Engineering the sialome of mammalian cells with sialic acid mimetics

Summary Mammalian glycans show a diversity in sialic acid capping, constituting the sialome. Sialic acids can be extensively modified chemically, yielding sialic acid mimetics (SAMs). Here, we present a protocol for detecting and quantifying incorporative SAMs using microscopy and flow cytometry, respectively. We detail steps for linking SAMS to proteins with western blotting. Lastly, we detail procedures for incorporative or inhibitory SAMs and how SAMs can be used for the on-cell synthesis of high-affinity Siglec ligands. For complete details on the use and execution of this protocol, please refer to Büll et al.1 and Moons et al.2


Specific inhibition of sialic acid capping
Step-by-step instructions for the detection of Golgiresident and cell surface sialoglycans Click chemistry protocol for sialoglycan functionalization of Siglec interactions SUMMARY Mammalian glycans show a diversity in sialic acid capping, constituting the sialome.Sialic acids can be extensively modified chemically, yielding sialic acid mimetics (SAMs).Here, we present a protocol for detecting and quantifying incorporative SAMs using microscopy and flow cytometry, respectively.We detail steps for linking SAMS to proteins with western blotting.Lastly, we detail procedures for incorporative or inhibitory SAMs and how SAMs can be used for the on-cell synthesis of high-affinity Siglec ligands.For complete details on the use and execution of this protocol, please refer to Bu ¨ll et al. 1 and Moons et al. 2

Timing: 1 h
The sialome consists of a diverse collection of sialylated glycans (sialoglycans).The structural complexity of the sialome arises from the type of sialic acid, glycosidic linkage, and glycoconjugate that carries the sialylated glycan.To probe sialic acid expression and their biological functions a large collection of SAMs has been synthesized with chemical modifications on one or more of the nine-carbon positions of the sialic acid backbone.Each SAM is designed to engineer and study various aspects of the sialome.SAMs generally enter the sialoglycan biosynthesis pathway, starting with formation of the respective CMP (cytidine monophosphate)-SAM nucleotide sugar by the enzyme CMAS (N-acylneuraminate cytidylyltransferase).CMP-SAMs are shuttled into the Golgi apparatus by the CMP-sialic acid transporter SLC35A1 and incorporation into glycans is catalyzed by the twenty sialyltransferase isoenzymes.These steps describe how to select a SAM from the many different options (Figure 1).Most SAMs can be grouped into two general classes: Inhibitory SAMs and incorporative SAMs.
Inhibitory SAMs interfere with one or more enzymes of the sialoglycan biosynthesis pathway and effectively halt the de novo production of sialoglycans.
Incorporative SAMs serve as substrates for the sialoglycan biosynthesis enzymes and are incorporated into sialoglycans.These can carry 'fixed' functional groups that cannot be further altered or 'flexible' groups that can be further modified once incorporated into sialoglycans on surface or secreted glycoproteins and glycolipids.
a. Select inhibitory SAMs such as fluorinated SAMs that act as potent metabolic inhibitors of sialyltransferases, for example Ac 5 Neu5Ac3F, for deletion of sialic acid capping. 3,4.Select incorporative SAMs for the introduction of functional groups into sialic acids.
Note: SAMs are available with either fixed functionality, such as 9-iodo-NeuAc, 5 or with flexible functionality, such as the azide or alkyne-modified SAMs.Flexible SAMs such as Ac 5 SiaNAz and Ac 5 SiaNPoc, can be further modified using bioorthogonal chemistry. 69][10][11] Note: Sialic acids are negatively charged and their ability to cross the cell membrane is poor and no active uptake mechanism has been reported in mammalian cells.SAMs are therefore frequently peracetylated (e.g.Ac 5 SiaNAz) which allows them to pass the cell membrane via passive diffusion.Inside the cells, the acetyl groups are removed by esterases and the active SAM is released.Generally, the released acetyl groups have no significant effect on the cells when using SAM concentrations up to 100 mM. 12 Note that non-acetylated SAM can be applied for metabolic engineering in some bacteria that express active sialic acid uptake mechanisms. 13te: Most SAMs have reversible effects that dilute with cell division and competing endogenous sialic acid biosynthesis.They can be supplemented to the culture medium for short and prolonged periods for temporal control of their effects. 14ternatives: De novo synthesis of sialic acids in mammalian cells starts from N-Acetylmannosamine (ManNAc) and is mediated by three enzymes (GNE, NANS, NANP).ManNAc derivatives such as the azide-containing Ac 4 ManNAz can also be used to introduce unnatural sialic acids into the sialoglycan biosynthesis pathway. 7,14,15However, ManNAc derivatives have to be tolerated by the three enzymes involved in the de novo synthesis, often have negative effects on cell proliferation, and they can be metabolized into other hexosamine sugars (e.g.GlcNAc). 7We therefore generally recommend SAMs for metabolic engineering of the sialome.

Application selection
Timing: 1 h SAMs allow to change the sialome of mammalian cells and are compatible with most cellular and biochemical assays.Therefore, multiple applications are available to apply SAMs in mammalian cells (Figure 1).These steps describe commonly used applications for selection.
2. If an inhibitory SAM is selected, e.g., Ac 5 Neu5Ac3F, the loss of sialic acid capping on cell surface glycans or secreted glycoproteins can be detected with glycan-binding proteins (GBPs).a. Select GBPs with specificity for sialoglycans to detect the loss of sialic acid-capping on cell surface or secreted glycans.
b. Cells treated with fluorinated SAMs are usually viable and can be applied in a wide range of cellular and biochemical assays to probe the role of sialoglycans herein.c.Select one of the many available adhesion, proliferation, migration, co-culture assays, or in vivo assays where many SAMs can be applied or any biochemical assay to probe the role of sialic acid-capping e.g., in protein function or glycan metabolism.3.If an incorporative, flexible SAM is selected, e.g., Ac 5 SiaNPoc, a wide range of applications becomes available.
Note: Applications include click chemistry-based strategies (copper(I)-catalyzed alkyneazide-cycloaddition (CuAAC)) with large functionalization potential of incorporated azide/ alkyne groups in sialoglycans by conjugation to alkyne/azide-containing molecules.Incorporative SAMs with fixed functionality can be directly assayed in cellular or biochemical assays.
a.For visualization of sialoglycans in cellular compartments of live or fixed cells, select click chemistry-based conjugation of sialoglycans to azide/alkyne-modified fluorophores.
b.For isolation/detection of sialoglycoproteins from the cell membrane or secretion, select click chemistry-based conjugation to azide/alkyne-biotin or other purification tags.c.For changing molecular interactions of sialoglycans with GBPs (e.g., Siglecs) or glycosidases, select click chemistry-based conjugation to azide/alkyne-modified small molecules that alter sialic acid -protein interactions.
Note: Depending on their modifications, SAMs can be used for a wide range of applications compatible with mammalian cells not limited to the examples provided in this protocol.
Note: Several GBPs are available with the ability to recognize sialoglycans or the loss thereof and can be applied to probe SAM-treated mammalian cells.For example, PNA (Peanut agglutinin) recognizes the beta-galactose units exposed after the loss of sialic acid capping.

Readout selection
Timing: 0.5 h Changes induced in the sialome of mammalian cells by SAMs can be assayed with several different methods and here we provide commonly used readouts for selection (Figure 1).Note: Several other readouts are available based on fluorescence, calorimetric assays or mass spectrometry.

Preparation of SAM working stocks
Timing: 1 h This step describes how SAMs provided as powder can be dissolved.

5.
To prepare working stocks from SAMs in their powder form, dissolve SAMs to 100 mM in cell culture-grade dimethyl sulfoxide (DMSO) and mix/vortex until the powder is fully dissolved.
CRITICAL: DMSO is hazardous for the skin at levels of 0.1% or higher.Take proper safety precautions and wear proper personal protection equipment.
Note: SAMs can be obtained from several commercial sources or are provided as community resource from several academic labs.
Note: Aliquots of the working stocks can be stored at À20 C in Eppendorf tubes.Most dissolved SAMs including Ac 5 Neu5Ac3F, Ac 5 SiaNAz, and Ac 5 SiaNPoc are stable for at least one year and tolerate multiple freeze-thaw cycles.
Alternatives: We recommend dissolving SAMs for working stocks in DMSO.Alternatively, SAMs can be dissolved in water, but their water solubility depends on their chemical composition and is different for every SAM.If needed, prepare high concentrations (e.g. 1 M) SAMs in DMSO and dilute stepwise in water until the desired concentration or saturation is reached.

Culturing of mammalian cells
Timing: 1-2 weeks Sialoglycans are assembled by virtually all mammalian cells and therefore SAMs can be applied to most established mammalian cells lines as well as ex vivo cultures of human tissue-derived cells.These steps describe the preparation of human embryonic kidney cell (HEK293) and HeLa cell cultures, both adherent cell lines, and human T-lymphoblast Jurkat cell cultures, a suspension cell line.
6. Make preparations to start up cells for culture.a. Warm the culture medium and 13 PBS in a water bath at 37 C. b.Sterilize the cell culture hood and all required supplies with 70% ethanol.7. Thawing of cryopreserved cells a. Fill 15-mL polystyrene tubes with 9 mL complete culture medium.b.Thaw cryovials containing HEK293 and Jurkat cells ($1 M cells) from liquid nitrogen storage in a water bath at 37 C until 50% has thawed.
Note: To reduce the chance of contamination, make sure to keep the O-ring and cap above the water surface.Thawing is a rapid process of approximately 2 minutes.The concentrations of DMSO in cryopreservation media can be toxic for the cells once thawed completely in a rapid manner, thus this should be prevented if possible.
c. Sterilize the cryovial and transfer the cells from the cryovials into the tubes.

Note:
The leftover liquid can be pipetted out by hand and added to the 15-mL tube.
8. Centrifuge the tubes at 300 3 g for 5 min at 20 C and remove supernatant by aspiration.9. Seeding cells for culture a. Resuspend the cell pellets with 12 mL pre-warmed culture medium and transfer to T75 flasks.b.Place the flasks in a horizontal manner for adherent cells and vertical for suspension cells into a cell culture incubator at 37 C with 5% CO 2 atmosphere.c.Culture the cells and check growth and adhesion daily.If required, change medium after 2-3 days.d.Passage the cells when they reach approx.80% confluency (adherent cells) and ca. 2 3 10 6 cells/mL (suspension cells).10.Passaging of adherent cells a.At confluence R80%, remove the culture medium by aspiration from the flask.b.Wash the cells carefully with 10 mL 13 PBS for 30 s and aspirate the supernatant.c.Add 1 mL trypsin-EDTA and incubate for 2-5 min at 37 C until the cells detach.d.Add 9 mL fresh culture medium, resuspend the cells, and transfer to a 15 mL tube.Centrifuge at 300 3 g for 5 min and remove supernatant.e. Resuspend the cells in 10 mL fresh pre-warmed culture medium and count viable cell numbers using trypan blue staining and the counting chamber.f.Seed required number of cells into the respective culture vessel.Passage at least 0.5 3 10 6 cells back into a T75.g.Place cultures in an incubator at 37 C with 5% CO 2 atmosphere.
CRITICAL: Some cell lines (e.g.HEK293) do not firmly adhere to culture plastic.Thus, liquid aspiration and addition steps must be performed by carefully adding liquids to the side of the flask and not directly onto the cells.CRITICAL: The passage number of mammalian cells should not exceed 25 before metabolic engineering experiments, because they can go into senescence or alter their metabolism and characteristics with increasing passage number.Always check cell viability, growth, and morphology before using them in an experiment.

Passaging of suspension cells.
a. Collect 10 mL cell suspension and count the cells to determine the density.b.If cells reach 2 3 10 6 cells/mL, transfer cells into a polystyrene tube and centrifuge at 20 C, 300 3 g, for 5 min.c.Remove the supernatant by aspiration and resuspend the cells in 10 mL fresh pre-warmed culture medium.d.Seed required number of cells into the respective culture dish containing at least 15 mL culture medium.Seed at least 1 3 10 6 cells back into the main culture to maintain the cells.e. Place cultures in an incubator at 37 C with 5% CO 2 atmosphere.
Note: It can take up to two weeks before suspension cells enter exponential growth after recovery from cryopreservation.Growing them with medium containing 20% fetal-bovine serum (FBS) for the first week can facilitate recovery.Confluency of suspension cells is difficult to assess by microcopy and should be done by counting.Optimum growing density is 1.0-1.5 3 10 6 cells/mL.CRITICAL: Most SAMs are effective after 1-3 days of culture, because this time is typically required for the sialome turnover.When seeding cells, the density should allow them to grow for that period without overgrowing.

Alternatives:
In principle, all mammalian cell lines and fresh tissue-derived cells can be used for this protocol.Check the cell-type specific characteristics and growth conditions in literature or databases such as the American Type Culture Collection (ATCC) or the European Collection of Authenticated Cell Cultures (ECACC).
Prepare the following 12.Pre-warm an incubator chamber or water bath to 37 C. 13.Pre-cool a plate/tube centrifuge to 4 C. 14. Initialize the CytoFLEX flow cytometer according to the manufacturer's instructions.This cytometer contains a 488 laser and a 510/20 bandpass filter.15.Setup the EVOS M5000 imaging system with ability to detect green, fluorescent dyes (excitation 490 nm, emission 510-525 nm) and DAPI (excitation 350 nm, emission 470 nm).
Alternatives: Setup a flow cytometer or fluorescence microscope with similar parameters.

Protocol
Alternatives: The CuAAC reaction buffer generates reactive oxygen (ROS) species that can damage cell membranes.To reduce the impact on cell viability, ROS-chelating agents e.g.
L-histidine or THPTA can be added to the buffer. 17ITICAL: Add sodium-L-ascorbate as last step before addition of the buffer to the samples.Sodium-L-ascorbate catalyzes Cu(II) into Cu(I) and for optimal reaction works best freshly prepared.These steps describe the treatment of mammalian cells with Ac 5 Neu5Ac3F that halts sialoglycan biosynthesis and the SAMs Ac 5 SiaNAz and Ac 5 SiaNPoc that allow introduction of clickable moieties into sialoglycans on surface and secreted glycoproteins and glycolipids.
Note: For example, prepare 1 mL medium containing 1 mM SAM and add to 9 mL culture medium to yield 100 mM final concentration.
3. Add the SAM to the cells at a 13 final concentration and gently swirl the culture dish to equally distribute the SAM.For maximum effect (sialoglycan inhibition or incorporation) we recommend 100 mM SAM for HEK293, HeLa, and Jurkat cells.
CRITICAL: Always include a volumetric DMSO control sample treated with equal concentrations of DMSO to ensure that the observed effects are not mediated by this solvent.
4. Culture the cells for the desired amount of time and selected application.
Note: 100 mM was found by us to be effective in most mammalian cell lines and types. 3,7owever, the effective concentration of SAM differs per cell line/type and depending on the application different concentrations are applicable.

Detection of cell-surface sialoglycans with GBPs
Timing: 6-8 h These steps describe the detection of cell-surface sialoglycans with GBPs in adherent HeLa cells treated with Ac 5 Neu5Ac3F.
5. Preparation of glass coverslips for culture.a. Sterilize culture competent glass coverslips by dipping them into 70% ethanol for 30 s and wash thoroughly with 13 PBS.b.Place one coverslip/well into a 12-well plate and add 0.5 mL poly-L-lysine coating solution.
Alternatives: Poly-L-Lysine is positively charged and enables attachment of net negatively charged cells to the cover slips preventing detachment during the staining procedure.Other reagents might also be suitable for coating.c.Incubate for 5 min at RT, remove the poly-L-lysine coating solution and thoroughly wash three times with 1 mL 13 PBS and aspirate all fluid.
CRITICAL: Remove all free poly-L-Lysine to avoid its binding to cells, which might interfere with adherence.Protocol 6. Seed HeLa cells as described (preparation step 10) and add 100 mM of Ac 5 Neu5Ac3F, or DMSO control (step 2 and 3).Culture treated cells for up to 72 h.
CRITICAL: When adding the cells, ensure that the coverslip is at the bottom of the well and not floating to have cells growing only on one side.
7. Fix the cells.a. Aspirate the supernatant and wash the wells twice with 1 mL 13 PBS.
Note: Supernatant can be stored for subsequent analysis of secreted glycoconjugates.Supernatant can be centrifuged at 300 3 g for 5 min at 20 C to pellet debris and stored at À20 C.
b. Remove the 13 PBS and add 0.5 mL fixation buffer and incubate for 10 min at RT.

Note:
Plates containing fixation buffer should not be placed back into the culture incubator, because the PFA might damage live cells in other well/plates due to cross contamination.
CRITICAL: Do not pipet directly on the cells, but against the side of the well to avoid detaching the cells.
Alternatives: When using a different method to fix cells on coverslips (methanol, acetone, or PEM buffer fixation), please check compatibility against a PFA control.
c. Remove the fixation buffer and discard in hazardous compounds waste.d.Wash the wells carefully three times with 1 mL 13 PBS.
CRITICAL: Wash thoroughly to remove all fixation buffer to prevent cross-linking of buffer proteins.
e. Remove the 13 PBS and add 1 mL blocking buffer.Incubate the wells for 1 h at 20 C or >3 h at 4 C.
Pause point: Coverslips with fixed cells can be stored in blocking buffer at 4 C for up to 2 months with 0.02% NaN 3 added.

Stain the cells with biotinylated MALII.
a. Wash the coverslip with 1 mL PBS-BSA and add 0.5 mL lectin staining solution containing 5 mg/mL biotinylated MALII.b.Incubate the samples for 1 h at 20 C or for >3 h at 4 C and wash three times with 1 mL PBS-BSA.
Note: Some lectins require divalent ions (Ca 2+ , Mg 2+ ) for binding and therefore should always be added to the lectin staining solution.
9. Detect bound MALII with immunofluorescence.a. Wash coverslips twice with PBS-BSA and incubate the samples for 30 min with 0.5 mL PBS-BSA containing 1 mg/mL streptavidin, Alexa Fluorä 488 conjugate protected from light at 20 C.
Note: Other secondary fluorescent streptavidin probes can be used, and samples can be stained with additional probes (e.g.antibodies) but ensure that these are compatible with the GBP.
b. Wash the coverslips twice with 1 mL PBS-BSA and once with 13 PBS followed by incubation with 0.5 mL 1 mg/mL DAPI in 13 PBS for 10 min at 20 C in the dark.
10. Wash the coverslips once with 1 mL 13 PBS and mount them with mounting medium onto a microscopy slide and seal the edges of the coverslip with transparent nail polish.11.Acquire representative images using the EVOS M5000 imaging system or alternative fluorescence microscopes.
(Sub)cellular localization of alkyne-tagged sialoglycans Timing: 6-8 h These steps describe how alkyne-tagged sialoglycans can be detected in the Golgi apparatus using bioorthogonal chemistry.
12. Seed HeLa cells on poly-L-lysine coverslips in 12-wells (step 5) and culture them for 2 h in the presence of 100 mM Ac 5 SiaNPoc or DMSO control as described in preparation step 10. 13.Fix the cells as described in step 7 and permeabilize the cells by adding 0.5 mL permeabilization buffer for 10 min at RT.
Pause point: Coverslips with fixed cells can be stored in blocking buffer at 4 C for up to 2 months with 0.02% NaN 3 added.This can only be done before permeabilization of the cells.
Note: Additional probes to stain extracellular targets can be used after fixation and before adding the permeabilization buffer.
14. Remove the permeabilization buffer and add 1 mL intracellular blocking buffer for 60 min at 20 C. 15.Wash the coverslips three times with 1 mL intracellular washing buffer and incubate with intracellular CuAAC solution for 45 min at 37 C.
CRITICAL: Thoroughly wash the samples with 13 PBS to remove all free protein from the blocking step, because free proteins can bind the copper and reduce the reaction efficiency.
16. Aspirate the CuAAC solution and wash the samples three times with 1 mL intracellular washing buffer.17.Incubate the samples with intracellular washing buffer containing 1 mg/mL streptavidin, Alexa Fluorä 488 conjugate protected from light at 20 C .
Note: Probes, e.g.antibodies against intracellular targets, can now be used for additional staining.
18. Wash three times with 1 mL intracellular washing buffer and once with 13 PBS.Perform DAPI staining and mounting as described in steps 9 and 10.
Pause point: Sealed samples can be stored in the dark at 4 C for up to 3 years.
19. Acquire representative images using the EVOS M5000 system or alternative fluorescence microscopes.

Timing: 4-6 h
These steps describe how the dose-dependent effects of Ac 5 Neu5Ac3F and Ac 5 SiaNPoc on the sialome of Jurkat cells can be quantified by flow cytometry using MALII and click chemistry-based detection of sialoglycans, respectively.
Alternatives: Different concentrations of SAM and different time points can be chosen.Keep in mind that a certain incorporation time, min.8-16 hours, is required to accumulate detectable amounts of alkyne label in surface and secreted sialoglycoproteins.
30.Lyse the cells.a. Gently wash the adherent cells twice with 1 mL 13 PBS (RT) and aspirate the supernatant.b.Add 400 mL ice-cold cell lysis buffer per well and detach the cells by repeated vigorous pipetting (hold plate at a 45 angle) or scraping with the pipette tip or cell scraper.c.Check that all cells are detached with a light microscope at 43 or 103 magnification.d.Hold the plate at a 45 angle and transfer the samples into 1.5 mL microcentrifuge tubes and incubate them on ice for 1 h and vortex for 10 s after 30 min.31.Vortex the sample tubes and centrifuge in a microcentrifuge at high speed (>10.000 3 g) for at least 10 min at 4 C. Transfer the supernatant containing soluble sialoglycoproteins to a clean 1.5 mL microcentrifuge tube.
Note: A small white pellet consisting of the insoluble components (e.g.membranes, DNA) should be visible in the tube.This can be used for subsequent analysis of insoluble molecules, by resuspending it in 2% SDS.
Pause point: Collected supernatants and cell lysates can be stored at À20 C for several years.32.Determine the protein concentration in the cell lysates using a BCA Protein Assay Kit following the manufacturers instruction.33.Load 30-50 mg of cell lysate per sample on a protein gel and run the gel 30 min at 60 V to run the samples out the slots and 60 min at 130 V in SD-PAGE running buffer to separate proteins by size.Transfer the proteins onto a nitrocellulose membrane for at least 60 min at 275 mA/ 100 V using ice cold 13 transfer buffer.
Optional: Directly after transfer, stain the nitrocellulose membrane with Ponceau S solution to detect the blotted lysates and confirm equal loading of protein.Rinse the blot in dH 2 O once and submerge the blot in Ponceau S solution in a fume hood.Incubate the blot for at least 5 min at 20 C gently shaking (30-50 rpm), after which the Ponceau S solution can be removed.Rinse the blot in dH 2 O till background of blot appears clear and acquire the image of the membrane.
Pause point: membranes can be dried and stored for at least 3 years at 20 C.
34. Place the membrane into a clean incubation tray with the protein side facing upwards and add 30 mL membrane blocking buffer and incubate shaking for 1 h at 20 C or >4 h at 4 C. 35.Discard the blocking buffer and wash the membrane three times for 5 min with 30 mL 13 PBS and once with 30 mL demi water shaking at 20 C.
CRITICAL: Thoroughly wash the membrane to remove all free protein from the blocking step, because free proteins can bind the copper and reduce the reaction efficiency.
36.Incubate the membrane in 30 mL membrane CuAAC solution for 45 min at 37 C shaking.37. Discard the click reaction buffer and wash the membrane four times for 5 min with 20 mL PBS-BSA shaking at RT. 38.Transfer the membrane into a 50 mL tube and add 4 mL PBS-BSA containing 1 mg/mL Streptavidin, Alexa Fluorä 647 conjugate and incubate on a tube roller for 1 h at 20 C or >4 h at 4 C protected from light.
Alternatives: Membranes can be stained with other probes and antibodies and secondary antibodies conjugated to compatible fluorophores to co-localize for example the sialoglycan and a specific protein signal.
39. Wash the membrane four times for 5 min with 20 mL PBS-T at 20 C and image the membrane with a fluorescent membrane imaging system.
On-cell synthesis of high affinity Siglec ligands

Timing: 4-6 h
Incorporative SAMs with azide or alkyne tags allow further chemical modification with click chemistry once incorporated into sialoglycans.These steps describe a method to chemically modify sialic acids at the cell surface of living cells, rendering them into high-affinity Siglec ligand expressing cells (HASLECs).CRITICAL: Thoroughly wash the cells to remove all medium proteins, because free proteins can bind the copper and reduce the reaction efficiency.
41. Remove the 13 PBS from the last wash step and resuspend the cells in 100 mL on-cell Siglec ligand synthesis buffer.Incubate the plate for 20 min at 37 C.
Note: Add the reaction buffer also to DMSO-treated cells to determine the background.
42. Centrifuge the plate with 500 3 g for 3 min at 4 C and discard the supernatant by flicking of the plate.Wash twice with 13 PBS-BSA by centrifugation and removing the supernatant by flicking.43.Resuspend the cells in 100 mL Siglec-3 staining buffer and incubate for 1 h at 4 C protected from light.
Note: Incubate one or more wells with PBS-BSA without recombinant Siglec-3/CD33 Fc chimera as negative control.
44. Centrifuge the plate with 500 3 g for 3 min at 4 C and discard the supernatant by flicking of the plate.Wash twice with 150 mL PBS-BSA by centrifugation and removing the supernatant by flicking.45.Resuspend the cell pellets in 100 mL PBS-BSA and pipet up and down to create single cell suspensions.
Alternatives: Cells can be stained with other probes/dyes (viability, surface marker), provided they do not interfere with Siglec binding and are compatible with the selected fluorophores.
Note: Immediately before and during the measurement, resuspend the cells by pipetting or using build-in shaking options to ensure that the cells are in solution.
46. Acquire the samples using the CytoFlex flow cytometer plate-reader function or a flow cytometer with similar parameters.For data analysis go to the quantification and data analysis step.
Alternatives: As an alternative to CuAAC, strain-promoted azide-alkyne cycloaddition (SPAAC) can be used for the bioorthogonal chemistry.This reaction is spontaneous and does not require copper and is more suitable for live-cell modifications.However, the copper-catalyzed azide and alkyne L39 conjugation forms a triazole that maybe required for high-affinity Siglec-3 binding.

EXPECTED OUTCOMES
Our protocol describes the application of SAMs to engineer the sialome of mammalian cells.The effects of the inhibiting SAM Ac 5 Neu5Ac3F that interferes with sialoglycan biosynthesis can be detected by reduced binding of MALII lectin to the cell surface of HeLa cells with fluorescence microscopy (Figure 2A).The incorporative SAM Ac 5 SiaNPoc is installed in the sialoglycans of HeLa cells within 2 h allowing detection after click reaction in the Golgi apparatus fluorescence microscopy (Figure 2B).Flow cytometry-based detection of cell surface bound MALII or click-chemistry-labeled sialoglycans forms a quantitative readout to determine dose-response curves that inform on the degree of sialoglycan synthesis inhibition or incorporation, respectively.Representative gating and quantification is shown for Jurkat cells treated with different concentration of Ac 5 Neu5Ac3F or Ac 5 SiaNPoc (Figure 3).Sialoglycoproteins in total cell lysate or in culture supernatant of HEK293 cells can be detected by incorporative SAMs with clickable groups and western blot analysis (Figure 4).Lastly, a representative example for on-cell synthesis of a high-affinity Siglec-3/CD33 ligand (L39) in HEK293 cells treated with Ac 5 SiaNPoc and analysis by flow cytometry is shown (Figure 5).

QUANTIFICATION AND STATISTICAL ANALYSIS
Timing: 1-2 h and removed to mammalian cell culture medium and thus their activity can be temporally controlled.However, there is a delay effect that needs to be considered, because from the point of addition and removal the de novo sialoglycan biosynthesis and turnover rates determine the speed of incorporation or inhibition.Also, it takes a certain time (ca.24 h) to build and deplete an intracellular pool of SAMs.Thus, effective concentrations and timing will depend on the cell-type specific sialoglycan biosynthesis and turnover rates.Additionally, SAM-based engineering utilizes the sialome generated by a cell and is therefore limited to the specific sialoglycans, glycoconjugates and glycoprotein/lipid of that cell.This could potentially influence the recognition by GBPs with specificity for linkage and glycoconjugate types.The nature of biosynthesized sialoglycans determines the products of the on-cell synthesis of high-affinity Siglec ligands that may thus produce different results in different cell types.

TROUBLESHOOTING Problem 1
After treatment, there seems to be no effect of SAMs on the mammalian sialome.

Potential solution 1
The cell type and concentration and incubation time can have large effects on the efficacy of SAMs.We advise to make a concentration range (e.g., 0-500 mM) and to incubate the cells for at least 48 h to give enough time for metabolic incorporation (step 1).For novel SAMs, it could be that they are not tolerized by the sialoglycan biosynthesis pathway.Known, validated inhibitory or incorporative SAMs should be included as positive controls.
Problem 2 GBP (including Siglec Fc proteins) binding to cells is low/weak.

Potential solution 2
This can have multiple reasons, for example that the used cell type does not produce the respective sialoglycan epitope needed for GBP binding or that the pre-complexing ratio of recombinant Siglec Fc protein to anti-human IgG detection antibody was not ideal.Alternatively, GBPs such as soluble recombinant Siglec Fc proteins can lose activity when stored at À20 C or À80 C for longer times and during repeated freeze-thaw cycles.GBP can differ in their binding potential between suppliers and batches.Concentrations should be evaluated prior to subsequent use.We recommend to aliquot such GBPs and to use them within 6 months after reconstitution and to avoid freeze-thaw cycles.Note that some GBPs require divalent ions (Ca 2+ and Mg 2+ ) for glycan binding.

Potential solution 3
This protocol makes use of the CuAAC reaction.Excess protein in the reaction buffer, for example BSA from blocking steps, lowers the efficacy of the reaction.We recommend thoroughly washing samples with 13 PBS before the click reaction to remove all free protein from blocking or staining steps.Letting the reaction proceed at 37 C for an additional 15-30 min may also enhance efficacy.Alkyne-and azide-biotin conjugates can lose activity during freezing-thawing and should be aliquoted to avoid multiple freeze-thaw cycles.

Problem 4
Cells shift in flow cytometry data analysis in FSC/SSC after click reaction (step 23, 41).

Figure 1 .
Figure 1.Chemical structure of sialic acid and the SAMs used in this protocol The scheme depicts step-wise selection of SAMs, applications, and readouts.

4 .
Select one of the following readouts to detect the effects of SAM.a. Select immunocytochemistry to probe changes in the sialome in live/fixed mammalian cells with structural information on the subcellular localization of sialoglycans.Samples can be analyzed by fluorescence-based microscopy.b.Select flow cytometry to obtain quantitative information on cell surface sialoglycan expression and binding levels to GBPs in cells treated with SAMs.c. Select western blot-based methods to yield information on how SAMs change the sialome on the level of individual surface and secreted sialoglycoproteins.

1 40.
Culture suspension Jurkat cells for 72 h with 100 mM Ac 5 SiaNPoc or DMSO control.a. Distribute the cells into a 96-well V-bottom plate with 0.1-0.2 3 10 6 cells/well.b.Centrifuge the plate in a plate centrifuge for 3 min, 500 3 g, at 4 C and discard the supernatant by flicking the plate.Wash twice by resuspension in 150 mL 13 PBS by centrifugation and flicking.

Figure 2 .
Figure 2. Detection of sialoglycans with immunocytochemistry (A) Representative fluorescence microscopy image of HeLa cells treated with DMSO and Ac 5 Neu5Ac3F for 3 days stained with MALII and DAPI.(B) Images show HeLa cells incubated for 2 h with DMSO or Ac 5 SiaNPoc followed click chemistry-based detection of intracellular alkyne-tagged sialoglycoproteins visible in the Golgi apparatus.

Figure 4 .
Figure 4.Western blot detection of sialoglycoproteins HEK293 cells were incubated for 3 d with DMSO, 10 mM or 100 mM Ac 5 SiaNAz and cell lysates and culture supernatant were subjected to western blot analysis.PonceauS staining shows equal protein loading and CuAAC visualizes sialoglycoproteins.

TABLE REAGENT
Poly-L-Lysine coating solution stored at 4 C or À20 C can be re-used ca. 10 times.Add PFA powder to 13 PBS and stir at 60 C in fume hood.Add NaOH dropwise until PFA is dissolved and fill up to final volume with 13 PBS.Filter solution to remove particles.CRITICAL: PFA is toxic!Take appropriate safety measures by wearing gloves and a lab coat and work under a fume-hood.Note: Prepare stock solutions of 1 M MgCl 2 and 1 M CaCl 2 in MilliQ.
(Continued on next page) STAR Protocols 4, 102330, September 15, 2023 CRITICAL: Heat-inactivate the FBS at 56 C for 30 minutes before usage.Do not freeze heat-inactivated FBS, but store at 4 C for up to 4 months.Alternatives: Additional medium components can be added to the culture medium, depending on the cell line/type used.Note:(Continued on next page) Note: The Western blot transfer buffer can be made with a final solution of 20% methanol, after which it can be reused 3-4 times.
Storage: À20 C (dry powder) in the dark.Prepare fresh before use.Storage: Prepare fresh (5-15 min before starting procedure).STAR Protocols 4, 102330, September 15, 2023 Storage: The hypotonic basis of the lysis buffer (NaCl, Tris-HCl, EDTA) can be stored at 20 C for up to 2 years.After addition of the other components, the lysis buffer can be stored at À20 C for up to 1 year.After preparation, keep on ice during the procedure.Storage: Can be stored at 4 C for up to 2 weeks.Alternatives:Note: Mix the recombinant Siglec protein and anti-human Fc antibody and incubate 20 min at 4 C to form complexes.Pre-complexing forms Siglec multimers with high binding avidity.Storage: Prepare fresh (20-30 min before addition to cells).STAR Protocols 4, 102330, September 15, 2023

Table 1 .
Triplicate values of normalized MALII binding, with raw MFI values normalized to DMSOThese values were used to generate Figure3.