Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment

Summary

N-Glycosylation is a common protein modification catalyzed by a series of glycosylation enzymes in the endoplasmic reticulum and Golgi apparatus. Here, based on a previously established Golgi α-mannosidase-I-deficient cell line, we present a protocol to investigate the enzymatic activity of exogenously expressed Golgi α-mannosidase IA in interphase and mitotic cells. We describe steps for cell surface lectin staining and subsequent live cell imaging. We also detail PNGase F and Endo H cleavage assays to analyze protein glycosylation.

For complete details on the use and execution of this protocol, please refer to Huang et al.¹

Graphical abstract

Highlights

• •Protocols for the analysis of Golgi mannosidase activity in cultured cells
• •Surface lectin staining to assess glycosylation in interphase cells
• •Lectin staining and live cell imaging to analyze glycosylation in mitotic cells
• •PNGase F and Endo H cleavage assays used to analyze protein glycosylation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

N-Glycosylation is a common protein modification catalyzed by a series of glycosylation enzymes in the endoplasmic reticulum and Golgi apparatus. Here, based on a previously established Golgi α-mannosidase-I-deficient cell line, we present a protocol to investigate the enzymatic activity of exogenously expressed Golgi α-mannosidase IA in interphase and mitotic cells. We describe steps for cell surface lectin staining and subsequent live cell imaging. We also detail PNGase F and Endo H cleavage assays to analyze protein glycosylation.

Before you begin

Overview

The Golgi apparatus is a central hub for protein trafficking, sorting, and post-translational modifications including glycosylation and phosphorylation.^2,3,4 It is generally believed that an ordered organization of glycosidases and glycosyltransferases in the subcompartments of the Golgi stack is required for sequential and accurate processing of N-glycans.³ Disruption of the Golgi structure impairs accurate glycosylation,^5,6 and glycosylation defects are often linked to Golgi structural disorganization in diseases.^7,8,9,10 But how the Golgi structure and its functions in post-translational modifications are coordinately regulated under physiological and pathological conditions remain largely unknown. For example, the Golgi undergoes severe fragmentation during cell division. The resulting trafficking block leads to an extended exposure of cargo molecules to Golgi enzymes.^4,11,12 This raises a question: how do cells avoid glycosylation defects during mitosis? Interestingly, we found that the Golgi enzyme MAN1A1 is highly phosphorylated at its cytoplasmic domain by CDK1 in mitosis, which inhibits MAN1A1 activity.¹ Previous studies demonstrated that CDK1 triggers mitotic Golgi fragmentation by phosphorylating and inhibiting several Golgi structure proteins, membrane tethers, and membrane fusion proteins such as GRASP65, GM130, and VCIP135.^12,13,14 These results together reveal an essential role for CDK1 in regulating both the structural organization and function of the Golgi apparatus in addition to cell cycle control.

This study contains a set of three short protocols to evaluate N-glycosylation in interphase and mitotic cells, which indicates the enzymatic activity of an exogenously expressed glycosylation enzyme (Figure 1).

• •For cell surface lectin staining of interphase cells, see steps 1–3 in “step-by-step method details”.
• •For live cell imaging of cell surface lectin staining at the onset of mitotic exit, see steps 4–6 in “step-by-step method details”.
• •For the Peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) cleavage assays of secreted glycoproteins (here we use a reporter glycoprotein His6-FLAG tagged lysosomal acid lipase sHF-LIPA, where sHF refers to an ER signal sequence with a His6-FLAG-tag), see steps 7–10 in “step-by-step method details”.

Figure 1.

Overall workflow of evaluation of N-glycosylation in interphase and mitotic cells

Individual or combination of these assays can be applied based on the research requirement.

Source of cell line

The wild type (WT) parental and MAN1 quadruple knockout CHO (QKO MAN1A1/1A2/1B1/1C1) cells were kindly gifted by Drs. Daniel N. Hebert (University of Massachusetts) and Henrik Clausen (University of Copenhagen, Denmark).¹⁵

Maintenance of CHO cell lines

• 1.
  Maintain WT and MAN1 QKO CHO cells in Gibco Minimum Essential Medium Alpha medium (MEM; ThermoFisher) supplemented with 10% Iron-supplemented bovine calf serum (Cytiva HyClone) and 1% Penicillin-Streptomycin (ThermoFisher) (complete medium).

  • a.
    Incubate cells at 37°C with 5% CO₂.

Note: All cells used should be tested negative for mycoplasma.

Poly-D-lysine coating of coverslip and glass-bottom dish

Timing: 80 min

• 2.Dissolve 5 mg poly-D-lysine in 100 mL autoclaved Milli-Q H₂O to prepare a 50 μg/mL solution.

Note: To improve coating efficiency, poly-D-lysine can be dissolved in 0.1 M borate buffer (1.24 g boric acid and 1.9 g sodium tetraborate in 400 mL water, pH 8.5) instead of Milli-Q H₂O.

• 3.For coverslips, place coverslips in a 10 cm petri dish without overlapping and add 10 mL 50 μg/mL poly-D-lysine solution. For a 2 cm glass-bottom dish, add 1.5 mL 50 μg/mL poly-D-lysine solution.
• 4.Rock for at least 1 h at room temperature or overnight in the cold room.
• 5.Remove poly-D-lysine solution and wash 3 times with autoclaved Milli-Q H₂O.

Note: Wash thoroughly to remove unbound poly-D-lysine and, if used, borate buffer residue. To accomplish this, discard the poly-D-lysine solution and add 10 mL of Milli-Q H₂O to the dish. Keep on a rocker for 10 min. Repeat the step for 3 times.

• 6.Allow to dry, which may take 30–60 min. Remove cover and expose to the hood UV light for 5–10 min to sterilize. Coated coverslips can be stored at room temperature for a few days before use.

Splitting and seeding cells

Timing: 15 min

• 7.Detach the CHO cells from a 10 cm culture dish with 1 mL 0.25% trypsin and 25 mM ethylenediaminetetraacetic acid (EDTA) in 1× Phosphate Buffered Saline (PBS) in a 37°C incubator for 2 min. Terminate the trypsinization with > 5 mL complete growth medium.
• 8.Resuspend the cells and transfer them to a 15 mL falcon centrifuge tube.
• 9.Spin at 200× g for 3 min to collect the cell pellet.
• 10.
  Split and seed the cells:

  • a.
    For splitting, split the cells at a 1:4 ratio and incubate cells in a new 10 cm plate with 10 mL fresh medium.
  • b.
    For seeding, refer to the table below.

Purpose of the experiment	Lectin stain imaging	Live cell imaging	PNGase F and Endo H cleavage assays
Plate/dish	6-well plate (per well) with Poly-D-lysine coated coverslip(s)	2 cm Poly-D-lysine coated glass-bottom dish	10 cm dish
Cells per well/dish	3 × 105	1.5 × 105	2 × 106

Transfection of cells

Timing: 25 min

• 11.
  Prepare the plasmid DNA-lipid complexes as shown on the following table using Lipofectamine™ 3000 following the manufacturer’s instructions with minor modifications.

  Purpose of the experiment	Lectin staining	Live cell imaging	PNGase F and Endo H cleavage assays
  Plate/dish	6-Well plate (per well)	2 cm Poly-D-lysine coated glass-bottom dish	10 cm dish
  Opti-MEM™ Medium	125 μL	100 μL	575 μL
  DNA per well	2 μg	1.6 μg	15 μg (10 μg sHF-LIPA+5 μg GFP or MAN1A1-GFP)
  P3000™ Reagent	3 μL	2 μL	20 μL
  Opti-MEM™ Medium	125 μL	100 μL	575 μL
  Lipofectamine™ 3000 Reagent	3 μL	2 μL	20 μL
  Total transfection mix:	258 μL	258 μL	1,540 μL
  Complete medium	2,000 μL	1,000 μL	10,000 μL

  • a.
    Prepare master mix of DNA by diluting DNA in OptiMEM™ Medium, then add P3000™ Reagent and mix well.
  • b.
    Dilute Lipofectamine™ 3000 Reagent in Opti-MEM™ Medium.
  • c.
    Prepare the transfection mix by mixing the two solutions and incubate at room temperature for 15 min.

    Note: The amount of Lipofectamine™ 3000 Reagent used here is lower than the manufacturer’s instruction to reduce cytotoxicity.

• 12.Add the transfection mix drop by drop to cells without disturbing the cells. Incubate cells in a CO₂ incubator for 6 h.
• 13.Discard the medium and replace it with fresh complete growth medium after 6 h incubation.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse monoclonal anti-Flag (clone M2, 1:2000)	Sigma-Aldrich	M1804
Mouse monoclonal anti-GFP (clone 1E10H7, 1:2000)	Proteintech	66002-1-Ig
Chemicals
Gibco Minimum Essential Medium Alpha	Invitrogen	12561056
Gibco Penicillin Streptomycin (10,000 U/mL)	Invitrogen	15140122
Iron-supplemented bovine calf serum	Hyclone	SH3007203
Gibco Opti-MEM I Reduced Serum Medium	Invitrogen	31985070
Lipofectamine 3000	Invitrogen	L3000015
Paraformaldehyde (PFA)	Thermo Fisher	AC41678-5000
Bovine serum albumin (BSA) Fraction V	Dot Scientific	DSA30075-100
Rhodamine labeled phaseolus vulgaris leucoagglutinin (PHA-L)	Vector Labs	RL-1112
Nocodazole	ThermoFisher	AC35824
Cycloheximide	AG Scientific	C1189
Gibco DMEM, high glucose, HEPES, no phenol red	Thermo Fisher	21063029
PNGase F	New England Biolabs	P0704S
Endo H	New England Biolabs	P0702S
30% acrylamide and bis-acrylamide solution, 37.5:1	Bio-Rad	1610159
N,N,N′,N′-Tetramethylethane-1,2-diamine (TEMED)	Thermo Fisher	PI17919
Ammonium persulfate (APS)	Thermo Fisher	BP179-100
Experimental models: Cell lines
MAN1 quadruple knockout CHO (QKO MAN1A1/1A2/1B1/1C1) cells	Lamriben et al.15	N/A
Wild type (WT) parental CHO cells	Lamriben et al.15	N/A
Recombinant DNA
pEGFP-N1-MAN1A1	This paper1	N/A
pEGFP-N1-MAN1A1 S12A	This paper1	N/A
pEGFP-N1-MAN1A1 S12E	This paper1	N/A
pHEK293Ultra-sHF-LIPA	Jin et al.16	N/A
Instruments
Nikon NIS-Elements C confocal microscope	Nikon	N/A
Software and algorithms
Nikon NIS-Elements analysis software	Nikon	N/A
Others
Sterile Filter System with PES membrane, 0.45 μm	CELLTREAT Scientific Products	229704
HisPurTM Ni-NTA Resin	Thermo Fisher	88222
Cytiva Amersham™ Protran™ Nitrocellulose Membranes 0.45 μm	Thermo Fisher	45-004-002

Materials and equipment

CHO culture medium
Reagent	Final concentration	Amount
Gibco Minimum Essential Medium Alpha	N/A	500 mL
Penicillin-Streptomycin	1% (v/v)	5 mL
Bovine calf serum	10% (v/v)	50 mL

Note: Store at 4°C for up to 4 weeks.

1× PBS, 500 mL
Reagent	Final concentration	Amount
NaCl	137 mM	4 g
KCl	2.7 mM	0.1 g
Na2HPO4	10 mM	0.71 g
KH2PO4	1.8 mM	0.12 g
Milli-Q H2O	N/A	Up to 500 mL

Note: Adjust pH by 1 M HCl or NaOH to 7.4. Store at room temperature.

4% PFA fixation buffer stock, 100 mL
Reagent	Final concentration	Amount
Paraformaldehyde	4% (w/v)	4 g
10 N NaOH	N/A	N/A
PBS (1×)	N/A	Up to 100 mL
Dilute to 1% before use
4% PFA stock	1% (w/v)	2.5 mL
PBS (1×)	N/A	7.5 mL

Note: Ideally PFA fixative should be prepared freshly before use. Alternatively, stock buffer can be prepared in advance and stored at 4°C for up to 4 weeks. To prepare 4% PFA stock, add 4 g PFA powder to 80 mL 1× PBS. Stir at 60°C with cover in a ventilation hood. Raise the pH of the mixture by adding NaOH drop by drop until a clear solution is formed. Cool the solution to room temperature and adjust the pH to 7.4 with HCl. Adjust the total volume to 100 mL with 1× PBS. Filter through a Sterile Filter System.

CRITICAL: PFA powder is flammable solid with acute oral and inhalation toxicity and is a potential carcinogen. It causes skin and respiratory irritation, serious eye damage and skin sensitization. PFA should not be handled until all safety precautions have been read and understood. Personal protective equipment should be worn, and a ventilation hood should be used when preparing PFA solutions.

• •20 mM NH₄Cl quenching buffer: dissolve 267.5 mg NH₄Cl in 80 mL 1× PBS. Adjust volume to 100 mL.
• •1% (w/v) PBSB blocking buffer: dissolve 1 g BSA in 100 mL 1× PBS. Stored at 4°C for up to 4 weeks.
• •2 μg/mL Rhodamine-PHA-L solution: add 1 μL of Rhodamine labeled PHA-L (2 mg/mL) into 999 μL of 1% PBSB.

His-binding buffer, 200 mL
Reagent	Final concentration	Amount
0.5 M Tris-HCl, pH 7.4	50 mM	20 mL
NaCl	150 mM	1.75 g
MgCl2	5 mM	0.2 g
1 M dithiothreitol (DTT) dissolved in Milli-Q H2O	2 mM	400 μL
imidazole	40 mM	0.54 g

Note: Store at 4°C. DTT is not stable in solution and should be added freshly before use.

• •His-elution buffer (200 mM imidazole in PBS): Add 1.36 g of imidazole to 80 mL 1× PBS. Adjust volume to 100 mL. Store at 4°C.

SDS-PAGE gel
Separating gel (10 mL)
Reagent	Final concentration	Amount
30% acrylamide and bis-acrylamide solution, 37.5:1	9%	3 mL
separating gel buffer (4X)	N/A	2.5 mL
N,N,N′,N′-tetramethylethane-1,2-diamine (TEMED)	0.07%	7 μL
Ammonium persulfate (APS) dissolved in Milli-Q H2O	1%	100 μL
Milli-Q H2O	N/A	4.5 mL

Stacking gel (5 mL)
Reagent	Final concentration	Amount
30% acrylamide and bis-acrylamide solution, 37.5:1	4%	0.65 mL
stacking gel buffer (4×)	N/A	1.25 mL
TEMED	0.07%	3.5 μL
APS	1%	50 μL
Milli-Q H2O	N/A	3.1 mL

Note: Ideally gels should be prepared freshly before use. Alternatively, the gels can be wrapped to keep moisture and stored at 4°C for no more than 1 week.

CRITICAL: TEMED releases irritating vapors which cause burns to eyes, skin and mucous membranes. Operate with personal protection equipment in an environment with good ventilation.

• •Separating gel buffer (4×): Add 18.17 g Tris-HCl and 0.4 g sodium dodecyl sulfate (SDS) to 80 mL Milli-Q H₂O. Adjust pH to 8.8 with HCl and adjust the volume to 100 mL. Store at room temperature.

CRITICAL: SDS is a detergent and a protein denaturant in the form of a light powder. It is toxic when in contact with skin, swallowed or inhaled, and causes irritation and damage to skin, eye, and respiratory system. SDS should be handled carefully only when all safety precautions have been read and understood. Personal protective equipment should be worn, and ventilation hood should be used.

• •Stacking gel buffer (4×): Add 6.06 g Tris-HCl and 0.4 g SDS to 80 mL Milli-Q H₂O. Adjust pH to 6.8 with HCl and adjust the volume to 100 mL. Store at room temperature.

6× loading buffer (10 mL)
Reagent	Final concentration	Amount
0.5 M Tris-HCl, pH 6.8	300 mM	6 mL
SDS	6% (w/v)	0.6 g
Bromophenol Blue	0.06% (w/v)	6 mg
glycerol	36% (v/v)	3.6 mL
Milli-Q H2O	N/A	Add up to 10 mL

CRITICAL: To accurately measure the amount of viscous pure glycerol, cut off the end of a pipette tip when pipetting and leave it in the solution while mixing. Ensure that the solution is thoroughly mixed before adding Bromophenol Blue to obtain a uniform solution.

Step-by-step method details

Part I. Cell surface lectin staining

Timing: 2 days (for steps 1 to 3)

Lectins are carbohydrate-binding proteins that can specifically interact with certain glycan branches of glycoproteins. Therefore, lectins are frequently used to identify specific post-translational modifications on glycoproteins,^6,17,18,19 which could thus be considered as glycosylation enzyme activity indicators.

Here, we introduce the lectin staining protocol using rhodamine-labeled PHA-L (rhodamine-PHA-L), which recognizes the tri- and tetra-antennary complex-type N-glycan. Researchers should choose the lectin(s) with desired binding affinity depending on their research purposes.²⁰

• 1.(Day 0) Seed WT and MAN1 QKO CHO cells on coverslips in 6-well plates as described in “Before you begin - splitting and seeding cells”. Incubate cells at 37°C and 5% CO₂ overnight (16–20 h).
• 2.(Day 1) Transfect CHO cells as described in “before you begin – Transfection of cells”.
• 3.
  (Day 2) Fixation and cell surface lectin staining.

  • a.
    Aspirate the medium.
  • b.
    Add 1.5 mL PBS per well to rinse the cells. Aspirate the PBS.
  • c.
    Add 1.5 mL of 1% (w/v) PFA fixation buffer per well at room temperature and incubate for 15 min.

    CRITICAL: Handle the PFA buffer in a ventilation hood.

  • d.
    Collect the PFA buffer with a pipette and dispose it in the aldehyde waste disposal.
  • e.
    Add 1.5 mL of PBS per well and wash thoroughly. Aspirate the PBS.
  • f.
    Add 1.5 mL of 50 mM NH₄Cl quenching buffer per well at room temperature and incubate for 10 min. Aspirate the quenching buffer.
  • g.
    Add 1.5 mL of PBS per well and wash thoroughly. Aspirate the PBS. Repeat this step for a total of three times.
  • h.
    Add 1.5 mL of PBSB blocking buffer per well at room temperature and incubate with gentle rocking for 30 min. Aspirate the blocking buffer.
  • i.
    Transfer coverslips with a fine-tip tweezer to a 24-well plate. Add 500 μL 2 μg/mL PHA-L in PBSB per well and incubate for 30 min with gentle rocking in a 4°C cold room.

    Note: Wrap the plate with aluminum foil to avoid light exposure during incubation.

  • j.
    Aspirate the PHA-L solution and transfer coverslips to a 6-well plate.
  • k.
    Add 1.5 mL of PBS per well and wash with gentle rocking for 5 min.
  • l.
    Repeat step 3k 3 times.

    Note: Extend or repeat washing if non-specific signal is detected.

  • m.
    Rinse the coverslips with Milli-Q H₂O.
  • n.
    Mount the coverslips on slides with Moviol.
  • o.
    Keep the slides in dark and allow the Moviol to dry completely. This normally takes overnight at 4°C.
  • p.
    Image with NIS-Elements C confocal microscope with a 60× oil lens and Z-stacks at 0.3 μm intervals.

    Pause point: Ideally, the staining and imaging process should be completed as soon as possible. Alternatively, mounted slides can be stored in dark at 4°C for 4 weeks or at −20°C for up to 3 months.

Part II. Live cell imaging of cell surface lectin stain at the onset of mitotic exit

Timing: 2 days (for steps 4 to 6)

In our study investigating mitotic regulation of N-glycosylation, we discovered that the enzymatic activity MAN1A1 undergoes cell cycle-dependent regulation.¹ However, because mitotic trafficking block prevents MAN1A1 substrates from being delivered to the cell surface, it is impossible to analyze the activity of MAN1A1 (and its phospho-deficient mutant) in mitosis by cell surface lectin staining of mitotic cells, as we did above in interphase cells.

Here, we introduce a live cell imaging protocol used to capture glycosylation changes at the cell surface upon the arrival of cargo proteins at the onset of mitotic exit when membrane trafficking resumes.¹

• 4.(Day 0) Seed MAN1 QKO CHO cells on glass-bottom dishes as described in “Before you begin - splitting and seeding cells”. Incubate cells at 37°C and 5% CO₂ overnight (16–20 h).
• 5.
  (Day 1) Transfection and nocodazole treatment to synchronize cells.

  • a.
    Transfect CHO cells as described in “before you begin – Transfection of cells”.
  • b.
    6 h after transfection, change medium to complete growth medium containing 150 ng/mL nocodazole.
  • c.
    Incubate cells at 37°C and 5% CO₂ for 18 h.

Note: Nocodazole treatment blocks cells to prometaphase. The concentration and time used for nocodazole blockage may vary depending on the cell line used. To determine the synchronization rate, a pilot experiment of Hoechst staining can be conducted to titrate the nocodazole treatment with the chosen cell line. The synchronized cells should be arrested at prometaphase. >90% of arrested cells should be observed in a successful nocodazole blockage.

• 6.
  (Day 2) Live cell imaging.

  • a.
    Change medium to imaging medium (DMEM, no phenol red, no fetal bovine serum added for short term imaging) containing 150 ng/mL nocodazole.
  • b.
    Place the glass-bottom dish in a live-cell imaging chamber at 37°C with 5% CO₂ coupled to a Nikon ECLIPSE Ti2 Confocal microscope.
  • c.
    Observe under a 60× oil objective to identify mitotic cells.
  • d.
    Save the locations of the mitotic cells.
  • e.
    Wash the cells extensively. In brief, cells are washed by adding excess (1 mL) imaging medium containing 100 μg/mL CHX to the empty space in the dish to avoid detaching cells and the medium is removed by a vacuum system. Repeat this step for three times, and incubate cells with imaging medium containing 100 μg/mL CHX and 2 μg/mL rhodamine-PHA-L.

    Note: Removing nocodazole thoroughly is critical for releasing cells from prometaphase. This step should be completed as fast as possible and try not to move the stage, and the live cell imaging program should be run immediately after double checking the saved positions and optimal z stack.

    CRITICAL: Be aware that mitotic cells can detach from the dish easily. Removal of medium should be practiced fast but cautiously.

  • f.
    Take images every 2 min with 5 stacks (0.8 μm/stack).
  • g.
    Process the generated videos with maximum intensity projection.

    Note: Time 0 is 5 min after nocodazole washout due to the time needed for the procedure and equipment setting.

Part III. PNGase F and Endo H cleavage of a secreted reporter glycoprotein

Timing: 4–5days (for steps 7 to 10)

PNGase F is an amidase which cleaves the innermost GlcNAc on asparagine residues with high mannose, hybrid, and complex oligosaccharides. Endo H is a glycosidase that cleaves N-linked mannose-rich oligosaccharides, but not complex oligosaccharides. Both enzymes are frequently used in protein glycosylation studies to deglycosylate glycoproteins.^21,22,23 Endo H is also commonly used to monitor intracellular protein trafficking through the secretory pathway.^24,25

Here, we introduce a protocol using the two enzymes to detect the glycosylation of a secreted reporter protein sHF-LIPA,¹ which indicates the enzymatic activity of the exogenously expressed MAN1A1 in a MAN1 QKO CHO cell line.

• 7.(Day 0) Seed WT and MAN1 QKO CHO cells as described in “before you begin - splitting and seeding cells”. Incubate cells at 37°C and 5% CO₂ overnight (16–20 h). In the following steps WT and MAN1 QKO CHO cells are analyzed in parallel.
• 8.(Day 1) Transfect CHO cells as described in “before you begin – Transfection of cells”.
• 9.(Day 2-4) Maintain the transfected cells.

Note: Check growing status and viability of the cells. Split on Day 2 if necessary. Depending on the proliferation rate of the cell line used, try to avoid medium change before collection day.

• 10.
  (Days 4–5) Collect conditioned media and perform PNGase F and Endo H cleavage assay.

  • a.
    Collect 10 mL conditioned medium to a 15 mL falcon centrifuge tube. Spin the medium at 1,000 g at 4°C for 3 min to remove dead cells.
  • b.
    Wash 200 μL of HisPurTM Ni-NTA resin with 1 mL His-binding buffer.

    CRITICAL: The Ni-NTA resin is supplied as 50% slurry in 20% ethanol. This washing step is required to remove ethanol extensively.

  • c.
    Collect the supernatant from 10a, mix with prewashed Ni-NTA resin and transfer to a new 15 mL tube. Incubate with gentle rotation at 4°C for 2 h.
  • d.
    Spin at 1000 g at 4°C for 3 min to collect the Ni-NTA resin.
  • e.
    Resuspend the resin with 1 mL His-binding buffer and transfer to 1.5 mL Eppendorf tube.
  • f.
    Wash the Ni-NTA resin with 1 mL His-binding buffer by gently inverting the tube 3–5 times. Spin at 1,000 g at 4°C for 3 min. Discard the supernatant carefully without disturbing the Ni-NTA resin pellet.

    Note: Use a swing-out rotor to pellet the resin at the bottom of the tube.

  • g.
    Repeat steps 10f 3 times.
  • h.
    Add 150 μL His-elution buffer and mix well with Ni-NTA resin. Spin at 1,000 g at 4°C for 10 min.
  • i.
    Collect 135 μL supernatant carefully without disturbing the resin. Add 15 μL of 10× glycoprotein denaturing buffer (New England Biolabs) and heat at 95°C for 5 min.
  • j.
    Prepare reaction solutions as follows:

    Untreated
    Denatured sample	45 μL
    10× GlycoBuffer	6 μL
    Add Milli-Q H2O to total:	60 μL

    Endo H treatment
    Denatured sample	45 μL
    10× GlycoBuffer 3	6 μL
    Endo H	0.5 μL (250 U)
    Add Milli-Q H2O to total:	60 μL

    PNGase F treatment
    Denatured sample	45 μL
    10× GlycoBuffer 2	6 μL
    10% NP-40	6 μL
    PNGase F	0.5 μL (250 U)
    Add Milli-Q H2O to total:	60 μL

    Incubate at 37°C on a shaker for 1 h.
  • k.
    Terminate reactions by adding 12 μL 6× SDS loading buffer and heating at 95°C for 3 min.
  • l.
    Run 5–10 μL of each sample on a 9% SDS-PAGE and transfer proteins to 0.45 μm nitrocellulose membranes. Probe sHF-LIPA with a mouse monoclonal anti-FLAG antibody.

Expected outcomes

Here, we outline the expected outcomes of the three protocols used to determine the enzymatic activity of the exogenously expressed MAN1A1 in a MAN1 QKO CHO cell line.

Part I. Cell surface lectin staining and imaging

Without permeabilization of the cell, PHA-L specifically binds to glycoproteins at the cell surface where rhodamine signals are expected to be detected exclusively. N-glycosylation is a sequential post-translational process; therefore, inhibition of upstream enzyme leads to a reduction of downstream product and accumulation of the substrate. Depletion of all four α-mannosidase I isoforms in CHO cells (i.e., the MAN1 QKO CHO cell line) abolishes the formation of the complex form of glycans, indicated by undetectable cell surface rhodamine signal (Figure 2). In our study investigating the enzymatic activity of MAN1A1, expression of WT MAN1A1 in MAN1 QKO CHO cells increased the PHA-L signal at the cell surface PHA-L, indicating that it is functionally active (Figure 3).

Figure 2.

Cell surface rhodamine-PHA-L staining of WT and MAN1 QKO CHO cells

Note that there is no rhodamine signal detected in the MAN1 QKO cells. Scale bar: 20 μm.

Figure 3.

Cell surface rhodamine-PHA-L staining of MAN1 QKO CHO expressing GFP or GFP-tagged WT MAN1A1

Note the recovery of cell surface rhodamine signal detected in the MAN1 QKO cells by expressing MAN1A1-GFP. Scale bar: 20 μm.

Part II. Live cell imaging of cell surface lectin stain at the onset of mitotic exit

Our study shows that the enzymatic activity of MAN1A1 is attenuated due to mitotic phosphorylation at serine-12 (S12).¹ Therefore, expression of a phospho-deficient mutant MAN1A1 S12A would lead to aberrant accumulation of complex-glycans in mitosis, indicated by a stronger cell surface signal of rhodamine-PHA-L compared to WT MAN1A1-expressing cells. However, it is impossible to measure the MAN1A1 activity by cell surface lectin staining of mitotic cells because MAN1A1 substrates in the Golgi cannot be delivered to the cell surface due to the mitotic trafficking block. To overcome this difficulty, we performed live cell imaging to capture glycosylation changes at the cell surface upon the arrival of cargo proteins at the onset of mitotic exit when membrane trafficking resumes. Our results show that the S12A mutant remains highly active in mitosis (Figure 4).

Figure 4.

The phosphorylation-deficient mutant MAN1A1 S12A remains active in mitosis

Shown are still images of chosen time points from live cell imaging (Methods videos S1, S2, S3, and S4). Note that the PHA-L signal in cells expressing MAN1A1 S12A appears earlier and stronger than that in WT MAN1A1-expressing cells, whereas this signal in S12E-expressing cells is comparatively lower. Scale bar: 20 μm. Also see Huang et al.¹Figure 6 for another set of representative images.

Part III. PNGase and Endo H cleavage of a secreted reporter glycoprotein

A simple method to assess the extent of glycosylation or deglycosylation is by mobility shifts of a reporter glycoprotein on SDS-PAGE gels. For sHF-LIPA, the deglycosylated form by PNGase F treatment is shifted down from the glycosylated form (Figure 5, lanes 2 and 5 vs. lanes 1 and 4, respectively). On the other hand, sHF-LIPA produced in WT CHO cells exhibits as an Endo H-resistant form (lane 3), indicating the formation of complex-type of N-glycans. When sHF-LIPA is secreted by MAN1 QKO cells, it displays as an Endo H-sensitive high-mannose form (Figure 5, lane 6 vs. 4) due to the lack of Golgi mannosidase I activity. Expression of WT and MAN1A1 S12A rescues sHF-LIPA glycosylation defects in MAN1 QKO cells, indicated by the resistance to Endo H treatment of sHF-LIPA (Figure 6, lane 4 and 6). However, expression of S12E MAN1A1 partially rescues sHF-LIPA glycosylation defects in MAN1 QKO cells, indicated by the production of a mixture of Endo H-resistant and sensitive forms of sHF-LIPA (Figure 6, lane 8).

Figure 5.

PNGase F and Endo H cleavage of secreted sHF-LIPA

MAN1 QKO cells are defective in producing complex-type glycans, indicated by the sensitivity of secreted sHF-LIPA to Endo H treatment. Adapted from Huang et al.¹Figure 5H.

Figure 6.

Expression of WT and MAN1A1 S12A rescues sHF-LIPA glycosylation defects in MAN1 QKO cells, indicated by the resistance to Endo H treatment of sHF-LIPA

Expression of S12E MAN1A1 partially rescues sHF-LIPA glycosylation defects in MAN1 QKO cells, indicated by the production of a mixture of Endo H-resistant and sensitive forms of sHF-LIPA.

Limitations

One limitation of using lectin staining in glycosylation studies is the choice of lectins to be used. It may not be an issue if the defective step in the N-glycosylation pathway is clear, as the study introduced in Part I. However, lectin staining could not be used individually to determine the defective step in the N-glycosylation pathway. Since the N-glycosylation is a series of sequential processes, defects in the upstream steps will cause failure of further process of the glycoproteins. For example, MAN1 QKO CHO cells are defective in producing complex-type glycans which are indicated by abolished PHA-L staining; however, negative PHA-L could indicate defects in any steps in the N-glycosylation pathway prior to the formation of complex-type oligosaccharides. Therefore, a combination of lectins recognizing different glycan branches may be necessary. Besides, lectin staining is quick, but is not as specific as antibodies and the result is also less linear.

One limitation of using Endo H in glycosylation studies is related to its cleavage specificity. Endo H is able to cleave the oligosaccharide on a glycoprotein trafficking through the ER to the Golgi before it is processed by the Golgi alpha-mannosidase II. Since all earlier oligosaccharide structures show Endo H sensitivity while later ones show resistance, the enzyme is widely used as a “landmark” in protein glycosylation and trafficking studies. However, Endo H cleavage assay could not provide more detailed information about which exact trafficking step the cargo protein is at when analyzed.

Troubleshooting

Problem 1

Low cell viability after transfection.

Potential solution

• 1.Increase the cell density before transfection. Confluency lower than 40% may lead to lower transfection efficiency and reduced cell viability afterward.
• 2.Reduce the amount of transfection reagent used. MAN1 QKO CHO cells can be sensitive to the toxicity of transfection reagents. Always change to complete growth medium 6 h after transfection.

Problem 2

Intracellular lectin staining signal is detected.

Potential solution

• 1.Permeabilization may be caused by the high concentration of PFA. Confirm the concentration of PFA used in the fixing buffer. Do not use PFA solution higher than 1%.
• 2.Alternatively, the fixation step can be done after the staining is completed. In this case, remove medium, rinse and incubate the cells with precooled 1× PBS for 15 min. Complete steps 3h-l on ice or in a cold room. Then complete steps 3c-g at room temperature. Rinse the coverslips with Milli-Q H₂O and mount them on slides.

Problem 3

The cells at the save locations disappear after nocodazole washout for live cell imaging.

Potential solution

• 1.Make sure the glass-bottom dishes are coated with poly-D-lysine before seeding the cells.
• 2.During wash, try to be as gentle as possible, avoid adding the medium onto the saved locations, and use the vacuum system to aspirate the medium. Never let the dish dry during all steps.

Problem 4

Mobility shift was not detected in the glycosylation assay using PNGase F and/or Endo H.

Potential solution

• 1.Confirm NP-40 is added into the reaction system when using PNGase F. PNGase is inhibited by the SDS in denaturing buffer, therefore it is essential to have NP-40 in the reaction mixture under denaturing conditions. Failure to include NP-40 in the denaturing protocol will result in loss of enzymatic activity.
• 2.The PNGase F and Endo H used in the introduced protocol was purchased from New England BioLabs with the commitment that “Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it.” According to New England BioLabs, one unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 10 μg of denatured RNase B in 1 h at 37°C in a total reaction volume of 10 μL. Increasing the amount of enzyme and prolonging the incubation should effectively increase the cleavage efficiency.
• 3.If there is a concern that enzymes are inactivated, following assay can be performed to confirm the activity of the enzymes as instructed by the manufacturer:

For the PNGase F activity: 10 μg of RNase B is denatured with 1X Glycoprotein Denaturing Buffer (0.5% SDS, 40 mM DTT) at 100°C for 10 min. After the addition of NP-40 and GlycoBuffer 2, a two-fold dilution of PNGase F is added and the reaction mix is incubated for 1 h at 37°C. Separation of reaction products are visualized by SDS-PAGE.

For the Endo H activity: 10 μg of RNase B is denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10 min. After the addition of 1X GlycoBuffer 3, a two-fold dilution of Endo H is added, and the reaction mix is incubated for 1 h at 37°C. Separation of reaction products is visualized by SDS-PAGE.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Yanzhuang Wang (yzwang@umich.edu).

Materials availability

The pEGFP-N1-MAN1A1 constructs are available upon request.

Data and code availability

This study did not generate any unique datasets or codes.