# γ-Cyclodextrin Increases Hydrolysis of Gangliosides by Sialidase from Arthrobacter ureafaciens: Hydrolysis of Gangliosides

Authors: Rie Mitsumori; Tomohisa Kato; Kenichi Hatanaka
Journal: International Journal of Carbohydrate Chemistry
 (2009)

---

## Abstract

Sialidase is a ubiquitous enzyme that catalyzes the hydrolytic removal of terminal sialic acid residues from oligosaccharides in glycolipids and glycoproteins. Ganglioside GM1 has been usually found to be resistant to various sialidases.Arthrobacter ureafaciens sialidase has been reported to remove sialyl residues of ganglioside GM1 in the presence of bile salts. However, bile salts are difficult to be removed, and disturb HPTLC analysis. Using γ-cyclodextrin (γ-CD) as a novel additive agent, ganglioside GM1 was efficiently hydrolyzed to asialo-GM1 by A. ureafaciens sialidase.


---

## Body

## 1. Introduction

Gangliosides are glycolipids
which contain sialic acid residues withα2-3, α2-6, and α2-8 linkages, and are primarily localized on the outer surface of mammalian cells.
The biologic functions of gangliosides are attributed to the acidic
carbohydrate chains [1] and have been found to be important for cell growth,
differentiation, cell-to-cell interactions, and signal transduction [2, 3]. The
linkages of sialic acid residues are enzymatically hydrolyzed by various sialidases
from viruses, bacteria, and mammals [4, 5]. While sialidase from leech, Macrobdella decora [6, 7], does not
cleave the sialic acid residues of GD1b and GT1b to form GM1, the major
gangliosides in mammalian brain, GD1a, GD1b, and GT1b are hydrolyzed into GM1
with most commercially available sialidases of bacterial origin, such as Vibrio cholerae and Clostridium perfringens [8, 9]. These bacterial sialidases cleave
the external NeuAcα2-3Gal in GD1a and GT1b, and NeuAcα2-8NeuAc
in GD1b, but not the internal NeuAcα2-3Gal
due to the steric hindrance by neighboring GalNAc [6, 10, 11]. However, three
isoenzymes (L, M1, M2) from Arthrobacter ureafaciens can hydrolyze the internal sialic acid
of GM1 under optimal condition with sodium cholate [7]. Recently, it was
reported that the C-terminal peptide of isoenzyme M2 is essential to prevent
the effect of the neighboring GalNAc and cleave the sialic acid at the internal
galactose residue [12]. Recently, it was reported thatγ-cyclodextrin
(γ-CD) was an efficient supporting material for glycosyltransferase reaction [13]. Therefore,
an effect of γ-CD on sialidase reaction is interesting.Recently, it was reported that asialo-GM1 contributes to various diseases
such as carcinogenesis, autoimmune disease, or
neurologic disease [14, 15]. It is hoped that understanding of action mechanisms
of various asialo gangliosides is useful for medical treatment
in future. In this paper, we attempted to prepare asialo gangliosides by using A. ureafaciens sialidase and γ-CD.


## 2. Materials and Methods

### 2.1. Materials

All organic solvents of analytical grade, ganglioside
GM1, crude ganglioside mixture (from rat brain), andA. ureafaciens sialidase (mixture of isoenzymes L, M1, M2 and S) were
purchased from Nacalai Tesque, Kyoto, Japan. α-cyclodextrin (α-CD), β-cyclodextrin (β-CD),
and γ-CD were purchased from WAKO, Osaka, Japan. Sep-Pak
C18 columns were purchased from Waters, Milford, MA, USA.


### 2.2. Sialidase Reaction

The reaction mixture for sialidase was comprised of 3μg
of ganglioside GM1 (or 10 μg
of crude ganglioside mixture), 0.1 M acetate buffer, pH 5.5, and 2–500 mU of A. ureafaciens sialidase, to a final
volume of 200 μL. Then the solution was incubated at
37°C for 24 hours [16]. After the reaction, the solution was treated with
a Sep-Pak C-18 cartridge immediately [17]. The entire sample was analyzed by high-performance
thin-layer chromatography (HPTLC). The addition of other components is mentioned
in the text.


### 2.3. HPTLC Analysis

Amphiphilic glycolipids were analyzed on an HPTLC plate
(Merck, Darmstadt, Germany). The developing solvent
used was chloroform-methanol-0.25% aqueous KCl (5:4:1, v/v). The separated
glycolipids were detected by spraying with resorcinol reagent and heating at
120°C for 15 minutes in an oven [17, 18]. Densitogram was obtained from HPTLC
by using Scion Image Software (Scion Corporation, http://www.scioncorp.com) to
quantify glycolipids.


### 2.4. Mass Spectral Analysis

Mass spectra were recorded on a high-capacity ion trap
mass spectrometer (HCTultra). At least ten scans were averaged to obtain each
spectrum. Samples were typically dissolved in chloroform/methanol (2:1, v/v) to
a concentration of 1 mg/mL, and diluted to 1/1000 with 95% acetonitrile. Sample
solution was injected to the ESI ion source by mechanical infusion at a flow
rate of 180μL/h. Analysis of neutral glycolipids
was carried out in positive ion mode. The following analysis parameters were
used: high voltage (HV) capillary voltage −4500 V, HV end plate offset voltage
−500 V, nebulizer gas pressure 15.0 psi (nitrogen), a flow rate of dry gas 7.0 l/min
(nitrogen, temperature 320°C), and skimmer voltage 40.0 V.


## 2.1. Materials

All organic solvents of analytical grade, ganglioside
GM1, crude ganglioside mixture (from rat brain), andA. ureafaciens sialidase (mixture of isoenzymes L, M1, M2 and S) were
purchased from Nacalai Tesque, Kyoto, Japan. α-cyclodextrin (α-CD), β-cyclodextrin (β-CD),
and γ-CD were purchased from WAKO, Osaka, Japan. Sep-Pak
C18 columns were purchased from Waters, Milford, MA, USA.


## 2.2. Sialidase Reaction

The reaction mixture for sialidase was comprised of 3μg
of ganglioside GM1 (or 10 μg
of crude ganglioside mixture), 0.1 M acetate buffer, pH 5.5, and 2–500 mU of A. ureafaciens sialidase, to a final
volume of 200 μL. Then the solution was incubated at
37°C for 24 hours [16]. After the reaction, the solution was treated with
a Sep-Pak C-18 cartridge immediately [17]. The entire sample was analyzed by high-performance
thin-layer chromatography (HPTLC). The addition of other components is mentioned
in the text.


## 2.3. HPTLC Analysis

Amphiphilic glycolipids were analyzed on an HPTLC plate
(Merck, Darmstadt, Germany). The developing solvent
used was chloroform-methanol-0.25% aqueous KCl (5:4:1, v/v). The separated
glycolipids were detected by spraying with resorcinol reagent and heating at
120°C for 15 minutes in an oven [17, 18]. Densitogram was obtained from HPTLC
by using Scion Image Software (Scion Corporation, http://www.scioncorp.com) to
quantify glycolipids.


## 2.4. Mass Spectral Analysis

Mass spectra were recorded on a high-capacity ion trap
mass spectrometer (HCTultra). At least ten scans were averaged to obtain each
spectrum. Samples were typically dissolved in chloroform/methanol (2:1, v/v) to
a concentration of 1 mg/mL, and diluted to 1/1000 with 95% acetonitrile. Sample
solution was injected to the ESI ion source by mechanical infusion at a flow
rate of 180μL/h. Analysis of neutral glycolipids
was carried out in positive ion mode. The following analysis parameters were
used: high voltage (HV) capillary voltage −4500 V, HV end plate offset voltage
−500 V, nebulizer gas pressure 15.0 psi (nitrogen), a flow rate of dry gas 7.0 l/min
(nitrogen, temperature 320°C), and skimmer voltage 40.0 V.


## 3. Results and Discussion

We attempted to prepare asialo
gangliosides by usingA. ureafaciens sialidase (mixture of isoenzymes L, M1, M2, and S) and sodium cholate. 10 μg
of crude ganglioside mixture (from rat brain) was hydrolyzed under various
conditions and the purified sample was analyzed by HPTLC. The addition of sodium
cholate into the reaction solution was effective for the cleavage of the
internal sialic acid residue of GM1. However, sodium cholate could not be
removed by using a Sep-Pak C18 cartridge and impeded HPTLC analysis (Figure 1(a)).
Generally, removal of detergents such as sodium cholate, sodium deoxycholate,
and sodium chenodeoxycholate is complicated. Recently, it was reported that γ-CD
was an efficient supporting material for the blotting of hydrophobic substrates
to water-soluble polymers and for the synthesis of neoglycolipids using
glycosyltransferase [13]. Therefore, a stimulating effect of γ-CD on sialidase reaction was investigated. As
shown in Figure 1(b), cleavage of the internal sialic acid residue of GM1 was effectively
promoted by  γ-CD and asialo-GM1 could be completely
separated from γ-CD by HPTLC. γ-CD
has the ability to include the hydrophobic part of compounds and the inclusion complex
becomes more soluble in the reaction solvent when compared with the
noncomplexed compounds. It has been reported that α-CD and dimethyl-β-cyclodextrin activate the hydrolysis of
ganglioside GM1 by acid β-galactosidases
[19]. The activation of the hydrolysis by sialidase may correlate with the
inclusion complex formation according to a mechanism suggested for the acid β-galactosidases
reactions.Effect of (a) sodium cholate and (b)γ-CD on hydrolysis of gangliosides by A. ureafaciens sialidase. 200 mU of sialidase was added (+) or not added (−) to the reaction
solution. Samples purified by using Sep-Pak C-18 cartridge were analyzed by
HPTLC with chloroform/methanol/0.25% aqueous KCl (5:4:1, v/v) as developing
solvent. The HPTLC plates were sprayed with resorcinol reagent and heated at
120°C to detect the separated glycolipids.
(a)(b)Todetermine the optimum γ-CD concentration for sialidase reaction, γ-CD
was added to the reaction solution at a final concentration of 0.01, 0.05, 0.25,
1.25, 5, or 10 mM. An increase of the concentration of γ-CD allows for ganglioside GM1 to be converted
to asialo-GM1 (Figure 2). GM1 completely hydrolyzed at concentrations of γ-CD
higher than 5 mM. α-CD and β-CD
were not as efficiently enhancing cleavage of the internal sialic acid as γ-CD
at a final concentration of 5 mM. The yields of asialo-GM1 produced in the
presence of α-CD and β-CD
were 3 and 10% of the yield of that produced in the presence of γ-CD, respectively. In the presence of 5 mM of γ-CD,
3 μg ganglioside GM1 has been completely digested by 100 mU of A. ureafaciens sialidase (Figure 3). The product of enzyme-catalyzed
desialylation from ganglioside GM1 was identified by MS/MS analyses. As shown
in Figure 4, the parent and fragment mass ions of asialo-GM1 were detected as monovalent
cations (m/z 1277.8: [M+Na]+ and m/z 912.7: [M-Gal-GalNAc+Na]+).
Under optimum condition described above, various kinds of gangliosides (GM1,
GD1a, GD1b, and DT1b) were completely deacylated by A. ureafaciens sialidase (Figure 5).Effect of the concentration ofγ-CD. 200 mU of sialidase was added (+), or not
added (−) to the reaction solution. HPTLC analysis was performed as described
in Figure 1. (a) Result of HPTLC analysis. (b) The amounts of ganglioside GM1
and asialo-GM1 after enzymatic reaction. □: ganglioside
GM1; ⧫: asialo-GM1.
(a)(b)Effect of the concentration of sialidase. 5 mM ofγ-CD
was added to reaction solution (+), or not added (−). HPTLC analysis was
performed as described in Figure 1. (a) Result of HPTLC analysis. (b) The
amounts of ganglioside GM1 and asialo-GM1 after enzymatic reaction. □: ganglioside GM1; ⧫: asialo-GM1.
(a)(b)Figure 4
Mass Spectra of the product from ganglioside GM1 after
sialydase reaction. Analysis of sample was carried out on high-capacity ion
trap mass spectrometer (HCTultra) in positive mode. The product was prepared by
scraping off the adsorbent at the corresponding spot site on the HPTLC plate.Figure 5
Desialylation of various kinds of ganglioside. HPTLC analysis
was performed as described in Figure1.γ-CD does not disturb HPTLC and HPLC analysis
and is useful for enzymatic desialylation. Nagashima et al. have
reported that γ-CD
is an efficient supporting material for the blotting of hydrophobic substrate
and for the synthesis of neoglycolipids using glycosyltransferase [13]. The
utility of γ-CD
is an efficient and widely useful method for chemical and enzymatic reactions
of glycolipids and (oligo)saccharide derivatives. Furthermore, asialo
glycolipid will be useful for study of a physiological function and the
industrial use by effective preparation methods.



---

*Source: 398284-2009-02-02.xml*