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

The overall goal of this technique is to generate cells or cell derived products with modified sialic acid expression. Therefore, cells are treated with N-acyl modified Mannosamines which are metabolized to the corresponding sialic acids and subsequently expressed on the cell surface. This method can help answer key questions in the Glycobiology field, such as the influence on glycoconjugates on receptor-ligand interactions.

The main advantage of this technique are that is easy to apply and purely cytotoxic. To demonstrate metabolic Glycoengineering, we will use N-acetylmannosamine, which is commercially available. Furthermore, we will demonstrate the synthesis and use of N-propionyl and N-butanoylmannosamine.

The biological effects of metabolic glycoengineering on the expression of polysialic acid is shown. First, dissolve 431.2 milligrams of Mannosamine hydrochloride in 10 milliliters of three Millimolar Sodium methoxide solution in a 15 milliliter glass bottle containing a stir bar. Cool the mixture on ice to zero degrees Celsius.

While stirring, slowly add 210 micrometers of propionyl chloride dropwise to the solution. Then, incubate the stirring mixture at zero degrees Celsius for four hours. Following this, transfer the solution into a 50 milliliter plastic tube and poke four to 8 holes into the cap of the tube with a thin needle.

Rapidly freeze the solution using liquid nitrogen. Then, lyophilize the sample until it has completely dried. After lyophilization, dissolve the dried product in ethyl acetate, methanol, and water.

Using a pipette, load the solution onto a previously prepared silica gel column. After eluting 500 milliliters of solvent, collect four milliliter fractions. Transfer the eluted fractions into a 15 milliliter plastic tube, and poke three to six holes into the cap of the tube with a thin needle.

After the freezing the solution with liquid nitrogen, lyophilize the sample until it has completely dried. Culture Kelly neuroblastoma cells in RPMI medium at 37 degrees Celsius and 5%carbon dioxide. Prior to application of the mannosamines, detach the Kelly neuroblastoma cells by incubating them for 10 minutes with Trypsin and EDTA.

After neutralizing the trypsin with RPMI medium, count the cells using a Neubauer chamber. To measure sialic acid monosaccharides, seat the cells in 500 microliters of medium into a 48-well tissue culture plate. For the analysis of polysialic acids, seat the cells in three milliliters of medium onto a six centimeter diameter cell culture dish.

Add the respective mannosamine derivatives, dissolved in RPMI, to the freshly plated cells, in a final concentration of five millimolar and incubate at 37 degrees Celsius and 5%carbon dioxide, replacing the medium containing the N-acyl modified Mannosamines every 24 hours. After the treatment, decant the medium and wash the cells with PBS. Detach the cells by incubating them for 10 minutes with Trypsin and EDTA.

Neutralize the trypsin by adding the same volume of cell culture medium to the detached cells. Following this, transfer the detached cells into 15 milliliter plastic tubes. Centrifuge the samples for three minutes at 500 times G.After discarding the supernatant, wash the cells three times with five milliliters of PBS.

Following centrifugation, resuspend the collected cell pellets in 500 microliters of ice cold HPLC lysis buffer. Ultrasonicate the samples with an ultrasonic processor needle three times for a period of 30 seconds at medium-high amplitudes, cooling the samples on ice for at least one minute in-between the cycles. After centrifuging the lysed cell samples, separate the supernatant from the membrane fractions in 1.5 milliliter plastic tubes.

For acid hydrolysis, add 150 microliters of one molar TFA solution to the separated membrane fractions. Incubate the samples for four hours at 80 degrees Celsius with shaking at 200 to 600 RPM. Next, transport the samples into 0.5 milliliter filter tubes with three kilodalton exclusion membranes.

Centrifuge the samples for approximately 30 minutes at 20, 000 times G and 21 degrees Celsius, until the upper phase volume is less than 20 microliters. Following this, discard the inserts and poke two to four holes into the caps of the tubes containing the ultrafiltrate with a thin needle. After the freezing the samples with liquid nitrogen, lyophilize them overnight.

Resuspend the dried samples in 10 microliters of 120 millimolar TFA solution, and add 50 microliters of DMB solution. Transfer the samples into dark 1.5 milliliter tubes to protect them from ultraviolet light. Incubate the cell membrane samples and standards for 1.5 hours at 56 degrees Celsius.

After incubation, add four microliters of 400 millimolar sodium hydroxide solution to each sample to stop the labeling reaction. For HPLC analysis, set the temperature of the column to 40 degrees Celsius and configure the fluorescence detector to 373 nanometers for excitation, and 448 nanometers for omission, respectively. Inject 10 microliters of sample volume, and separate the probes for 15 minutes at a 0.5 milliliters per minute flow rate with methanol, Acetonitrile, and water as the eluant.

For mass spectrometry analysis, inject 20 microliters of each collected HPLC sample into an LC mass selective detector system. In the evaluation software of the LC mass selective detector system, select the peak of interest in the total ion chromatogram. View the mass spectrum of the resolved peak and display the positive mass-to-charge ratio between 300 and 700.

Add one milliliter of western blot lysis buffer to the previously prepared pellets of glycoengineered cells, and vortex. Incubate the samples for 30 minutes on ice, vortexing every five minutes. Following this, centrifuge the samples for 1.5 hours at 20, 000 times G and four degrees Celsius.

Then, collect the supernatant containing the proteins from the lysed cells into 1.5 milliliter tubes. Prepare the samples for SDS-page by adding 10 microliters of Laemmli sample buffer to 90 microliters of each protein fraction. Boil the samples for five minutes.

After loading the samples onto an 8%SDS-acrylamide gel, run the gel at 25 milliamps for two hours at room temperature. When finished, carefully remove the glass cover from the gel and cut out the upper part of the gel containing the loading pockets. Next, place the gel on a 0.2 micro nitrocellulose membrane, previously soaked in western blot buffer.

Transfer the proteins to the nitrocellulose according to the recommendation of the system. After removing the nitrocellulose membrane from the blotting system, stain the blot with Ponceau red to visualize the proteins and thereby to control the quality of the transfer. Then, transfer the nitrocellulose membrane into a plastic chamber and add 10 milliliters of blocking solution.

After incubating the nitrocellulose membrane, decant the blocking solution, and add monoclonal anti-polysialic acid 735 antibody in PBS. After incubation, wash the membrane at least three times for five minutes with PBS. Now, add polyclonal anti-Mouse IgG secondary antibody coupled to horseradish peroxidase, to the nitrocellulose membrane.

After incubating and washing the membrane, transfer it onto a plate of an appropriate imager. Then, detect the signal according to the manufacturer's instructions. DMB labeled N-glycolyl neuraminic acid, elutes between seven and nine minutes.

And, DMB labeled N-acetyl neuraminic acid elutes between 10 and 12 minutes. Several smaller peaks observed in the chromatogram represent unreacted DMB and reaction intermediates. The HPLC chromatograms of the cell lysates show several undefined peaks, which represent internal impurities and DMB by-products from reaction with other alpha keto acids present in the cell lysates.

Treatment with N-acetyl Mannosamine often leads to depletion of N-glycolylneuraminic acid on the cell surface. In chromatograms of lysed cells treated with N-propionyl neuraminic acid or N-butanoyl neuraminic acid, peaks are observed that indicate the appearance of the corresponding non-natural sialic acids. The reaction of DMB with sialic acid species leads to an increase in molecular mass of 116.2 dalton, compared to the sialic acid species that are not labeled with this dye.

Besides the potentated ion species, the sodium adducts are also observed. DMB-Neu5Gc elutes shortly after the unreacted or partly reacted DMB species, which explains the appearance of undefined peaks in the mass spectrum of DMB-Neu5Gc. Treatment of Kelly neuroblastoma cells with N-propionylmannosamine or N-butanoylmannosamine leads to reduced expression of polysiatic acid on the cell surface.

Treatment with N-acetylmannosamine on the other hand, leads to increased polysiatic acid expression. By applying this technique, cells with utter sialylation can be generated within several days. Such glycoengineered cells can be used in further experiments.

For example, to study receptor-ligand interactions or as shown here, to inhibit the expression of polysiatic acid. The procedure of metabolic engineering presented here is optimized for sialic acid experiments. However, the same procedure can be also used in animal experiments.

We hope that our presented protocol and this video production is a help for the authors to bring metabolic glycoengineering into the labs to analyze the function and also the biological role of sialic acid and sialic acid receptors in biological systems. Organic chemists could synthesize N-acyl modified mannosamines with more complex structures and use these as suggested in our protocol. Following our protocol, all samples should be analyzed immediately after fluorescent labeling.

Consequently, there should be no delay between HPLC and ACMS analyzers.