Methods for Analysis of Unusual Forms of O-Glycosylation

  • Aleksandra Nita-Lazar
  • Robert S. Haltiwanger
Part of the Methods in Molecular Biology book series (MIMB, volume 347)

Abstract

The identification of the novel forms of O-linked glycosylation, O-fucose, and O-glucose requires the development of new methods for their analysis. Here we describe approaches to analyze these novel O-glycans. The major method involves metabolic radiolabeling of recombinant glycoproteins expressed in Lec1 Chinese hamster ovary (CHO) cells. The glycoproteins are purified from the media and the stoichiometry of modification is determined by comparing protein levels (by immunoblot) and incorporated radioactivity (by fluorography). The O-glycans are subsequently released by alkali-induced β-elimination, and released saccharides are analyzed using a combination of chromatography and exoglycosidase digestion. With these methods, we can determine both stoichiometry and the structure of the glycans on the expressed proteins. We have begun to utilize mass spectrometry in addition to metabolic radiolabeling methods to analyze these structures.

1 Introduction

The novel forms of protein O-glycosylation, O-fucose, and O-glucose are notably different from classical O-glycans. Unlike mucin-type O-glycans, these have relatively short and unbranched structures. Both O-fucose and O-glucose can exist as either mono- or oligosaccharide (OS) species. O-fucose can be elongated to two types of OSs, suggesting that two separate O-fucose pathways exist (1). One of the O-fucose OSs is the tetrasaccharide Sia-α2,3/6-Gal-β1,4-GlcNAc-β1,3-Fuc-α1-O-Ser/Thr. To date this structure has been found exclusively on proteins containing epidermal growth factor (EGF)-like repeats (2,3). The second O-fucose OS is the disaccharide Glc-β1,3-Fuc-α1-O-Ser/Thr, which was found on proteins containing thrombospondin type 1 repeats (TSRs; seerefs.4, 5, and 5a). O-glucose, detected so far only on proteins containing EGF repeats, can be elongated to the trisaccharide Xyl-α1,3-Xyl-α1,3-Glc-β1-O-Ser (6).

The O-fucose and O-glucose modifications on EGF repeats have generated a great deal of excitement recently owing to the importance of O-fucose in Notch signaling (7, 8, 9). EGF repeats are small protein domains characterized by six conserved cysteine residues forming three disulfide bonds that provide the distinct fold (10). They are found in many cell-surface and secreted proteins, and many EGF repeats contain the consensus sites for O-fucosylation (C2X4–5S/TC3; seerefs.11 and 12) and/or O-glucosylation (C1XSXPC2; seeref.13). The extracellular domain of Notch1 contains 36 tandem EGF repeats (14) with multiple O-fucosylation and O-glucosylation consensus sites (15). A number of studies have demonstrated that the enzyme responsible for the addition of O-fucose to EGF repeats, protein O-fucosyltransferase 1 (O-FucT-1; seerefs.16 and 17), is essential for proper Notch function in both Drosophila melanogaster (18,19) and mice (20). O-FucT-1 knockout mice show an embryonic lethal phenotype characteristic of a Notch signaling defect (20). Recent work has shown that O-FucT-1 localizes to the endoplasmic reticulum and may play a role in folding and quality control during Notch receptor biosynthesis (21,22). Fringe, a known modulator of Notch function (23, 24, 25), is an O-fucose-specific β1,3-N-acetylglucosaminyltransferase, catalyzing the committed step in O-fucose tetrasaccharide biosynthesis (26,27). These and other studies have demonstrated the importance of O-fucose saccharides in the Notch signaling pathway (for reviews seeref.7).

Like EGF repeats, TSRs are small cysteine-knot motifs containing six conserved cysteine residues forming three disulfide bonds (although in a different pattern than EGF repeats). TSRs are also characterized by conserved Trp, Ser, and Arg residues (28). They are found in a number of cell-surface and secreted proteins and appear to play important roles in protein-protein interactions. They can be modified with the O-fucose disaccharide Glc-β1,3-Fuc on the sequence C1XX(S/T)C2G (seerefs.4 and 5a). The O-fucosylation site in thrombospondin-1 is present at a putative CD-36 binding site, suggesting a possible role for the glycan in regulation of this interaction (29, 30, 31). Recent studies in our laboratory have demonstrated that a novel enzyme, O-FucT-2, is responsible for the addition of O-fucose to TSRs (31a).

We have developed specific methods to analyze both O-fucose and O-glucose glycans. These methods have been adapted from more traditional methods of O-glycan analysis (32, 33, 34, 35), although several modifications have been used to enhance radiolabeling of the desired saccharides. For instance, we use Chinese hamster ovary (CHO) cells with mutations in more common glycosylation pathways developed by Dr. Pamela Stanley (Albert Einstein College of Medicine) to minimize incorporation of radiolabel into unwanted structures. For metabolic radiolabelings of O-fucose saccharides, we take advantage of Lec1 cells (36,37). These cells lack GlcNAc transferase 1 and cannot make complex or hybrid-type N-glycans. The majority of [3H]-fucose is incorporated into N-glycan structures in CHO cells (38); the use of Lec1 cells eliminates this “background,” allowing simple analysis of minor structures such as O-fucose saccharides (1). To enhance radiolabeling of O-glucose saccharides we use [3H]-galactose as the precursor. Galactose is rapidly converted to uridine 5′-diphosphate (UDP)-galactose upon entering the cell, which is subsequently epimerized to UDP-glucose (39). Thus the UDP-glucose pool in the cell becomes radiolabeled without incorporating label into all of the products of glucose metabolism. Lec1 cells can be used for the same reasons described above for the O-fucose radiolabelings, although [3H]-galactose can also be incorporated into mucin-type O-glycans in Lec1 cells (40). Alternatively we have used Lec8 cells, which have a defect in the Golgi UDP-galactose transporter (41). Because the UDP-galactose is inefficiently transported into the Golgi in Lec8 cells, the incorporation of [3H]-galactose is dramatically reduced. We have successfully used both Lec8 and Lec1 cells to study O-glucose modifications on EGF repeats (seeref.15 and Nita-Lazar and Haltiwanger, in preparation). Radiolabeling experiments can be performed in other cell lines (e.g., HeLa, Cos-1, 293T), although the high level of radiolabel incorporated into N-glycans can obscure the signal from these more minor O-glycans. This background can be significantly reduced by removal of N-glycans with peptide N-glycosidase F. Nonetheless, these extra steps reduce yields and complicate the analysis (42).

Either endogenous or recombinant glycoproteins produced in CHO cells can be metabolically labeled with radioactive monosaccharides (1,15). We typically transfect (either stably or transiently) plasmids encoding proteins of interest into the appropriate CHO cell line to produce sufficient material for analysis. The constructs contain amino terminal signal sequences to ensure secretion and C-terminal tags (e.g., MYC and His6) for detection and purification purposes (26,43). The protein is then purified from cell lysates or the media using appropriate antibodies (e.g., against the protein itself or against a tag on a recombinant protein) or Nickel (Ni)-Sepharose if the protein has an His6-tag. At this point, radiochemical purity needs to be established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or fluorography. A simple β-elimination followed by acid hydrolysis can be used to compare the extent of the radioactivity directly bound to the protein (yielding an alditol) vs that linked to other sugars (yielding an aldose; seerefs.1 and 15). If some of the radiolabel is not in the form of the alditol, the percentage of alditol can be calculated and used to determine what percentage of total radioactivity was directly linked to the protein (as either O-fucose or O-glucose). The extent of glycosylation can then be determined by comparing protein levels (using immunoblots) to the level of radioactivity (using fluorography, normalized with percent alditol if necessary [43]). As this is only a relative extent of glycosylation, it can only be used to compare the extent of glycosylation within a specific experiment (e.g., comparison of wild-type and glycosylation site mutants [43]).

The structures of the radioactive O-glycans can then be examined after release from the protein by alkali-induced β-elimination. The released OSs are analyzed by size-exclusion chromatography (Superdex) in combination with glycosidase digestions to determine the size and sequence of the OS chain (15). Linkages between sugars can be confirmed using high-performance anion-exchange chromatography (HPAEC) analysis (Dionex) in combination with standard compounds. We have used this approach successfully to determine the structure of O-fucose and O-glucose glycans in EGF repeats (15) and O-fucose in TSRs (31a). A flowchart describing these steps is shown in Fig. 1. Recently we have begun to utilize mass spectrometry (MS) to analyze the structures of O-fucose and O-glucose glycans. Although MS methodologies are extremely sensitive and require no radioactivity, they cannot be used on saccharides of unknown composition or structure. Many excellent reviews describing the use of MS to analyze OS structure have been published in this series and in this book (seerefs.44 and 45 and Chapters 6 and 8).
Fig. 1.

Flow chart of the methods used in structural analysis of O-linked glycans.

2 Materials

2.1 Reagents and Tools

  1. 1.

    Radioactive (3H) sugars (L-[6-3H]fucose and D-[6-3H]galactose; American Radiolabeled Chemicals, St. Louis, MO).

     
  2. 2.

    Lec1 and Lec8 mutant CHO cells (American Type Culture Collection, Manassas, VA).

     
  3. 3.

    Alpha-minimal essential medium (α-MEM) cell culture media (Invitrogen, Carlsbad, CA) with 10% bovine calf serum (BCS) and 10 mM of penicillin/streptomycin.

     
  4. 4.

    OptiMem I cell culture media (Invitrogen, Carlsbad, CA).

     
  5. 5.

    Dowex-50, H+-form (Bio-Rad, Hercules, CA).

     
  6. 6.

    Acetone (cold, kept at −20°C).

     
  7. 7.

    1-mL and 10-mL disposable syringes and 18-gage needles.

     
  8. 8.

    Disposable columns (Bio-Rad).

     
  9. 9.

    Sep-Pak C18 (Waters, Milford, MA).

     
  10. 10.

    100% Methanol.

     
  11. 11.

    MilliQ water (or water of similar purity).

     
  12. 12.

    Superdex™peptide gel filtration column (Pharmacia Biotech/GE Healthcare, Piscataway, NJ) connected to the high performance liquid chromatography (HPLC) capable of 0.5 mL/min flow, with refractive index detector for standardization.

     
  13. 13.

    Dionex DX300 HPLC system equipped with pulsed amperometric detection (PAD-2 cell) and CarboPac MA-1 column (Dionex Corp., Sunnyvale, CA).

     
  14. 14.

    Nickel-nitrilotriacetic acid (Ni-NTA) Sepharose beads (Qiagen, Valencia, CA).

     
  15. 15.

    Nitrocellulose membrane (Bio-Rad).

     
  16. 16.

    Appropriate antibodies for Western blot protein detection.

     
  17. 17.

    En3Hance autoradiography enhancer (Perkin Elmer, Boston, MA). Caution: Corrosive!

     
  18. 18.

    Exoglycosidases: N-acetylneuraminidase I (α2,3-specific sialidase) from Glyko, Inc. (San Leandro, CA); β-galactosidase (Diplococcus pneumoniae) from Roche Molecular Biochemicals (Indianapolis, IN); β-hexosaminidase (jack bean) from Sigma.

     
  19. 19.

    Standards: dextran D-4133 (Sigma-Aldrich, St. Louis, MO) hydrolyzed according to ref.46, monosaccharides (Sigma), alditol sugar standards prepared by reduction of the corresponding sugar as in ref.47, and disaccharide standards synthesized as described in ref.15.

     

2.2 Stock Solutions

  1. 1.

    Radioimmunoprecipitation assay (RIPA) buffer: 50 mM of Tris-HCl (pH 8.0), 150 mM of NaCl, 1% NP40, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate (SDS).

     
  2. 2.

    0.1 M of ethylenediaminetetraacetic acid (EDTA), pH 8.0.

     
  3. 3.

    2 M of NaBH4 and 100 mM of NaOH (β-elimination solution) made fresh.

     
  4. 4.

    4 M of acetic acid.

     
  5. 5.

    50% Slurry of Dowex-50 in 20% methanol.

     
  6. 6.

    2 M of trifluoroacetic acid (TFA).

     
  7. 7.

    1 M of NaCl.

     

3 Methods (see Note 1)

3.1 Metabolic Labeling of Cells in Culture

  1. 1.

    Seed one plate (100-mm diameter) with 106 of cells stably transfected with the glycoprotein of interest, or transfect the cells transiently before radiolabeling.

     
  2. 2.

    Grow the cells to approx 50% confluency.

     
  3. 3.

    Change the medium to 5 mL of α-MEM (seeNote 2) containing 20 µCi/mL of [3H]fucose or [3H]galactose.

     
  4. 4.

    Incubate the cells for 48 h, collect the media, and purify the radioactively labeled recombinant glycoprotein (seeNote 3).

     

3.2 Protein Purification (see Note 4)

  1. 1.

    Centrifuge the harvested medium to pellet the cell debris.

     
  2. 2.

    Add 100 µL of Ni-NTA beads (50% slurry, washed thoroughly with RIPA) per 5 mL of media.

     
  3. 3.

    Incubate by rotating for 1 h at 4°C.

     
  4. 4.

    Centrifuge at 5000g and discard the supernatant.

     
  5. 5.

    Wash three times with 1 mL of RIPA buffer. Remove the supernatant with an 18-gage needle using a 1-mL syringe each time.

     
  6. 6.

    Elute with 250 µL of 100 mM EDTA.

     
  7. 7.

    Centrifuge and collect the supernatant.

     
  8. 8.

    Count 5 µL in the liquid scintillation counter for quantification purposes.

     
  9. 9.
    Analyze a portion of the sample by SDS-PAGE-fluorography to check the radiochemical purity:
    1. a.

      Load an equal amount of radioactivity (at least 2000 cpm) in each sample onto the 10% polyacrylamide gel.

       
    2. b.

      Perform fluorography using En3Hance (seeNote 5) according to the manufacturer’s instructions.

       
     
  10. 10.

    Perform analysis of the portion of radiolabel directly linked to protein as O-glycan vs other glycans (seeNote 6).

     

3.3 Analysis of Relative Extent of Glycosylation (Stoichiometry) by Western Blot and Fluorography

  1. 1.

    Load an equal amount of radioactivity (at least 2000 cpm) onto two SDS-polyacrylamide gels of appropriate pore size for the protein being analyzed.

     
  2. 2.

    With the first gel, perform Western blot and probe with appropriate antibodies.

     
  3. 3.

    With the second gel, perform fluorography using En3Hance (seeNote 5) according to the manufacturer’s instructions.

     
  4. 4.

    Scan both films (making sure film is in the linear range) and determine a ratio of radioactivity (from fluorography) to protein (from Western blot). Normalize the scan from fluorography using the percentage of alditol (seeNote 6) as 100%.

     

3.4 Release of O-Glycans by Alkali-Induced β-Elimination

  1. 1.

    Precipitate purified protein (∼10,000–25,000 cpm) with acetone using 4 vol of chilled acetone for 1 vol of the protein solution and incubating at least 2 h at −20°C.

     
  2. 2.

    Pellet the precipitate by centrifugation at maximum microcentrifuge speed at 4°C, remove the supernatant with the 18-gage needle, and air-dry the pellet.

     
  3. 3.

    Add 500 µL of β-elimination solution to the acetone-precipitated protein pellet (seeNote 7).

     
  4. 4.

    Incubate at 55–65°C for 18–24 h.

     
  5. 5.

    Cool and neutralize by slowly adding 4 M of acetic acid (dropwise) on ice until the pH is below 6.0 (seeNote 8).

     
  6. 6.

    Desalt by passing the sample through 3 mL of Dowex-50 (H+) resin (50% slurry) in a disposable Bio-Rad column with an 18-gage needle attached (Dowex-50 should be washed with 15 mL of water before loading the sample). Collect the flow-through in a 50-mL conical tube.

     
  7. 7.

    Wash with 10 mL of water. Add to the flow-through.

     
  8. 8.

    Pass the sample through the Sep-Pak C18 column (Sep-Pak must be preconditioned with 10 mL of 100% methanol and then with 10 mL of water) using a 10-mL syringe. Collect the flow-through containing OSs in a 50-mL conical tube.

     
  9. 9.

    Freeze and lyophilize the flow-through.

     
  10. 10.

    Remove residual borate by performing methanol dry-down. Add sufficient 100% methanol to the sample to resuspend, mix well, and evaporate in the vacuum centrifuge on the medium heat setting (about 60°C). Repeat twice with 0.5 mL of methanol or until white powder no longer diminishes.

     
  11. 11.

    Dissolve the sample in 100 µL of water and determine the radioactivity recovered (CPM concentration) using 5 µL of the sample.

     

3.5 Analysis of OS Structure

3.5.1 Size-Exclusion Chromatography (seeNote 9)

This is performed as described in ref.1.
  1. 1.

    HPLC-Superdex run conditions: run the sample (about 10,000 cpm) through the Superdex column and flow 0.5 mL/min for 45 min (seeNote 10).

     
  2. 2.

    Collect 90 fractions (0.5 min/fraction; each fraction is 250 µL).

     
  3. 3.

    Analyze the radioactivity in aliquots (size depends on total amount of radioactivity) using a liquid scintillation counter.

     

3.5.2 Exoglycosidase Digestions

  1. 1.

    Pool radioactive chromatographic peaks from size fractionation on Superdex column.

     
  2. 2.

    Dry the pooled fraction in the vacuum centrifuge.

     
  3. 3.

    Resuspend in 50 µL of sodium acetate buffer, pH 5.5.

     
  4. 4.

    Add appropriate exoglycosidases: e.g., N-acetyl neuraminidase I (α2,3 specific sialidase; 10 mU), β1,4-galactosidase (5 mU), or β-hexosaminidase (250 mU). Include mock-digested controls (without enzyme).

     
  5. 5.

    Incubate for 18 h at 37°C.

     
  6. 6.

    Terminate the reaction by diluting the samples with 50 µL of water and heating for 5 min at 100°C.

     
  7. 7.

    Subject the samples to chromatography on the Superdex column (seeSubheading 3.5.1.).

     

3.5.3 Charge Analysis of OSs

This is performed as described in ref.38.
  1. 1.

    Dry the OS fraction digested or mock digested with sialidase (seeSubheading 3.5.2.) in the vacuum centrifuge.

     
  2. 2.

    Resuspend the sample in 500 µL of 2 mM Tris-base.

     
  3. 3.

    Pass through a 330-µL QAE-Sephadex column equilibrated with 2 mM of Tris-base.

     
  4. 4.

    Collect the flow-through (unbound fraction, 0 mM of NaCl representing neutral species).

     
  5. 5.

    Elute with increasing amounts of NaCl, releasing species with negative charges: 20 mM of NaCl, 1 negative charge; 70 mM of NaCl, 2 negative charges; 100 mM of NaCl, 3 negative charges; 140 mM of NaCl, 4 negative charges; and 1 M of NaCl, 5 or more negative charges. Collect 0.5-mL fractions.

     
  6. 6.

    Monitor the fractions by scintillation counting.

     

3.5.4 Acid Hydrolysis

  1. 1.

    Resuspend lyophilized OSs in 2 M of TFA.

     
  2. 2.

    Heat at 100°C for 2 h.

     
  3. 3.

    Dry in a Speed Vac evaporator.

     
  4. 4.

    Resuspend in water for HPAEC.

     

3.5.5 High-Performance Anion-Exchange Chromatography

  1. 1.

    Mix radioactive samples after acid hydrolysis with appropriate standards: 1 nmol for each fucitol, fucose, and glucose for [3H]fucose-labeled sample, or 1 nmol for each galactosaminitol, glucose, glucitol, galactose, and galactitol for [3H]galactose-labeled sample.

     
  2. 2.

    Subject samples to chromatography on CarboPac MA-1 column at 0.4 mL/min using the following gradients: 0–11 min, 0.1 M of NaOH; 11–21 min, 0.1–0.7 M of NaOH; 21–40 min, 0.7 M of NaOH.

     
  3. 3.

    Follow the standards by PAD-2 cell.

     
  4. 4.

    Collect 0.5-min (0.2-mL) fractions.

     
  5. 5.

    Monitor the fractions for radioactivity by scintillation counting.

     

4 Notes

  1. 1.

    Caution: Remember to work in a space assigned for radioactivity, wear appropriate protection (gloves, lab coat), and properly dispose of radioactive waste.

     
  2. 2.

    Opti-MEM (Invitrogen) serum-free can be used to avoid contamination with serum proteins, but it can reduce the incorporation of radioactivity.

     
  3. 3.

    Cell-associated proteins can be purified from the cell lysates using the same method (15).

     
  4. 4.

    We use cells expressing His6-tagged recombinant mouse Notch1 fragments (EGF 1–5, 6–10, 11–15, 16–18, 19–23, 24–28, and 29–36) and purify them using Ni-NTA affinity chromatography.

     
  5. 5.

    Caution: This reagent is very corrosive. Wear gloves and execute caution.

     
  6. 6.

    To check whether the radiolabel (fucose or glucose) is directly attached to the protein in O-linkage (e.g., O-fucose or O-glucose) or attached through other sugars, perform acid hydrolysis (seeSubheading 3.4.) on a portion of OS immediately after alkali-induced β-elimination (seeSubheading 3.4.), omitting the desalting and methanol blow-down steps; perform HPAEC (seeSubheading 3.4.) in the presence of fucitol or fucose (for glycans labeled with [3H]fucose, radioactivity migrating together with fucitol indicates O-fucose) and glucitol, glucose, galactitol, and galactose (for glycans labeled with [3H]galactose, radioactivity migrating together with glucitol indicates O-glucose). Seeref.1 for more details.

     
  7. 7.

    Physically break up the protein pellet to solubilize.

     
  8. 8.

    Neutralization is usually complete after approx 10 drops. Add one drop every 5 min and keep the tube lid open to release hydrogen. Vortex before measuring pH with broad-range pH paper.

     
  9. 9.

    Size markers are generated by mild acid hydrolysis of dextran, according to Kobata (46).

     
  10. 10.

    To be certain that the Superdex column does not become contaminated, inject 100 µL of 1 M NaCl, collect the fractions, and monitor radioactivity by scintillation counting. If radioactivity is released by this procedure, the column must be washed with 1 M of NaOH for 4–5 h and then re-equilibrated with water.

     

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Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Aleksandra Nita-Lazar
    • 1
  • Robert S. Haltiwanger
    • 1
  1. 1.Department of Biochemistry and Cell BiologyState University of New York at Stony BrookStony Brook

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