Iozzo Renato V. Proteoglycan Protocols

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36. Hamai, A., Hashimoto, N., Mochizuki, H., Kato, F., Makiguchi, Y., Horie, K., and Suzuki,

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38. West, L. A., Roughley, P., Nelson, F. R., and Plaas, A. H. (1999) Sulfation heterogeneity in

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Glycan Sequencing of HS and Heparin Saccharides 129

129

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Integral Glycan Sequencing

of Heparan Sulfate and Heparin Saccharides

Jeremy E. Turnbull

1. Introduction

The functions of heparan sulfate (HS) are determined by specific saccharide motifs

within HS chains. These sequences confer selective protein-binding properties and

the ability to modulate protein bioactivities (1,2). HS chains consist of an alternating

disaccharide repeat of glucosamine (GlcN; N-acetylated or N-sulfated) and uronic

acid (glucuronic [GlcA] or iduronic acid [IdoA]). The initial biosynthetic product

containing N-acetylglucosamine (GlcNAc) and GlcA is modified by N-sulfation of

the GlcN, ester (O)-sulfation (at positions 3 and 6 on the GlcN and at position 2 on the

uronic acids) and by epimerization of GlcA to IdoA. The extent of these modifications

is incomplete, and their degree and distribution varies in HS between different cell

types. In HS chains, N- and O-sulfated sugars are predominantly clustered in

sequences of up to 8 disaccharides separated by N-acetyl-rich regions with a low

sulfate content (3).

Sequence analysis of HS saccharides has presented a daunting analytical problem

and until very recently sequence information had been obtained for only relatively

short saccharides from HS and heparin. Gel chromatography and high-performance

liquid chromatography (HPLC) methods have been employed to obtain information

on disaccharide composition (3,4). Other methods such as nuclear magnetic resonance

(NMR) spectroscopy and mass spectroscopy (5–9) have provided direct sequence

information, but are difficult for even moderately sized oligosaccharides and in

the case of NMR requires large amounts of material (micro-moles). However, the

scene has changed rapidly in the last few years with the availability of recombinant

exolytic lysosomal enzymes. These exoglycosidases and exosulphatases remove spe-

cific sulfate groups or monosaccharide residues from the nonreducing end (NRE) of

saccharides (10). These can be used in combination with PAGE separations to derive

direct information (based on band shifts) on the structures present at the nonreducing

end of GAG saccharides (11; see Fig. 1 for an example).

130 Turnbull

Integral glycan sequencing (IGS), a PAGE-based method using the exoenzymes,

was recently developed as the first strategy for rapid and direct sequencing of

heparan sulfate and heparin saccharides (11). Its introduction has been quickly fol-

lowed by a variety of similar approaches using other separation methods including

HPLC and MALDI mass spectrometry (12–14). In IGS, an oligosaccharide (previ-

ously obtained from the polysaccharide by partial chemical or enzymatic degrada-

tion and purification) is labeled at the reducing terminal with a fluorescent tag. This

is subjected to partial nitrous acid degradation to give a ladder of evenly numbered

oligosaccharides (di-, tetra-, hexa-, etc.), each bearing a fluorescent tag at its reduc-

ing-end terminus. Portions of this material are then treated with a variety of highly-

specific exolytic lysosomal enzymes (exosulfatases and exoglycosidases), which act

only at the nonreducing end of each saccharide if it is a suitable substrate. The vari-

ous digests are then separated on a high-density polyacrylamide gel and the posi-

tions of the fragments are detected by excitation of the fluorescent tag with an

ultraviolet (UV) transilluminator. Band shifts due to the different treatments permit

the sequence to be read directly from the banding pattern (see Fig. 1 for an example).

This novel strategy allows direct readout sequencing of a saccharide in a single set

of adjacent gel tracks in a manner analogous to DNA sequencing (11). IGS provides

for the first time a rapid approach for sequencing HS saccharides, and it has proved

invaluable in recent structure–function studies (15). It should be noted that this meth-

odology is designed for sequencing purified saccharides, not whole HS prepara-

tions. Clearly, a critical factor in all sequencing methods is the availability of

sufficiently pure oligosaccharide starting material. HS and heparin saccharides can

be prepared following selective scission by enzymic (or chemical) reagents and iso-

lation by methods such as affinity chromatography (4). Final purification usually

requires the use of strong anion-exchange HPLC (15; see Chapter 14).

2. Materials

1. 2-aminobenzoic acid (2-AA; Fluka Chemicals).

2. Formamide.

3. Sodium cyanoborohydride (>98% purity).

4. Distilled water.

5. Oven or heating block at 37°C.

6. Desalting column (Sephadex G-25; e.g., HiTrap

desalting columns, Pharmacia).

7. Centrifugal evaporator.

8. 200 mM HCl.

9. 20 mM sodium nitrite. 1.38 mg/mL in distilled water; prepare fresh.

10. 200mM sodium acetate, pH 6.0: 27.2 g/L sodium acetate trihydrate, pH to 6.0 using

acetic acid.

11. Enzyme buffer: 0.2 M Na acetate, pH 4.5. Make 0.2 M sodium acetate (27.2g/L sodium

acetate trihydrate) and 0.2 M acetic acid (11.6 mL/L) and mix in a ratio of 45 mL to 55 mL

respectively.

12. Enzyme stock solutions (typically at concentrations of 500 mU/mL, where 1U = 1 µmol

substrate hydrolyzed per minute). Available from Glyko (Novato, CA).

13. Vortex tube mixer.

14. Microcentrifuge.

Glycan Sequencing of HS and Heparin Saccharides 131

15. Acrylamide stock solution (T50%/C5%). Caution: acrylamide is neurotoxic. Wear gloves

(and a face mask when handling powdered forms). It is convenient to use pre-mixed

acrylamide-bis, such as Sigma A-2917. Add 43 mL of distilled water to the 100 mL bottle

containing the premixed chemicals and dissolve using a small stirrer bar (~2 h). Final

volume should be ~80 mL. Store the stock solution at 4°C. Note that it is usually necessary

to warm gently to redissolve the acrylamide after storage.

16. Resolving gel buffer stock solution: 2 M Tris-HCl, pH 8.8. 242.2 g/L Tris base, pH to 8.8

with HCl.

17. Stacking gel buffer stock solution: 1 M Tris-HCl, pH 6.8. 121.1 g/L Tris base, pH to 6.8

with HCl.

18. Electrophoresis buffer: 25 mM Tris-HC1, 192 mM glycine, pH 8.3. 3 g/L Tris base, 14.4 g/L

glycine, pH to 8.3 if necessary with HCl.

19. 10% ammonium persulfate in water (made fresh or stored at –20°C in aliquots).

20. TEMED.

21. Vertical slab gel electrophoresis system (minigel or standard size).

22. DC power supply unit (to supply up to 500–1000 V and 200 mA).

23. UV tansilluminator (312-nm maximum emission wavelength).

24. Glass UV bandpass filter larger than gel size (type UG-11 or M-UG2).

25. CCD imaging camera fitted with a 450-nm (blue) band -pass filter.

3. Methods

3.1. Derivatization of Saccharides with the Fluorophore 2AA

HS and heparin saccharides can be labeled by reaction of their reducing aldehyde

functional group with a primary amino group (reductive amination). For sulfated

saccharides anthranilic acid (2-aminobenzoic acid; 2-AA; ref. 11) has been found to

be effective for the IGS methodology. 2-AA conjugates typically display an excita-

tion maxima in the range 300–320 nm, which is ideal for visualization with a com-

monly available 312 nm UV source (e.g., transilluminators used for visualizing

ethidium bromide stained DNA). Emission maxima are typically in the range 410–420

nm (bright violet fluorescence). The approach described below allows rapid labeling

and purification of tagged saccharide from free tagging reagent, gives quantitative

recoveries, and the product is free of salts that might interfere with subsequent enzymic

conditions. For saccharides in the size range hexa- to dodecasaccharides, approxi-

mately 2–3 nmol of purified starting material is the minimum required (~2–10 µg).

1. Dry down the purified saccharide (typically 2–20 nmol) in a microcentrifuge tube by

centrifugal evaporation.

2. Dissolve directly in 10–25 µL of formamide containing freshly prepared 400 mM 2-AA

(54.8 mg/mL) and 200 mM reductant (sodium cyanoborohydride; 12.6 mg/mL) and incu-

bate at 37°C for 16–24 h in a heating block or oven. (Caution: the reductant is toxic and

should be handled with care.) The volume used should be sufficient to provide a 500- to

1000-fold molar excess of 2-AA over saccharide (see Note 1).

3. Remove free 2-AA, reductant, and formamide from the labeled saccharides by gel filtra-

tion chromatography (Sephadex G-25 Superfine). Dilute the sample (maximum 250 µL

of reaction mixture) to a total of 1 mL with distilled water (see Note 2).

4. Load onto two 5 mL HiTrap

desalting columns (Pharmacia) connected in series. Alter-

natively, it is possible to use self-packed columns of other dimensions.

132 Turnbull

5. Elute with distilled water at a flow rate of 1 mL/min and collect fractions of 0.5 mL.

Saccharides consisting of four or more monosaccharide units typically elute in the void

volume (approximately fractions 7–12).

6. Pool and concentrate these fractions by centrifugal evaporation or freeze-drying.

3.2. Treatment of Saccharides with Nitrous Acid

Low-pH nitrous acid cleaves only at linkages between N-sulfated glucosamine and

adjacent hexuronic acid residues (16,17; see also Chapter 34). Under controlled con-

ditions nitrous acid cleavage can be used to create a ladder of bands that correspond to

the positions of internal N-sulfated glucosamine residues in the original intact saccha-

ride (11). To achieve this a series of different reaction stop points are pooled to pro-

duce a partial digest with a range of different fragment sizes.

1. Dry down 1–2 nmol of labeled saccharide by centrifugal evaporation.

2. Redissolve in 80 µl of distilled water and chill on ice.

3. Add 10 µl of 200 mM HCl and 10 µl of 20 mM sodium nitrite (both prechilled on ice) and

incubate on ice.

4. At a series of individual time points (typically 0.5, 1, 2, and 3 h), remove an aliquot and

stop the reaction by raising the pH to approximately 5.0 by the addition of 1/5 volume of

200 mM sodium acetate buffer, pH 6.0 (see Note 3).

5. Pool the set of aliquots and either use directly for enzyme digests or desalt as described

under Subheading 3.1.

3.3. Treatment of Saccharides with Exoenzymes

The basic approach for treatment of HS/heparin samples with exoenzymes is

described below. Details of the specificities of the exoenzymes are given in Table 1.

Although these enzymes have differing optimal pH and buffer conditions, in general

it is possible to use them under the single set of conditions given here, simplifying

multiple enzyme treatments (see Note 4).

1. Dissolve the sample (typically 10–200 pmol of saccharide) in 10 µL of H

O in a

microcentrifuge tube.

2. Add 5 µL of exoenzyme buffer, 1 µL of 0.5 mg/mL bovine serum albumin, 2 µL of

appropriate exoenzyme (0.2–0.5 mU) and distilled water to bring the final volume to 20 µL.

3. Mix the contents well on a vortex mixer, and centrifuge briefly to ensure that the reactants

are at the tip of the tube.

4. Incubate the samples at 37°C for 16 h in a heating block or oven.

3.4. PAGE Separation of Saccharides

Polyacrylamide gel electrophoresis (PAGE) is a high-resolution technique for the sepa-

ration of HS and heparin saccharides of variable sulfate content and disposition. It provides

a level of resolution for oligosaccharides larger than tetrasaccharides that is superior to gel

filtration or anion-exchange HPLC (18,19). It is possible to obtain improved resolution

using gradient gels. However, these are more difficult to prepare and use routinely and in

most cases adequate resolution can be obtained with isocratic gels (see Note 5).

Oligosaccharide mapping by PAGE is a rapid and reproducible method for the simulta-

neous comparison of multiple samples. It thus provides a simple but powerful approach for

separating the saccharide products generated in the sequencing process.

Glycan Sequencing of HS and Heparin Saccharides 133

3.4.1. Preparing the PAGE Gel

1. Assemble the gel unit (consisting of glass plates and spacers, etc.).

2. Prepare and degas the resolving gel acrylamide solution without ammonium persulfate or

TEMED. To make a 30% acrylamide gel solution for a 16 cm ×12cm × 0.75 mm gel, 16

mL are required. Mix 9.6 mL of T50%/C5% acrylamide stock with 3 mL of 2 M Tris pH

8.8 and 3.4 mL of distilled water.

3. Add 10% ammonium persulfate (30 µl) and TEMED (10 µL) to the gel solution, mix

well, and immediately pour into the gel unit.

4. Overlay the unpolymerized gel with resolving gel buffer (375 mM Tris-HCl, pH 8.8,

diluted from the 2 M stock solution) or water-saturated butanol. Polymerization

should occur within ~30–60 min. The gel can then be used immediately or stored at

4°C for 1–2 wk.

3.4.2. Electrophoresis

1. Immediately before electrophoresis, rinse the resolving gel surface with stacking gel

buffer (0.125 M Tris-HC1 buffer, pH 6.8, diluted from the 1 M stock solution).

2. Prepare and degas the stacking gel solution (for 5 ml, mix 0.5 mL of T50%/C5%

acrylamide stock with 0.6 mL of 1 M Tris pH 6.8 and 3.9 mL of distilled water).

Table 1

Exoenzymes for Sequencing Heparan Sulfate and Heparin

Enzyme

Substrate specificity

Sulfatases

Iduronate-2-sulfatase IdoA(2S)

Glucosamine-6-sulfatase GlcNAc(6S), GlcNSO

(6S)

Sulphamidase (glucosamine N-sulfatase) GlcNSO

Glucuronate-2-sulfatase GlcA(2S)

Glucosamine-3-sulphatase GlcNSO

(3S)

Glycosidases

Iduronidase IdoA

Glucuronidase GlcA

α-N-Acetylglucosaminidase GlcNAc

Bacterial exoenzymes

∆-4,5-Glycuronate-2-sulfatase ∆-UA(2S)

∆-4,5-Glycuronidase ∆-UA

Enzyme availability: Glucuronidase is widely available commercially as purified enzyme.

Recombinant iduronate-2-sulphatase, iduronidase, glucosamine-6-sulphatase, sulfamidase, and

α-N-acetylglucosaminidase are available from Glyko (Novato, CA). Glucuronate-2-sulfatase and

glucosamine-3-sulfatase have only been purified from cell and tissue sources to date. The bacterial

exoenzymes are available from Grampian Enzymes, Nisthouse, Harray, Orkney, Scotland; e-mail

grampenz@aol.com.

The specificities are shown as the nonreducing terminal group recognised by the enzymes. Sulfatases

remove only the sulfate group, whereas the glycosidases cleave the whole nonsulfated monosaccharide.

134 Turnbull

3. Add 10% ammonium persulfate (10 µL) and TEMED (5 µL). Immediately pour on to the

top of the resolving gel and insert the well-forming comb.

4. After polymerization (~15 min), remove the comb and rinse the wells thoroughly with

electrophoresis buffer.

5. Place the gel unit into the electrophoresis tank and fill the buffer chambers with electro-

phoresis buffer.

6. Load the oligosaccharide samples (5–20 µL depending on well capacity, containing ~10%

(v/v) glycerol or sucrose in 125 mM Tris-HCl, pH 6.8, carefully into the wells with a

microsyringe. Marker samples containing bromophenol blue and phenol red should also

be loaded into separate tracks.

7. Run the samples into the stacking gel at 150–200 V (typically, 20–30 mA) for 30–60 min,

followed by electrophoresis at 300–400 V (typically 20–30 mA and decreasing during

run) for approximately 5–8 h (for a 16 cm gel). Heat generated during the run should be

dissipated using a heat exchanger with circulating tap water, or by running the gel in a

cold room or in a refrigerator.

8. Electrophoresis should be terminated before the Phenol red marker dye is about 5 cm

from the bottom of the gel. (At this point, disaccharides should be 3–4 cm from the bot-

tom of the gel.)

Fig. 1. Principles of integral glycan sequencing and an example. (A) Fluorescence detection

of different amounts of a 2AA-tagged heparin tetrasaccharide run on a 33% minigel. (B)

Exosequencing of a 2AA-tagged heparin tetrasaccharide with lysosomal enzymes and separation

of the products on a 33% minigel (15 pmol per track). Band shifts following the exoenzyme

treatments shown reveal the structure of the nonreducing end disaccharide unit (track 1,

untreated). I2Sase, iduronate-2-sulfatase; Idase, iduronidase; G6Sase, glucosamine-6-sulfa-

tase; Nsase, sulfamidase. (C) Schematic representation of IGS of a hexasaccharide (pHNO2,

partial nitrous acid treatment). (D) Actual example of IGS performed on a purified heparin

hexasaccharide, corresponding to the scheme in (C), using the combinations of pHNO

and

exoenzyme treatments indicated (track 1, untreated, 25 pmol; other tracks correspond to ~200

pmol/per track of starting sample for pHNO

digest). The hexasaccharide (purified from bovine

lung heparin) has the putative structure IdoA(2S)-GlcNSO

(6S)-IdoA(2S)-GlcNSO

(6S)-

1999 National Academy of Sciences, USA. From ref. (11).

Glycan Sequencing of HS and Heparin Saccharides 135

3.5. Imaging the Gels

Effective gel imaging requires a CCD camera that can detect faint fluorescent band-

ing patterns by capturing multiple frames. Systems commonly used for detection of

ethidium bromide stained DNA can usually be adapted with appropriate filters as

described in Note 6.

1. Place a UV filter (UG-1, UG-11, or MUG-2) onto the transilluminator, and fit a 450-nm

blue filter onto the camera lens.

2. Remove the gel carefully from the glass plates after completion of the run and place on

the UV transilluminator surface wetted with electrophoresis buffer. Also wet the upper

surface of the gel to prevent gel drying and curling.

3. Switch on transilluminator and capture image using CCD camera. Exposure times are

typically 1–5 s depending on the amount of labeled saccharide (see Note 7).

3.6. Interpreting the Data

The sequence of saccharides subjected to IGS can be read directly from the banding

pattern by interpreting the band shifts due to removal of specific sulfate or sugar

Fig. 2. IGS of a heparin hexasaccharide of known structure. Α heparin hexasaccharide with

the structure DHexA(2S)-GlcNSO

(6S)-IdoA-GlcNAc (6S)-GlcA-GlcNSO

(6S), was 2AA-

tagged and subjected to sequencing on 16cm 33% gel. (A) IGS of hexasaccharide using the

combinations of pHNO

and exoenzyme treatments indicated (track 1, untreated, 20 pmol;

other tracks correspond to ~90 pmol/per track of starting sample for pHNO

digest). NAG,

N-acetylglucosaminidase. (B) Determining the sequence of the nonreducing disaccharide unit

of the hexasaccharide using the I2Sase, G6Sase, and mercuric acetate (MA) treatments shown

From ref. (11).

136 Turnbull

moieties. Figure 1 shows an actual example and a schematic representation. First,

bands generated by the partial nitrous acid treatment indicate the positions of N-sul-

fated glucosamine residues in the original saccharide (see Fig. 1C, track 2). Lack of a

band at a particular position indicates the presence of an N-acetylated glucosamine

residue (an example of this is shown in Fig. 2). Such saccharides can be sequenced

with the additional use of the exoenzyme N-acetylglucosaminidase, which removes

this residue and allows further sequencing of an otherwise “blocked” fragment. Fol-

lowing the nitrous acid treatment, the “ladder” of bands is then subjected to various

exoenzyme digestions. The presence of specific sulfate or sugar residues can be

deduced from the band shifts that occur (see Fig. 1C, tracks 3–5). Figure 3 shows an

example of a decasaccharide from HS which has been purified by SAX-HPLC and

sequenced using IGS.

Usually the band shifts are downwards, due to the lower molecular mass and thus

higher mobility of the product. However, it should be noted that occasionally upward

shifts occur, probably due to subtle differences in charge/mass ratio (for examples, see

Figs. 1B, 2B and 3C). Note also that minor “ghost” bands sometimes appear after the

nitrous acid treatment. They are probably due to loss of an N-sulfate group, and nor-

mally these do not affect interpretation of the shifts in the major bands (11).

Fig. 3. Purification and IGS of a HS decasaccharide. (A) SAX-HPLC of a pool of HS

decasaccharides derived by heparitinase treatment of porcine mucosal HS. For details of this

technique, see Chapter 14. The arrowed peak was selected for sequencing. (B) IGS of the puri-

fied HS decasaccharide on a 16 cm 33% gel using the combinations of pHNO

and exoenzyme

treatments indicated (track 1, untreated, 20 pmol; other tracks correspond to ~400 pmol/per track

of starting sample for pHNO

digest). (C) Determining the sequence of the nonreducing disac-

charide unit of the HS decasaccharide using the mercuric acetate (MA) and G6Sase treatments

emy of Sciences, USA. From ref. (11).

Glycan Sequencing of HS and Heparin Saccharides 137

If the saccharide being sequenced was derived by bacterial lyase treatment, it will

have a ∆-4,5-unsaturated uronate residue at its nonreducing terminus. If this residue has

a 2-O-sulfate attached, this can be detected by susceptibility to I2Sase (see Fig. 2B), but

the sugar residue itself is resistant to both Idase and Gase. Its removal is required in order

to confirm whether there is a 6-O-sulfate on the adjacent non-reducing end glucosamine

(see Figs. 2B and 3C for examples). However, bacterial enzymes which specifically

remove the ∆-4,5-unsaturated uronate residues (and the 2-O-sulfate groups that may be

present on them) are now available commercially (see Table 1). Alternatively, they can

be removed chemically with mercuric acetate (20; see Figs. 2B and 3C).

In addition to the basic sequencing experiment, it is wise to confirm agreement of

the data with an independent analysis of the disaccharide composition of the saccha-

ride (see Chapter 14). It can sometimes be difficult to sequence the reducing terminal

monosaccharide, due to it being a poor substrate for the exoenzymes. In these cases it

has proved more effective to analyze the terminal 2AA-labeled disaccharide unit in

comparison to 2AA-labeled disaccharide standards (11).

4. Notes

1. Using large excesses of reagent as described, saccharides derived from HS and heparin by

bacterial lyase scission generally couple with 2-AA with efficiencies in the range 60–70%. In

contrast, saccharides derived from HS and heparin by low-pH nitrous acid scissioning (i.e.,

having an anhydromannose residue at their reducing ends) label more efficiently (~70–80%

coupling efficiency).

2. Unwanted reactants and solvent can also be removed from labeled saccharides by methods

such as dialysis, but the rapid gel filtration chromatography step described above using the

HiTrap desalting columns is convenient and usually allows good recoveries of loaded

sample, particularly for 2-AA-labeled saccharides (~80%).

3. It is useful to perform some trial incubations to test for optimal time points needed to gener-

ate a balance of all fragments in the partial nitrous acid digestion. With longer saccharides

(octasaccharides and larger) it is observed that the largest products are generated quickly

and thus a bias toward shorter incubations is required as saccharide length increases.

4. The enzyme conditions should provide for complete digestion of all susceptible residues.

This is important to the sequencing process, since incomplete digestion would create a more

complex banding pattern and would give a false indication of sequence heterogeneity. It is

useful to run parallel controls with standard saccharides to enable monitoring of reaction

conditions. When combinations of exoenzymes are required, these can be incubated simul-

taneously with the sample. If necessary, the activity of one enzyme can be destroyed before

a secondary digestion with a different enzyme by heating the sample at 100°C for 2–5 min.

5. Adequate separations, particularly over limited size ranges of saccharides, can be obtained

using single concentration gels, typically in the range 25–35% acrylamide. Improvements in

resolution can be made by using longer gel sizes. Different voltage conditions (usually in

the range 200–600 V) and running times are required for different gel formats, and should

be established by trial and error with the particular samples being analyzed. Gels up to 24 cm

in length can usually be run in 5–8 h using high voltages, whereas with longer gels it is more

convenient to use lower voltage conditions and run overnight. We have also found that

minigels can also be used effectively for separation of HS/heparin saccharides (see Fig. 1).

Note that it is also possible to run Tris-acetate gels with a Tris-MES electrophoresis buffer

(see Fig. 1; 11).