 °C.
    4. Decant the blocking solution and add monoclonal anti-polySia 735 antibody (1 µg/mL) in PBS. Incubate the membrane for 1 h at RT or overnight at 4 °C. After incubation, wash the membrane at least three times for 5 min with PBS.
    5. Add polyclonal anti-mouse IgG secondary antibody (1 µg/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.
    6. Transfer the membrane onto a plate of an appropriate imager. Detect the signal according to the manufacturer's instructions.

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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...

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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).

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
CellsSigma-Aldrich92110411
RPMI mediumSigma-AldrichR8758
75 ml tissue culture flasksGreiner690175
48-well platesCorning3548
FCSPAAA15-102
Pen/StrepGibco15140-122
TrypsineGibco15400-054
EDTARothX986.1
TrisServa37190.03
SDSRoth2326.2
SDS-PAGE equipment (tanks, glassware etc., machineVWRSDS Gel/Blot
AcrylamideRoth3019.1
Protein ladderFisher Scientific267620
NitrocelluloseGE Healthcare10600002
Ponceau redRoth5938.2
Milk powderRothT145.3
ECLMilliporeWBLUF 0500
0.5 ml Centrifugal Filter Unit with 3 kDa membraneMerck-MilliporeUFC500324
15 mL centrifuge tubesSigma-Aldrich (Corning)CLS430791-500EA
2-MercaptoethanolSigma-AldrichM6250-10ML
2-PropanolSigma-Aldrich34965-1LHPLC gradient grade
4,5-Methylenedioxy-1,2-phenylenediamine dihydrochlorideSigma-AldrichD4784-50MG
48 well, flat bottom tissue culture plateSigma-Aldrich (Corning)CLS3548-100EA
50 mL centrifuge tubesSigma-Aldrich (Corning)CLS430829-500EA
AcetonitrileSigma-Aldrich34967-1LHPLC gradient grade
Aprotinin from bovine lungSigma-AldrichA1153-10MGlyophilized powder, 3-8 TIU/mg solid
Butyryl chlorideSigma-Aldrich109614-250G
C18 RP columnPhenomenex00F-4435-E0110 Å, 3 µm particle size, 4.6 x 150 mm
D-Mannosamine hydrochlorideSigma-AldrichM4670-1G
Dulbecco`s Phosphate Buffered Salt SolutionPAN BiotechP04-36500
Ethylenediaminetetraacetic acidSigma-AldrichE9884-100G
Formic acidSigma-Aldrich56302-50ML-GL
Hydrochloric acid solutionSigma-AldrichH1758-100ML36.5-38.0%, in water
LeupeptinSigma-AldrichL2884-10MG
MethanolCarl-RothT169.2HPLC gradient grade
N-Acetyl-D-mannosamineSigma-AldrichA8176-250MG
N-Acetylneuraminic acidSigma-AldrichA0812-25MG
N-Glycolylneuraminic acidSigma-AldrichG9793-10MG
Phenylmethanesulfonyl fluorideSigma-AldrichP7626-250MG
Propionyl chlorideSigma-AldrichP51559-500G
Safe-Lock Tubes, 1.5 mL, amber (light protection)Eppendorf30120191
Safe-Lock Tubes, 1.5 mL, colorlessEppendorf30120086
Sodium bisulfite solutionSigma-Aldrich13438-1L-R-D40%, in water
Sodium chlorideSigma-Aldrich746398-500G-D
Sodium hydroxideSigma-Aldrich795429-500G-D
Sodium hydroxide solutionSigma-Aldrich319511-500ML1.0 M, in water
Sodium methoxideSigma-Aldrich164992-5G
Trifluoroacetic acidSigma-AldrichT6508-100ML-D
Tris hydrochlorideSigma-AldrichT5941-100G
Trypsin 0.25 %/EDTA 0.02 % in PBSPAN BiotechP10-019100
WaterCarl-RothT905.1HPLC gradient grade
Silica Gel 60Carl-Roth9779.1
HPLCAgilent
ESI-MSD-SystemAgilent

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Angata, T., Varki, A. Chemical diversity in the sialic acids and related alpha-keto acids: An evolutionary perspective. Chem Rev. 102, 439-469 (2002).
  2. Schauer, R., Schoop, H. J., Faillard, H. On biosynthesis of glycolyl group of N-glycolylneuraminic acid. oxidation of N-acetyl group to glycolyl group. Hoppe-Seylers Zeitschrift Fur Physiologische Chemie. 349, (1968).
  3. Irie, A., Koyama, S., Kozutsumi, Y., Kawasaki, T., Suzuki, A. The molecular basis for the absence of N-glycolylneuraminic acid in humans. J Biol Chem. 273, 15866-15871 (1998).
  4. Kelm, S., et al. Use of sialic acid analogues to define functional groups involved in binding to the influenza virus hemagglutinin. Eur J Biochem. 205, 147-153 (1992).
  5. Colley, K. J., Kitajima, K., Sato, C. Polysialic acid: Biosynthesis, novel functions and applications. Crit Rev Biochem Mol Biol. 49, 498-532 (2014).
  6. Rillahan, C. D., et al. Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome. Nat Chem Biol. 8, 661-668 (2012).
  7. Wratil, P. R., et al. A Novel Approach to Decrease Sialic Acid Expression in Cells by a C-3-modified N-Acetylmannosamine. J Biol Chem. 289, 32056-32063 (2014).
  8. Nieto-Garcia, O., et al. Inhibition of the key enzyme of sialic acid biosynthesis by C6-Se modified N-acetylmannosamine analogs. Chem Sci. 7, 3928-3933 (2016).
  9. Keppler, O. T., et al. UDP-GlcNAc 2-epimerase: A regulator of cell surface sialylation. Science. 284, 1372-1376 (1999).
  10. Kayser, H., et al. Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors. J Biol Chem. 267, 16934-16938 (1992).
  11. Keppler, O. T., et al. Biosynthetic modulation of sialic acid-dependent virus-receptor interactions of two primate polyoma viruses. J Biol Chem. 270, 1308-1314 (1995).
  12. Laughlin, S. T., Bertozzi, C. R. Imaging the glycome. Proc Natl Acad Sci U S A. 106, 12-17 (2009).
  13. Wratil, P. R., Horstkorte, R., Reutter, W. Metabolic Glycoengineering with N-Acyl Side Chain Modified Mannosamines. Angewandte Chemie International Edition. 55, 9482-9512 (2016).
  14. Horstkorte, R., et al. Selective inhibition of polysialyltransferase ST8Siall by unnatural sialic acids. Exp Cell Res. 298, 268-274 (2004).
  15. Gnanapragassam, V. S., et al. Sialic Acid Metabolic Engineering: A Potential Strategy for the Neuroblastoma Therapy. PLOS ONE. 9, 10(2014).
  16. Agard, N. J., Prescher, J. A., Bertozzi, C. R. A strain-promoted 3+2 azide-alkyne cycloaddition for covalent modification of blomolecules in living systems. J Am Chem Soc. 126, 15046-15047 (2004).
  17. Dehnert, K. W., et al. Imaging the Sialome during Zebrafish Development with Copper-Free Click Chemistry. ChemBioChem. 13, 353-357 (2012).
  18. Prescher, J. A., Dube, D. H., Bertozzi, C. R. Chemical remodelling of cell surfaces in living animals. Nature. 430, 873-877 (2004).
  19. Neves, A. A., et al. Imaging sialylated tumor cell glycans in vivo. Faseb Journal. 25, 2528-2537 (2011).
  20. Vogt, J., et al. Homeostatic regulation of NCAM polysialylation is critical for correct synaptic targeting. Cell Mol Life Sci. 69, 1179-1191 (2012).
  21. Qiu, L., et al. A novel cancer immunotherapy based on the combination of a synthetic carbohydrate-pulsed dendritic cell vaccine and glycoengineered cancer cells. Oncotarget. 6, 5195-5203 (2015).
  22. Erikson, E., et al. Mouse Siglec-1 Mediates trans-Infection of Surface-bound Murine Leukemia Virus in a Sialic Acid N-Acyl Side Chain-dependent Manner. J Biol Chem. 290, 27345-27359 (2015).
  23. Klein, A., Diaz, S., Ferreira, I., Lamblin, G., Roussel, P., Manzi, A. E. New sialic acids from biological sources identified by a comprehensive and sensitive approach: liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of SIA quinoxalinones. Glycobiology. 7, 421-432 (1997).
  24. Laemmli, U. K. Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature. 227, (1970).
  25. Orozco-Solano, M. I., Priego-Capote, F., Luque de Castro, M. D. Ultrasound-assisted hydrolysis and chemical derivatization combined to lab-on-valve solid-phase extraction for the determination of sialic acids in human biofluids by µ-liquid chromatography-laser induced fluorescence. Analytica Chimica Acta. 766, 69-76 (2013).
  26. Hara, S., Takemori, Y., Yamaguchi, M., Nakamura, M., Ohkura, Y. Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids. Anal Biochem. 164, 138-145 (1987).
  27. İzzetoğlu, S., Şahar, U., Şener, E., Deveci, R. Determination of sialic acids in immune system cells (coelomocytes) of sea urchin, Paracentrotus lividus, using capillary LC-ESI-MS/MS. Fish Shellfish Immunol. 36, 181-186 (2014).
  28. Wolf, S., et al. Chemical Synthesis and Enzymatic Testing of CMP-Sialic Acid Derivatives. ChemBioChem. 13, 2605-2615 (2012).
  29. Sonnenburg, J. L., Altheide, T. K., Varki, A. A uniquely human consequence of domain-specific functional adaptation in a sialic acid-binding receptor. Glycobiology. 14, 339-346 (2004).
  30. Oetke, C., et al. Evidence for efficient uptake and incorporation of sialic acid by eukaryotic cells. Eur J Biochem. 268, 4553-4561 (2001).
  31. Buttner, B., et al. Biochemical engineering of cell surface sialic acids stimulates axonal growth. J Neuro. 22, 8869-8875 (2002).
  32. Kontou, M., Weidemann, W., Bork, K., Horstkorte, R. Biological Chemistry. 390, 575(2009).
  33. Tanaka, F., et al. Expression of polysialic acid and STX, a human polysialyltransferase, is correlated with tumor progression in non small cell lung cancer. Cancer Res. 60, 3072-3080 (2000).
  34. Cheng, B., Xie, R., Dong, L., Chen, X. Metabolic Remodeling of Cell-Surface Sialic Acids: Principles, Applications, and Recent Advances. ChemBioChem. 17, 11-27 (2016).
  35. Collins, B. E., Fralich, T. J., Itonori, S., Ichikawa, Y., Schnaar, R. L. Conversion of cellular sialic acid expression from N-acetyl- to N-glycolylneuraminic acid using a synthetic precursor, N-glycolylmannosamine pentaacetate: inhibition of myelin-associated glycoprotein binding to neural cells. Glycobiology. 10, 11-20 (2000).
  36. Schwartz, E. L., Hadfield, A. F., Brown, A. E., Sartorelli, A. C. Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine. Biochimica Et Biophysica Acta. 762, 489-497 (1983).
  37. Jones, M. B., et al. Characterization of the cellular uptake and metabolic conversion of acetylated N-acetylmannosamine (ManNAc) analogues to sialic acids. Biotechnol Bioeng. 85, 394-405 (2004).
  38. Bayer, N. B., et al. Artificial and Natural Sialic Acid Precursors Influence the Angiogenic Capacity of Human Umbilical Vein Endothelial Cells. Molecules. 18, 2571-2586 (2013).
  39. Schmidt, C., Stehling, P., Schnitzer, J., Reutter, W., Horstkorte, R. Biochemical engineering of neural cell surfaces by the synthetic N-propanoyl-substituted neuraminic acid precursor. J Biol Chem. 273, 19146-19152 (1998).
  40. Laughlin, S. T., Bertozzi, C. R. In Vivo Imaging of Caenorhabditis elegans Glycans. ACS Chem Biol. 4, 1068-1072 (2009).
  41. Laughlin, S. T., Baskin, J. M., Amacher, S. L., Bertozzi, C. R. In vivo imaging of membrane-associated glycans in developing zebrafish. Science. 320, 664-667 (2008).
  42. Chang, P. V., et al. Copper-free click chemistry in living animals. Proc Natl Acad Sci U S A. 107, 1821-1826 (2010).
  43. Gagiannis, D., Gossrau, R., Reutter, W., Zimmermann-Kordmann, M., Horstkorte, R. Engineering the sialic acid in organs of mice using N-propanoylmannosamine. Biochimica Et Biophysica Acta-General Subjects. 1770, 297-306 (2007).
  44. Rong, J., et al. Glycan Imaging in Intact Rat Hearts and Glycoproteomic Analysis Reveal the Upregulation of Sialylation during Cardiac Hypertrophy. J Am Chem Soc. 136, 17468-17476 (2014).
  45. Galuska, S. P., et al. Quantification of Nucleotide-Activated Sialic Acids by a Combination of Reduction and Fluorescent Labeling. Anal Chem. 82, 4591-4598 (2010).
  46. Harms, E., Reutter, W. Half-Life Of N-Acetylneuraminic Acid In Plasma-Membranes Of Rat-Liver And Morris Hepatoma 7777. Cancer Res. 34, 3165-3172 (1974).
  47. Kolarich, D., Lepenies, B., Seeberger, P. H. Glycomics, glycoproteomics and the immune system. Curr Opin Chem Biol. 16, 214-220 (2012).
  48. Preidl, J. J., et al. Fluorescent Mimetics of CMP-Neu5Ac Are Highly Potent, Cell-Permeable Polarization Probes of Eukaryotic and Bacterial Sialyltransferases and Inhibit Cellular Sialylation. Angewandte Chemie-International Edition. 53, 5700(2014).
  49. Macauley, M. S., et al. Systemic Blockade of Sialylation in Mice with a Global Inhibitor of Sialyltransferases. J Biol Chem. 289, 35149-35158 (2014).
  50. Jorge, P., Abdul-Wajid, A. Sialyl-Tn-KLH, glycoconjugate analysis and stability by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Glycobiology. 5, 759-764 (1995).
  51. Lin, S. L., Inoue, Y., Inoue, S. Evaluation of high-performance anion-exchange chromatography with pulsed electrochemical and fluorometric detection for extensive application to the analysisof homologous series of oligo- and polysialic acids in bioactive molecules. Glycobiology. 9, 807-814 (1999).
  52. Pan, Y. B., Chefalo, P., Nagy, N., Harding, C., Guo, Z. W. Synthesis and immunological properties of N-modified GM3 antigens as therapeutic cancer vaccines. J Med Chem. 48, 875-883 (2005).
  53. Chefalo, P., Pan, Y. B., Nagy, N., Guo, Z. W., Harding, C. V. Efficient metabolic engineering, of GM3 on tumor cells by N-phenylacetyl-D-mannosamine. Biochemistry. 45, 3733-3739 (2006).
  54. Lee, S., et al. Chemical Tumor-Targeting of Nanoparticles Based on Metabolic Glycoengineering and Click Chemistry. ACS Nano. 8, 2048-2063 (2014).
  55. Koulaxouzidis, G., Reutter, W., Hildebrandt, H., Stark, G. B., Witzel, C. In vivo stimulation of early peripheral axon regeneration by N-propionylmannosamine in the presence of polysialyltransferase ST8SIA2. J Neural Transm. , 1-9 (2015).
  56. Gnanapragassam, V. S., et al. Correction: Sialic Acid Metabolic Engineering: A Potential Strategy for the Neuroblastoma Therapy. PLOS ONE. 11, e0154289(2016).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Sialic Acid ModificationN acyl MannosaminePolysialic Acid AnalysisHigh Performance Liquid ChromatographyMass SpectrometryWestern BlotCell Culture TreatmentFluorescent LabelingHPLC Analysis

Related Articles