Enzyme assay of GAG glycosyltransferases for heparan sulfate
EXT1 and EXT2 genes encode heparan sulfate (HS) co-polymerases, and the genetic defects of which result in hereditary multiple exostoses in humans. Both EXT1 and EXT2 exhibit GlcNAc transferase (GlcNAcT) and GlcA transferase (GlcAT) activities required for the HS synthesis (Fig. 1). EXT1 and EXT2 form a stable complex and the complex is thought to be the biologically relevant form of enzymes. In addition, three highly homologous EXT-like genes, EXTL1-EXTL3, have been cloned (Fig. 1). Both EXTL1 and EXTL3 have been shown to exhibit GlcNAcT activities likely involved in the biosynthesis of HS (Fig. 1). EXTL1 exerts only GlcNAcT-II activity, which may be involved in HS chain elongation, whereas EXTL3 possesses activity transferring the first GlcNAc residue to tetrasaccharide-linkage region (so-called GlcNAcT-I activity) in addition to GlcNAcT-II activities. EXTL2 exhibits the GlcNAcT-I activities involved in the initiation of HS biosynthesis (Fig. 1). Although all EXTL proteins show glycosyltrasferase activities associated with HS biosynthesis, their roles in the HS biosynthesis remain unclear.
EXT gene family
Substrate: GlcAβ1-3Galβ1-O-C2H4NH-benzyloxycarbonyl (chemically-synthesized linkage region analog)*
Donor: UDP-[3H]GlcNAc (60 Ci/mmol) (PerkinElmer Life Sciences)
* Acceptor substrates for GlcNAcT-I, GlcAβ1-3Galβ1-O-C2H4NH-benzyloxycarbonyl, were kindly provided from Prof. Jun-ichi Tamura (Tottori University).
Substrate: N-Acetylheparosan oligosaccharides derived from the capsular polysaccharide of Escherichia coli K5,
GlcAβ1-4GlcNAcα1-(4GlcAβ1-4GlcNAcα1)n with the non-reducing terminal GlcA
GlcNAcα1-(4GlcAβ1-4GlcNAcα1)n with the non-reducing terminal GlcNAc
Donor: UDP-[U - 14C]GlcA (285.2 mCi/mmol) (PerkinElmer Life Sciences)
Substrate: GlcAβ1-3Galβ1-O-C2H4NH-benzyloxycarbonyl (chemically-synthesized linkage region analog)* or
Donor: UDP-[3H]GlcNAc (60 Ci/mmol) and UDP-[U - 14C]GlcA (285.2 mCi/mmol) (PerkinElmer Life Sciences)
Nova-Pak C18 column (3.9 x 150 mm) (Waters)
2300TR liquid scintillation counter (PerkinElmer)
GlcNAcT-II activities and HS-GlcAT-II activities
Syringe column (TERUMO SS-01T) packed with Superdex G-25 superfine (GE Healthcare)2)
Superdex Peptide HR 10/30 or Superdex 200 10/300 GL (GE Healthcare)
The following reaction mixtures of a total volume of 20 μl are prepared.
10 μl of enzyme source (see Comment *)
100 mM MES-NaOH (pH 6.5)
10 mM MnCl2
1 mM ATP-2Na
GlcAβ1-3Galβ1-O-C2H4NH-benzyloxycarbonyl (250 nmol)
250 μM UDP-[3H]GlcNAc (approx. 1.1 x 106 dpm)
10 μl of enzyme source
GlcAβ1-4GlcNAcα1-(4GlcAβ1-4GlcNAcα1)n (20 μg) (see Comment **)
10 mM MgCl2
5 mM CaCl2
171 μM ATP-2Na
GlcNAcα1-(4GlcAβ1-4GlcNAcα1)n (20 μg) (see Comment **)
250 μM UDP-[14C]GlcA (approx. 1.4 x 105 dpm)
GlcAβ1-3Galβ1-O-C2H4NH-benzyloxycarbonyl (100 nmol)
250 μM UDP-[3H]GlcNAc (approx. 5.5x 105 dpm)
250 μM UDP-GlcA
Reaction mixtures of GlcNAcT-I, GlcNAcT-II, and HS-GlcAT-II are incubated at 37 ˚C for 4 h.
Polymerization reaction is performed at 37 ˚C overnight.
Each enzyme activity is measured according to the following method.
GlcNAcT-I activities (Fig. 2)3)
GlcNAcT-I reaction products
↓ HPLC analysis on a Nova-Pak C18 column.
↓ the effluent fractions are analyzed for radioactivity.
GlcNAcT-II and HS-GlcAT-II activities (Fig. 3)
i) Preparation of syringe columns
Syringe columns (TERUMO SS-01T)
↓ pack with Sephadex G-25 (superfine) equilibrated with 0.2 M NH4HCO3.
↓ centrifuge at 800 rpm for 5 min.
↓ centrifuge again at 2,000 rpm for 5 min.
Samples (20 μl)
↓ add 30 μl of 0.2 M NH4HCO3.
Samples (50 μl)
↓ apply to the packed syringe column.
↓ centrifuge at 2,000 rpm for 5 min.
↓ add 50 μl of 0.2 M NH4HCO3 to the top of the syringe columns.
↓ measure by scintillation counting.
Polymerization activities (Fig. 4)
The reaction products
↓ analyze using a Superdex Peptide HR 10/30 column (see Comment *).
↓ measure radioactivity using a liquid scintillation counter.
Each reaction product is confirmed.
GlcNAcT-I reaction products (Fig. 2)
↓ pool and evaporate.
GlcNAcT-I reaction products (about 36 pmol)
↓ digest with 10 mIU of heparitinase I (Seikagaku Kogyo) in a total volume of
50 μl of 20 mM sodium acetate buffer (pH 7.0) containing 2 mM calcium
acetate at 37 ˚C overnight.
↓ analyze digested or undigested samples on a Nova-Pak C18 column.
The radioactivity of the GlcNAcT-I-reaction products is released by digestion
with heparitinase I and eluted in the flow-through fraction as free [3H]GlcNAc.
GlcNAcT-II reaction products
GlcNAcT-II reaction products (about 36 pmol)
100 μl of 20 mM sodium acetate buffer (pH 7.0) containing 2 mM calcium
↓ analyze by 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 heparitinase I and shifted to the free [3H]GlcNAc position.
Polymerized products (Fig. 4)
Polymerized products (about 70 pmol as disaccharide)
↓ digest with 6 mIU of heparitinase I (Seikagaku Kogyo) in a total volume of 100 μl of
20 mM sodium acetate buffer (pH 7.0) containing 2 mM calcium acetate at 37 ˚C
↓ analyze using a Superdex peptide HR 10/30 column.
The radioactivity peak of the polymerized products eluted near the void volume is
degraded by digestion with heparitinase I and shifted to the elution position of the
Figure & Legends
Fig. 1 The five cloned members of the EXT gene family.
(A) Schematic structures of five EXT gene family are shown. Highly conserved regions are indicated by gray bars. White bars show DXD motif. a.a., amino acid; TM, transmembrane domain. In addition, glycosyltransferase activities detected in EXT members are summarized. (B) The biosynthesis of HS in mammals. Mammalian HS is biosynthesized by EXT1/EXT2 polymerase complex. The significance of EXTL1, EXTL2, and EXTL3 in the biosynthesis of HS remains unclear.
Fig. 2 Measurement of GlcNAcT-I activities.
GlcNAcT-I-reaction products were subjected to HPLC analysis using a Nova-Pak C18 column. The separated fractions (2 ml each) were measured for radioactivities. White arrow, UDP-[3H]GlcNAc; Black arrow, [3H]GlcNAca1-4GlcAb1-3Galb1-O-C2H4NH-benzyloxycarbonyl.
Fig. 3 GlcNAcT-II and HS-GlcAT-II assays using a syringe column.
Fig. 4 Analysis of polymerized HS chains.
(A) The polymerization reaction products were analyzed on a Superdex peptide column .
(B) Molecular size of HS chains was determined by gel filtration chromatography on a Superdex 200 column. White arrowheads indicate the elution positions of commercial dextrans of known molecular weights.