Enzyme assay of glycosaminoglycan glycosyltransferases for heparan sulfate

Materials

1. GlcAβ1-3Galβ1-O-C₂H₄NH-benzyloxycarbonyl (chemically synthesized linkage region analog) (Note 1)

2. N-Acetylheparosan oligosaccharides derived from the capsular polysaccharide of Escherichia coli K5, GlcAβ1-4GlcNAcα1-(4GlcAβ1-4GlcAcα1)n with the nonreducing terminal GlcA

3. N-Acetylheparosan oligosaccharides derived from the capsular polysaccharide of Escherichia coli K5, GlcNAcα1-(4GlcAβ1-4GlcNAcα1)n with the nonreducing terminal GlcNAc

4. GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser-Gly-Trp-Pro-Asp-Gly (chemically synthesized) (2)

5. UDP-[³H]GlcNAc (1,000–2,000 GBq/mmol) (NET434050UC, PerkinElmer Life Sciences, Waltham, MA)

6. UDP-[U-¹⁴C]GlcA (10 GBq/mmol) (NEC414050UC, PerkinElmer Life Sciences)

7. MES-NaOH (pH 6.5)

8. MnCl₂

9. ATP-2Na

10. UDP-GlcA

11. MeOH

12. NH₄HCO₃

13. Heparinase III (EC4.2.2.8) (IBEX or Sigma-Aldrich)

14. Sodium acetate buffer

15. Calcium acetate

Instruments

1. Nova-Pak C18 column (3.9 × 150 mm) (Waters, Milford, MA)

2. Chromatography system (FPLC, ÄKTA, and so on)

3. 2300TR liquid scintillation counter (PerkinElmer, Waltham, MA)

4. Syringe column (TERUMO SS-01T) packed with Superdex G-25 superfine (Cytiva, Tokyo, Japan) (3)

5. Superdex peptide HR 10/30 column (Cytiva)

6. Superdex 200 10/300 GL (Cytiva)

7. High-performance liquid chromatography (HPLC)

8. SpeedVac vacuum concentrator (Thermo Fisher Scientific, Waltham, MA)

Methods

1.

Preparation of recombinant soluble enzyme proteins is mentioned in the methods described in the chapter “Enzyme assay of glycosaminoglycan glycosyltransferases for chondroitin sulfate”.

2.

Assay for GlcNAcT-I activity (Figure 2) (4)

a.

The reaction mixture for GlcNAcT-I activity (a total volume of 20 μL) contains 10 μL of enzyme source (Note 2), 100 mM of MES-NaOH (pH 6.5), 10 mM of MnCl₂, 1 mM of ATP-2Na, 250 nmol GlcAβ1-3Galβ1-O-C₂H₄NH-benzyloxycarbonyl, and 250 μM of UDP-[³H]GlcNAc (approximately 1.1 × 10⁶ dpm).

b.

The enzyme reaction is performed at 37°C for 4 h.

c.

The 3H-labeled GlcNAcT-I reaction products are separated by HPLC analysis on a Nova-Pak C18 column using MeOH as the eluent at a flow rate of 1.0 mL/min.

d.

The collected fractions (2 mL each) are analyzed using a liquid scintillation counter.

3.

Assay for GlcNAcT-II activity (Figure 3)

a.

The reaction mixture for GlcNAcT-II activity (a total volume of 20 μL) contains 10 μL of enzyme source, 100 mM of MES-NaOH (pH 6.5), 10 mM of MnCl₂, 1 mM of ATP-2Na, 20 μg GlcAβ1-4GlcNAcα1-(4GlcAβ1-4GlcNAcα1)n (Note 3), and 250 μM of UDP-[³H]GlcNAc (approximately 1.1 × 10⁶ dpm).

b.

The enzyme reaction is performed at 37°C for 4 h.

c.

The ³H-labeled GlcNAcT-II reaction products are analyzed by syringe columns (TERUMO SS-01T)

i.

Pack with Sephadex G-25 (superfine) equilibrated with 0.2 M NH₄HCO₃. Centrifuge the column at 800 rpm for 5 min, followed by the second centrifuge at 2,000 rpm for 5 min.

ii.

Add 30 μL of 0.2 M NH₄HCO₃ to the reaction products. Apply the samples to the packed syringe column and centrifuge at 2,000 rpm for 5 min.

iii.

Add 50 μL of 0.2 M NH₄HCO₃ to the top of the syringe columns and centrifuge at 2,000 rpm for 5 min.

iv.

Measure radioactivity of the eluates using a scintillation counter.

4.

Assay for HS-GlcAT-II activity (Figure 3)

a.

The reaction mixture for HS-GlcAT-II activity (a total volume of 20 μL) contains 10 μL of enzyme source, 100 mM of MES-NaOH (pH 6.5), 10 mM of MnCl₂, 1 mM of ATP-2Na, 20 μg GlcNAcα1-(4GlcAβ1-4GlcNAcα1)n (Note 3), and 250 μM of UDP-[U-¹⁴C]GlcA (approximately 1.6 × 10⁵ dpm).

b.

The enzyme reaction is performed at 37°C for 4 h.

c.

The ¹⁴C-labeled HS-GlcAT-II reaction products are analyzed by syringe columns as described in Method 3-c.

5.

Assay for polymerization activity (Figure 4)

a.

The reaction mixture for polymerization activity (a total volume of 20 μL) contains 10 μL of enzyme source, 100 mM of MES-NaOH (pH 6.5), 10 mM of MnCl₂, 1 mM of ATP-2Na, 100 nmol GlcAβ1-3Galβ1-O-C₂H₄NH-benzyloxycarbonyl, 250 μM of UDP-[³H]GlcNAc (approximately 5.5 × 10⁵ dpm), and 250 μM of UDP-GlcA.

b.

The enzyme reaction is performed at 37°C for overnight.

c.

The ³H-labeled polymerization products are separated by gel filtration using a Superdex Peptide HR 10/30 column (Note 4) using NH₄HCO₃ gradient as the eluent at a flow rate of 0.4 mL/min.

d.

The collected fractions (0.4 mL each) are analyzed using a liquid scintillation counter.

6.

The reaction products are characterized taking advantage of the substrate specificities of respective glycosidases

a.

GlcNAcT-I-reaction products (3) (Figure 2)

i.

Pool and concentrate the reaction products by SpeedVac

ii.

Digest with 10 mUnits of heparinase III (EC4.2.2.8) (IBEX or Sigma-Aldrich) (Note 5) in a total volume of 50 μL of 20 mM of sodium acetate buffer (pH 7.0) containing 2 mM of calcium acetate at 37°C overnight.

iii.

Analyze the digested or undigested samples using HPLC on a Nova-Pak C18 column.

The radioactivity of the GlcNAcT-I-reaction products is released by digestion with heparinase III and eluted at the elution position of free [³H]GlcNAc.

b.

GlcNAcT-II-reaction products (3)

i.

Pool and concentrate the reaction products by SpeedVac.

ii.

Digest the reaction products with 10 mUnits of heparinase III (EC4.2.2.8) (IBEX or Sigma-Aldrich) in a total volume of 100 μL of 20 mM of sodium acetate buffer (pH 7.0) containing 2 mM of calcium acetate at 37°C overnight.

iii.

Analyze the digested samples by gel filtration on a Superdex peptide HR 10/30 column.

The radioactivity peak of the GlcNAcT-II-reaction products eluted near the void volume is released by digestion with heparinase III and shifted to the free [³H]GlcNAc position.

c.

Polymerization products (Figure 4)

i.

Pool and concentrate the polymerized products by SpeedVac.

ii.

Digest with 6 mUnits of heparinase III (EC4.2.2.8) in a total volume of 100 μL of 20 mM of sodium acetate buffer (pH 7.0) containing 2 mM of calcium acetate at 37°C overnight.

iii.

Analyze the digested samples by gel filtration using a Superdex peptide HR 10/30 column.

The radioactivity peak corresponding to the polymerized products is observed near the void volume (Figure 4). This peak is shifted to the elution position of unsaturated disaccharides by digestion with heparitinase III, when heparosan polymer is synthesized by polymerization reactions.

Notes

1. Acceptor substrates for GlcNAcT-I and GlcAβ1-3Galβ1-O-C₂H₄NH-benzyloxycarbonyl were kindly provided by Prof. Jun-ichi Tamura (Tottori University).

2. Each EXT protein is expressed in COS-1 or COS-7 cells as a soluble form fused to Protein A. Forty-eight h after transfection, the secreted Protein A-tagged proteins are purified with IgG-Sepharose beads (Cytiva). The enzyme-bound beads are used for enzyme assays. Additionally, co-expression of EXT1 and EXT2 is required for the polymerization reaction (2).

GlcNAcT and GlcAT assays using crude cell lysates as an enzyme source have been reported (5).

3. Oligosaccharide acceptors for GlcNAcT-II and HS-GIcAT-II can be prepared as described previously (6). K5 polysaccharides are partially N-deacetylated with hydrazine and subjected to deaminative cleavage with nitrous acid at pH 3.9. The resulting mixture of oligosaccharides was fractionated by gel filtration chromatography. The decasaccharide fraction was used as a substrate for GlcNAcT-II. The digestion of a tetradecasaccharide fraction with β-D-glucuronidase yielded tridecasaccharides with nonreducing terminal GlcNAc residues, which are suitable substrates for HS-GlcAT-II.

4. To determine the molecular size of polymerized HS chains, a Superdex 200 10/300 GL column should be used instead of a Superdex Peptide HR 10/30 column.

5. Unit is defined as follows: one unit of the enzyme is the amount required for the eliminative cleavage of typical CS or HS, yielding ultraviolet-absorbing materials corresponding to 1 μmol Δ^4,5-hexuronate residues/min at 37°C, pH 7.0, as calculated with a value of 5,500 (7) for the molar extinction coefficient of Δ^4,5-hexuronated oligosaccharides.