Revelation of Antiviral Activities by Artificial Sulfation ofa Glycosaminoglycan from a Marine Pseudomonas

Ahmad Shamsuddin Ahmad,1 Masahiro Matsuda,1,* Shiro Shigeta,2 and Koichi Okutani1

1Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan2Faculty of Medicine, Fukushima Medical School, Fukushima 960-1247, Japan

Abstract: Sulfated derivatives of a glycosaminoglycan containing L-glutamic acid produced by a marine Pseu-

domonas species, No. 42 strain, were prepared by the method of dicyclohexyl-carbodiimide-mediated reaction.

Both low and high degrees of sulfation of the polysaccharides (products A1 and A2, respectively) were inves-

tigated for their antiviral activities against influenza virus type A (FluV-A) and B (FluV-B) in MDCK cells. Both

preparations showed antiviral activity against FluV-A at the 50% antiviral effective concentration of 17.3 and

5.2 µg/ml, respectively, whereas they had no antiviral activity against FluV-B. No cytotoxicity of either product

was noted against MDCK cells at the 50% cytotoxic concentration of 100 µg/ml.

Key words: marine Pseudomonas, glycosaminoglycan, antiviral activity, artificial sulfation

INTRODUCTION

In recent years many reports on the effects of both naturally

occurring and artificially synthesized sulfated polysacchari-

des on several viruses in vitro, including human immuno-

deficiency viruses, have been published (Baba et al., 1988a;

Hirabayashi et al., 1989; Okutani, 1992; Okutani and

Shigeta, 1993; Hasui et al., 1995). However, no information

on the sulfated polysaccharide of a novel structure such as

glycosaminoglycan having amino acids as a constituent was

reported. As described previously a marine bacterium Pseu-

domonas sp., strain No. 42, which was originally isolated

from seawater, produced glycosaminoglycan containing L-

glutamic acid when grown on seawater agar medium

(Worawattanamateekul et al., 1992). This polysaccharide

was composed of linear repeating units having N-acetyl-D-

glucosamine (D-GlcNAc), D-glucuronic acid (D-GlcUA),

and L-glutamic acid (L-Glu) in a molar ratio of 2:1:1, as well

as O-acetyl groups (Shamsuddin et al., 1998).

In the present report we describe the synthesis of the

sulfated derivatives of this glycosaminoglycan and their an-

tiviral activities against influenza virus in vitro.

MATERIALS AND METHODS

Preparation and Purification of the Polysaccharide

Pseudomonas sp. No. 42, which was isolated from the sea-

water of the Seto Inland Sea, Japan, was grown on seawater

agar plates containing 0.5% peptone, 0.1% yeast extract,

and 1.5% agar supplemented with 3% sucrose for 4 days at

25°C (Worawattanamateekul et al., 1992). Viscous polysac-

charide produced on the agar plates containing cellular ma-

terials was collected by scraping from the agar surface and

suspended in 1% phenol solution. After stirring overnight

at 4°C, the bacterial cells were removed by filtration

through a Whatman GFF filter. The polysaccharide wasReceived April 4, 1998; accepted July 24, 1998.

*Corresponding author. Fax: 087-891-3021; e-mail: matsuda@ag.kagawa-u.ac.jp

Mar. Biotechnol. 1, 102–106, 1999

© 1999 Springer-Verlag New York Inc.

precipitated with 2 vol of ethanol. The crude polysaccharide

obtained was dissolved in water and purified by precipita-

tion with cetyltrimethyl-ammonium bromide (Cetavlon).

The precipitated Cetavlon-polysaccharide complex was col-

lected by filtration and redissolved in 4 M NaCl solution.

The polysaccharide component was recovered by precipita-

tion with 2 vol of ethanol, followed by filtration. This ma-

terial was dissolved in water and dialyzed against deionized

water, followed by freeze-drying to afford the sodium salt of

the polysaccharide. The yield of this polymer was 250 mg/L

of the medium (native polysaccharide).

Sulfation of the Polysaccharide

Sulfation of the polysaccharide was achieved essentially by

the method of Takano et al. (1996). Briefly, the native poly-

saccharide (910 mg) was dissolved in water and treated with

Amberlite IR-120 (H+ form), followed by dialysis against

deionized water. The polysaccharide solution was neutral-

ized with pyridine and freeze-dried (780 mg). The resulting

pyridinium salt of the polysaccharide was dissolved in N,N-

dimethylformamide (DMF, 50 ml), then we added N,N8-

dicyclohexylcarbodiimide (DCC) dissolved in DMF (3 g/40

ml) and sulfuric acid dissolved in DMF (0.2 ml/5 ml). After

the reaction mixture was stirred for 1 hour (partial sulfa-

tion) or 3 hours (oversulfation) at 0°C under nitrogen gas,

crushed ice was added to the reaction mixture, then it was

neutralized with sodium hydroxide solution, followed by

dialysis, and freeze-dried (750 mg). The sulfation procedure

was repeated on this sulfated polymer by the same method

as described above. The polysaccharide was purified by

chromatography on DEAE-cellulose column using stepwise

elution with 0.01 M phosphate buffer (pH 7.0) containing 0

to 2.0 M NaCl. Each fraction was collected and dialyzed

against deionized water, followed by freeze-drying.

Analytical Methods

Electrophoresis was performed on cellulose acetate strips

(Sartorius 11200, 57 × 145 mm) in 0.2 M calcium acetate

buffer (pH 7.5) or in 0.05 M sodium borate buffer (pH 9.4).

Polysaccharide and monosaccharide bands were visualized

with 0.1% alcian blue in 10% acetic acid or alkaline silver

nitrate reagent, respectively.1H-Nuclear magnetic resonance (NMR) spectra were

obtained with a Jeol Alpha 400 spectrometer (400 MHz) at

75°C. The samples were dissolved in D2O containing 3-tri-

methylsilyl-propionic acid sodium salt (TSP) as the internal

reference.

Paper chromatography (PC) was carried out by the

descending method on Whatman No. 1 paper with ethyl

acetate–acetic acid–formic acid–water (18:3:1:4) as the sol-

vent. Sugars were detected with alkaline silver nitrate, p-

anisidine hydrochloride, or ninhydrin reagents.

Sugars were also analyzed by high-performance liquid

chromatography (HPLC) with a Hitachi 655 HPLC

equipped with a refractive index detector on a Wakopak

WBT 130E column (Wako Pure Chemicals, 7.8 × 300 mm)

using water as a mobile phase at 60°C at a flow rate of 0.5

ml/min.

The mean molecular weight of the polysaccharide was

determined with a Hitachi 655 HPLC equipped with a re-

fractive index detector on an Asahipak GFA 7M column

(Asahi Chemicals, 7.6 × 500 mm). For minimizing the as-

sociation effect of the polysaccharide solution, aqueous 0.1

M NaCl solution was used as a mobile phase at 30°C and at

a flow rate of 0.4 ml/min. The molecular size was calibrated

with pullulans of various molecular weights (Shodex Stan-

dard Kit P-82, Showa Denko).

Component Analyses

For the analyses of amino acids and amino sugars, the poly-

saccharide was hydrolyzed with 4 N HCl for 12 hours at

100°C, followed by a concentration to dryness over P2O5

and NaOH in vacuo. The residue was analyzed by PC, elec-

trophoresis, and an amino acid analyzer.

Sulfate content was determined with a Shimadzu HIC-

6A ion chromatograph equipped with a Shimpack IC-A1

column (4.6 × 100 mm) and a conductivity detector using

2.5 mM phthalic acid containing 2.4 mM tris(hydroxy-

methyl) aminomethane (pH 4.0) as a mobile phase at 40°C

after hydrolysis of the sample.

Uronic acids were identified by both PC and electro-

phoresis after hydrolysis of the polysaccharide with 2 M

TFA for 12 hours at 100°C. Uronic acid content was deter-

mined by the carbazole–sulfuric acid method (Dische,

1947).

Viruses and Cell Culture

Ishikawa/7/82 (H3N2) strain of influenza virus type A

(FluV-A) and Singapore/222/79 strain of influenza virus

type B (FluV-B) were grown on Madin-Darby canine kid-

ney (MDCK) cells.

The growth medium for MDCK cells consisted of Ea-

gle’s minimum essential medium (MEM, Nissui Pharma-

ceutical Co., Tokyo) supplemented with 10% heat-

Antiviral Polysaccharide from Marine Pseudomonas 103

inactivated fetal calf serum (FCS) (Cellculture Lab., Cleve-

land, Ohio), 2 mM L-glutamine, 50 U/ml penicillin G, and

20 µg/ml gentamicin. The viability of cells was determined

colorimetrically by the MTT method, which is based on the

mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (Pouwels et al., 1988).

Antiviral Assays

The inhibitory effects of the test compounds on influ-

enza virus replication were monitored by the inhibition of

virus-induced cytopathic effect in MDCK cells. The test

compounds were diluted and 100-µl samples of various

concentrations of the test sample were plated in a round-

bottomed microtiter tray. One hundred microliters of the

trypsinized cells (1 × 104 cells) and 100 µl of 100 MTT-

TCID50 (1 MTT-TCID50 being the 50% cytopathic dose for

cell culture determined by the MTT method) of the virus

were added to each well, and the plates were then centri-

fuged (700 × g) for 5 minutes at room temperature, then

incubated at 37°C in 5% CO2. After a 5-day incubation, the

number of viable cells was determined by the MTT method.

The 50% antiviral effective concentration (EC50) and

the 50% cytotoxic concentration (CC50) of the test sample

were determined.

RESULTS AND DISCUSSION

Preparation of the Sulfated Polysaccharides

The native polysaccharide was homogeneous in electropho-

retic analysis (Shamsuddin et al., 1998). Both products of

partial sulfation and oversulfation (products A1 and A2,

respectively) gave main fractions in DEAE-cellulose column

chromatographies upon elution with 1.0 M NaCl buffers

(Figure 1). Both products (A1 and A2) were found to be

homogeneous in electrophoresis and gel-filtration analysis

(Figure 2) and had the molecular weight of 130 and 150

kDa, respectively. Components of products A1 and A2 were

GlcNAc, GlcUA, glutamic acid, and sulfate in a molar ratio

of 2:1:1:4 and 2:1:1:8, respectively (Table 1). This indicates

that both products had the same monosaccharide constitu-

ents as those of the unsulfated native polysaccharide, except

containing sulfate groups and the loss of O-acetyl groups

judging from NMR analyses (Figure 3). Although there

were great differences in molecular weight between the na-

tive and sulfated polysaccharides, no difference in the main

components between both products was observed.

Antiviral Effect of the Polysaccharide

The effects of the sulfated polysaccharides on viral replica-

tion are shown in Table 2. The unsulfated polysaccharide

(native polysaccharide) had no antiviral activity. The sul-

Figure 1. Fractionation of the sulfated polysaccharide on a DEAE-

cellulose column. The samples (96 mg for partially sulfated poly-

saccharide, 117 mg for oversulfated polysaccharide) were dissolved

in 0.01 M phosphate buffer and applied to the column (2.0 × 32

cm). Fractions (each 300 ml) from the column were dialyzed

against deionized water, followed by freeze-drying. Fractions

eluted with 1.0 M NaCl buffers from the partially sulfated and

oversulfated polysaccharides were expressed as products A1 and

A2, respectively.

104 Ahmad Shamsuddin Ahmad et al.

fated polysaccharides, however, showed antiviral activity

against the FluV-A but not against FluV-B (EC50 > 100

µg/ml for both A1 and A2). These results indicate that the

products A1 and A2 had antiviral activities against FluV-A

comparable to those of ribavirin (1-b-D-ribofuranosyl-

1,2,4-triazole-3-carboxamide), dextran sulfate 500,000

(EC50 = 1.0 µg/ml), and dextran sulfate 8000 (EC50 = 5.8

µg/ml) (Hasui et al., 1995). However, for the polysaccharide

with a lower degree of sulfation (product A1), the inhibitory

effect was noted only at a higher concentration, perhaps

being related to the degree of sulfation. Although the

mechanism has not yet been clarified, both products A1 and

A2 had the general properties of the sulfated polysacchari-

des such that they had weak or no antiviral activity against

FluV-B (Hasui et al., 1995). The native polysaccharide con-

tained no sulfate group at all, whereas products A1 and A2,

contained one and two sulfate groups per sugar residue

(including glutamic acid), respectively. These sulfate values

are similar to those of other sulfated polysaccharides, such

as heparin (1.25 sulfate groups per sugar residue) and dex-

tran sulfate 7000 (2–3 sulfate groups per sugar residue)

Figure 2. GPC profiles of the sulfated polysaccharides. Molecular

sizes of the polysaccharides were calculated as described in the text.

Products A1 and A2 are the same as in Figure 1. Native indicates

native polysaccharide.

Table 1. Component Analyses of the Sulfated Polysaccharides

Polysaccharide*

Components†

GlcN Glu GlcUA Sulfate

A1 2.0 1.0 1.0 4.3

A2 2.2 1.0 1.0 8.0

*Products A1 and A2 are the same as in Figure 1.

†Components are expressed as the relative molar ratio. GlcN indicates

glucosamine; Glu, glutamic acid; GlcUA, glucuronic acid.

Figure 3. 1H-NMR spectrum of the sulfated polysaccharide. The

polysaccharides were dissolved in D2O and measured at 75°C with

TSP as the internal reference. Products A1 and A2 are the same as

in Figure 1. Native, indicates native polysaccharide. Arrows indi-

cate the position of methyl portion of O-acetyl groups.

Antiviral Polysaccharide from Marine Pseudomonas 105

(Takemoto and Fabisch, 1964). The inhibitory effects of

various polyanions on viruses have suggested that these

substances influence the primary electrostatic attachment of

the virus to the cell prior to viral penetration (Baba et al.,

1988b; Nahmias et al., 1964).

In sum, the introduction of sulfate groups into the

glycosaminoglycan containing L-glutamic acid produced by

marine Pseudomonas sp. No. 42 strain showed anti-FluV-A

activity, and the level of the activity seems to be related to

the degree of sulfation. Products A1 and A2 had no cyto-

toxicity against MDCK cells at the 50% cytotoxic concen-

tration of 100 µg/ml.

ACKNOWLEDGMENTS

This work was partly supported by a Grant-in-Aid for Sci-

entific Research from the Ministry of Education, Science,

and Culture of Japan. The authors thank Dr. S. Tajima

(Kagawa University) for his helpful comments during the

development of this study.

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Table 2. Antiviral Activities and Cytototoxicities of the Sulfated

Polysaccharides

Samples*

EC50 (µg/ml)

CC50 (µg/ml)FluV-A FluV-B

Native >100 >100 >100

A1 16.0–18.6 >100 >100

A2 5.2 >100 >100

Ribavirin 3.1–5.7 8.9–9.2 >100

*Products A1 and A2 are the same as in Figure 1. Native indicates native

polysaccharide.

106 Ahmad Shamsuddin Ahmad et al.