Journalof Applied Phycology 8: 155-161, 1996. © 1996 KluwerAcademic Publishers. Printedin Belgium.
155
Annual variation in composition and in vitro anti-HIV-1 activity of the sulfated glucuronogalactan from Schizymenia dubyi (Rhodophyta, Gigartinales) N. Bourgougnonl,*, M. Lahaye 2 , B. Quemener 2 , J-C Chermann3 , M. Rimbert 4 , M. Cormaci5 , G. Furnaris & J-M Komprobst 4 1
Laboratoirede Biologie et Biochimie Marines, P6le Sciences et Technologie, Universite de La Rochelle, avenue Marillac,17042 La Rochelle, France (email: nathalie.bourgougnon@biolo. univ-lr.fr) 2 Laboratoirede Biochimie et Technologie des Glucides, INRA BP 527, 44026 Nantes Cedex 03, France (email:
[email protected]) 3 INSERM U322, Campus Universitairede Luminy BP33, 13273 Marseille, Cedex 09 France 41SOMer, Facultede Pharmacie,1 rue Gaston Veil, 44035 Nantes, Cedex 01 France 5 Dipartimentodi Botanica, Universitadi Catania,Sicily, Italy (*Authorfor correspondence) Received 26 April 1996; revised 22 May 1996; accepted 28 May 1996
Key words: Schizymenia dubyi, sulfated polysaccharide; antiviral; HIV-1
Abstract An account is given of the annual variation in composition and in vitro anti-HIV-1 activity of the water-soluble sulfated glucuronogalactan from Schizymenia dubyi collected in Sicily. No marked variation seasonal chemical or physico-chemical features was observed, with the exception of L-galactose residues and viscosity. Evaluation of the anti-HIV- effect of the polysaccharide indicated that syncitial formation and HIV-associated reverse transcriptase in vitro were completely suppressed at 5 /jgml-' by alga collected during the summer/spring period. This high anti-HIV- 1 activity corresponded with a decrease in L-galactose, viscosity and sulfate content of the polysaccharide. Introduction Marine algal cell walls contain a large quantity of characteristic mucilaginous polysaccharides which have recently been demonstrated to reveal some biological activities (Noda et al., 1989; Given et al., 1990; Indergaard & Ostgaard, 1991; Witvrouw et al., 1994). Red algal cell-walls contain large amounts of sulfated galactans such as agars and carrageenans, generally built on the repeating alternating 1,3-linked /D-galactopyranose and 1,4-linked anhydro-3,6-L- or D-a-galactopyranose. Different repeating structures have been identified on the basis of the sulfation pattern and of the substitution by methoxyl, glycosyl and/or pyruvate groups (Craigie, 1990; Usov, 1992; Knutsen et al., 1994). A sulfated polysaccharide isolated from Schizymenia pacifica (Kylin) Kylin (Gigartinales, Gymmnophlaeaceae) with chemical characteristics close to that of carrageenans inhibits in vitro
Human Immunodeficiency Virus (HIV) replication and reverse transcriptase (Nakashima et al., 1987a, b). The sulfated glucuronogalactan from Schizymenia dubyi (Chauvin ex Duby) J. Agardh collected in Sicily differed from the other generally encountered red algae by its unusually high content of glucuronic acids (Bourgougnon et al., 1993). Indeed, the gametophytic S. dubyi (Sicily) was shown to contain a sulfated polysaccharide composed of 1/0.75/1.3 of galactose, glucuronic acid and sulfate groups. 45% of total galactose was in a L-form and no 3,6-anhydrogalactose was detected (Bourgougnon et al., 1996a). This polysaccharide marked in vitro specific activity against several viruses, including HIV-1 (Bourgougnon et al., 1993; 1996b). The present work relates to the seasonal variation of its chemical composition and its relationship to the in vitro anti-HIV activity of S. dubyi sulfated glucuronogalactan.
156 Materials and methods Materials Gametophytic Schizymenia dubyi samples were collected on the east coast of Sicily, at St Maria La Scala (Catania) in April, May, June and September 1993 and at St Giovanni Li Cuti (Catania) in June, July, November and December 1993, January, February and March 1994. The alga was collected at the second location because of its scarce quantity at the first station during these periods. Generalmethods Extractionprocedure Sulfated polysaccharide from air-dried seaweed powder was extracted in hot distilled water and precipitated with 950 ethanol (Bourgougnon et al., 1993). Due to the low amount of available algae, only one extraction was performed. Thus, statistical analysis of yields with regard to sample was not possible. The composition of neutral sugars and sulfate content were previously described by Bourgougnon et al. (1996b). Uronic acids, 3, 6-ankydrogalactose, ash and D-galactosecontents Uronic acids in the above acid hydrolysates were measured by colorimetry according to the method of Ahmed & Labavitch (1977) automatized by Thibault (1979) and using glucuronic acid as standard. 3,6-Anhydrogalactose content was determined by colorimetry with the resorcinol reagent (Yaphe & Arsenault, 1965). Protein content was measured by the Kjeldahl method (nitrogen content x 6.25). Ash values were determined gravimetrically after incineration of samples at 550 C for 16 h, followed by 2 h at 900 OC. D-galactose content was determined by p-galactose dehydrogenase bioassay (Cat. No. 176303, Boehringer Mannheim, France). Viscosimetry The intrinsic viscosity of polysaccharide was determined at 40 C in 150 mM NaCI with an automatic capillary Ubbelholde Viscometer (Amtec, France) and the intrinsic viscosity was derived from Kraemer and Huggins equations by extrapolation to infinite dilutions (Billmeyer, 1984).
Virus and cells The virus strain used was HIV- 1I(HIV- 1-LAV Pasteur strain). The MT4 cells were grown in RPMI 1640 medium containing 10% FCS (Dutscher) and PSN antibiotics (Whittaker), 1% glutamine (Whittaker) and pug ml- ' polybrene (Whittaker) (Barr6-Sinoussi et al., 1983). Cytotoxicity assay To assess the effect of polysaccharide on uninfected MT4 cells, varying concentrations of polymer in the maintenance medium were added to the MT4 cell line at 37 C with 5% CO 2 during one hour in a 96-well microtiter plate containing 3 x 105 MT4 cells for 100 u1 of compounds (in duplicate). The cells were then readjusted to 3 x 105 MT4 ml- l in a 24-well microtiter plate containing various concentrations of polysaccharide. After 8 days the cells were diluted to 1/3 with the corresponding dilution of compound. Cell viability was measured by the cells exclusion with trypan blue. Determination of antiviralactivity of sulfatedpolysaccharide Determination of activity against HIV-1 was based on measuring the protective effect of sulfated polysaccharide against virus-induced cytopathogenicity in MT4 cells (syncitia formation) and on measuring reverse transcriptase activity on cell free supernatant. Four to six days after infection, syncitia formation was observed, preceeding cell death. MT4 cells were incubated in maintenance medium at 37 °C during one hour in a 96-well microtiter plate containing various concentrations of sulfated polysaccharide (3 x 104 cells for 100 pi of compound). 100 pul of HIV-1 (dilution 10- 3) suspension was added to each well. One hour after incubation, infected cells were washed three times with maintenance medium and then cocultured at 3 x 105 cells ml - l in a 24-well microtiter plate containing various concentrations of the polysaccharide. Every three or four days, cell suspensions were diluted three times and the cell concentration adjusted to 3 x 105 cells mlmaintenance medium in the presence of the polysaccharide. Three days after culture, infected MT4-HIV1 cells with and without sulfated polysaccharide and uninfected MT4 cells were observed for syncitial formation every one or two days. For the antiretroviral assay, MT4 cells were infected with HIV-1 (100 TCIU ml- ). Cultures were incubated in the presence of varying concentrations of polysaccharide, and the number of
157 viable cells was recorded after 8 days by the method described by Rey et al. (1987).
Results and discussion Annual variationsof the anti-HIV activity of the sulfatedpolysaccharidesfromS. dubyi The in vitro anti-HIV activities of the water-soluble polysaccharide of Schizymenia dubyi are shown in Tables 1, 2, and 3. The determination of antiviral activity with HIV-1 was based on the measure of the protective effect of sulfated polysaccharide against the virus-induced cytopathogenicity in MT4 cells during 8 days. The syncitial formation was completely suppressed with 5 /tg ml-' of galactan extracted from S. dubyi collected from April to July. Furthermore, this polysaccharide inhibited the HIV-1 reverse transcriptase in cell free supernatant at concentrations as low as 5 Mig ml- 1 without cytotoxicity on MT4 cells. The polymer extracted from the alga collected from January to March (Table 1) inhibited the HIV1 reverse transcriptase at slighlty high concentrations (10 /ig ml-l). The syncitial formation was completely suppressed at this concentration of galactan and was delayed at 5 /ig ml-'. No cytotoxicity toward MT4 cells was found at these concentrations but the polysaccharide was more toxic than the polymer from the April to July collections (Table 2). Polysaccharide extracted from algae collected in September to December inhibited the HIV- 1 reverse transcriptase and syncitial formation at concentrations as low as 25 jg ml- l. No cytotoxicity towards MT4 cells was found at these concentrations (Table 3). Comparison of the variation of anti-HIV activities enables us to establish a seasonal variation of greater efficiency and less cytotoxicity of the sulfated polysaccharide from S. dubyi. From April to July, a decrease of cytotoxicity with an increase of activity was observed. Inhibition of syncitia formation and HIV reverse transcriptase vary in the same way. Annual variationsof the chemical composition of the sulfatedpolysaccharidesfromS. dubyi The monthly yield and chemical composition of the water-soluble polysaccharide from Schizymenia dubyi is shown in Tables 4 and 5. The maximum yield (26%) was obtained in April and corresponds to a period during which gametophytes were abundant. During summer, thalli were covered up withs diatoms and
began to wither. The minimum yield was 18% and was obtained from algae collected in July. Sugar analysis showed that the polysaccharide consisted mostly of galactose (Table 4), the content of which varied from 29.4% mol in winter to 40.6% mol in summer. The Lgalactose form appeared at the beginning of summer (21% in May), reached a maximal level in September (42%) and decreased to an undetectable level in spring (Table 5). The content of xylose (1.0-2.0% molar) and uronic acids (13.5-18.9% molar) were constant during the year but the small amount of glucose (0.4-2.1% molar) disappeared in autumn. The sulfate content varied during the year (41.9 to 54.3% molar). These results differed from values obtained from the sulfated glucuronogalactan isolated from S. dubyi collected in May 1991. In particular, the latter polysaccharide contained opposite the molar ratio of sulfate to uronic acid (15.6%-33.7% dry weight) (Bourgougnon et al., 1993). The content of residual proteins was constant during winter and autumn (2.3-3.4% dry weight) but decreased from April (1.5%) to a nondetectable level in July. No marked variation in the chemical composition was noticed between sulfated glucuronogalactan extracted from algae collected at the two different locations. The intrinsic viscosity of the polysaccharide remained constant throughout the year (310-355 ml g-l) except for lower (251-159 ml g- 1) and higher (532 ml g-l) values in June and November/January, respectively (Table 6). Thus, the molecular weight of the sulfated glucuronogalactan remains relatively constant over the year. Following the suggestion that polyanionic compounds related to suramin may inhibit HIV replication through interaction with the virus adsorption step, chemically sulfated polysaccharides such as dextran sulfate, heparin, and pentosan polysulfate or polysaccharides extracted from algae, such as fucoidan or carrageenan, have recently proved to be highly potent and selective inhibitors of HIV-1 replication, in vitro (Nakashima et al., 1987a, b; Baba et al., 1990a, b; Itoh et al., 1990; Schols et al., 1990; Mc Clure et al., 1992; Damonte et al., 1994; Witvrouw et al., 1994). Since virus-cell binding was based on the interaction between the glycoprotein gp120 of the virus envelope and the CD4 receptor of the target cells, these polymers may also interact with gpl20 and CD4 receptor to block giant cell (syncitium) formation between HIV- 1-infected cells and uninfected CD4-positive cells (Pauwels et al., 1991; Baba et al., 1990a, b; Witvrouw et al., 1994). After 8 days of treatment, sulfated polysaccharide from S. dubyi completely blocked giant cell
158 Table I. Evaluation of anti-HIV- I activity of sulfated polysaccharide from Schizymenia dubyi: inhibition of anti-HIV- 1 associated reverse transcriptase (cpm ml - l in duplicate) and syncitia formation for January to March 1994. (-: absence of syncitia; +: presence of syncitia; ++: syncitia; T: cellular death; TOX: toxicity). Concentration g ml- l
January RT
Syncitia
February RT
Syncitia
March RT
100 50 25 10 5
TOX TOX 660/300 384/524 260,248/224,554
TOX TOX -/TOX ++
TOX TOX 482/304 1,116/808 9,324/6,080
TOX TOX +
TOX TOX TOX 876/3,212 445,540/226,236
Control MT4
980
-
980
-
980
Control MT4/HIV-1
201,370/242,822
++/T
201,370/242,822
++/T
201,370/242,822
Syncitia TOX TOX - (+) +
++/T
Table 2. Evaluation of anti-HIV- activity of sulfated polysaccharide from Schizymenia dubyi: inhibition of anti-HIV- associated reverse transcriptase (cpm ml- l in duplicate) and syncitia formation for April to June 1993. (-: absence of syncitia; +: presence of syncitia; ++: syncitia; T: cellular death; TOX: toxicity). Concentration ,ug ml-'
April* RT
Syncitia
May* RT
Syncitia
June RT
Syncitia
100 50 25 10 5
130/188 154/188 174/330 260/1,012 320/528
-
178/182 134/150 166/198 292/564 338/564
-
162/140 124/188 182/202 284/100 858/458
-
Control MT4
424
-
424
-
980
Control MT4/HIV-1
237,302/195,920
++
237,302/195,920
++
237,302/195,920
formation and reverse transcriptase at a concentration of 5 jig ml-1. Cellular protection was total. In comparison, other sulfated algal polysaccharides, such as fucoidan, -carrageenan and A-carrageenan, inhibited virus-induced cytopathogenicity by 50% (IC50) with concentrations of 2.8, 12 and 1.9 pg ml- respectively, after 5 days of incubation (Baba et al., 1990b; Montefiori et al., 1990). For chemically sulfated dextran, a concentration of 100 /zg ml-' was required to achieve complete inhibition of syncytia. The antiretroviral activity of natural polysaccharides extracted from various sources has been also investigated (Nakashima et al., 1987a, b; Sydow & Klocking; 1987; Beress et al., 1993; Bourgougnon et al., 1993). Dextran sulfate inhibits HIV RT activity at a 50% inhibitory concentration (IC50) of approximately 2 ulg ml- t. Alginate and fucoidan fractions extracted from Fucus vesiculosus L. (Fucophyceae) inhibited HIV-RT to 96%, at concentrations which
++
were not toxic (Beress et al., 1993). A-carrageenan (SAE: Seaweed Algae Extract) extracted from Schizymenia pacifica selectively inhibited the RT of Avian Myeloblastosis Virus (AMV) and the replication of HIV. However, the inhibition of HIV infection by SAE may not be entirely due to the inhibition of RT. The sulfated polysaccharide may suppress HIV infection by interfering with virus adsorption as well as by inhibiting RT. Like the sulfated polysaccharide from S. dubyi, SAE immediately inhibited RT activity when added to an assay mixture after the start of the reaction. More than 90% of cells were viable in the cultures exposed to SAE, while almost all the MT4 cells in the control culture had died by 10 days after HIV infection (Nakashima et al., 1987a, b). The mechanism of anti-HIV action of sulfated polysaccharides has been explained as being an inhibition of virus attachment to the cell surface (Baba et al., 1990a, b; Witvrouw et al., 1994). A similar structure-activity relationship
159 Table 3. Evaluation of anti-HIV-1 activity of the sulfated polysaccharide from S. dubyi Inhibition of anti-HIV-I associated reverse transcriptase (cpm ml- in duplicate) and syncitia formation for June to December 1993. (-: absence of syncitia; +: presence of syncitia; ++: syncitia; T: cellular death; TOX: toxicity). * Station St Maria. ConenlaiM
I
July
June
Septrmber*
November RT
Synciia
December RT
Syncilia
TOX
TOX
TOX
TOX -
728/650 708/996
RT
Syncili.
RT
Syncitia
RT
186/162 270/176 204/298 450/388 220/230
-
390/594 310/378 422/458 350/1,096 880/950
--
TOX TOX 788/754
--
778/1634 1,034/548 870/828 74,768/35,354 ++ 124,952/185,87 ++IT
3,640/24,206 (+) 234,736/229,414 ++/T
158,198/230,592 ++ 259,052/389,570 ++rr
Connol MT4
990
-
-
990
990 -
990
-
Control
55,494/86,824
++/T
++/T
55,494186,824
55,494/186.824
++T
g ml100 50 250 10 5
90 55,494186,824
Syncia
++rT
55,494186,824
++T
Table 4. Monthly chemical composition of water-soluble polysaccharide from S. dubyi (%molar, excepted for yield, protein and ash values expressed in % dry weight). * Stations St Maria La Scala; ND: non detected. Month
Yield
Protein
Ash
Galactose
Glucose
Xylose
Uronic acids
Sulfate
3,6 A-G
April* 93 May* 93 June 93 June* 93 July93 Sept.* 93 Nov. 93 Dec. 93 Jan. 94 Feb. 94 March 94
26 20 19 19 18 24 24 23 21 20 24
1.5 2.1 1.5 1.6 ND 3.3 2.4 2.8 3.4 2.3 3.4
25.2 17.2 21.8 19.7 24.4 28.9 26.4 26.0 25.0 24.2 24.2
36.4 39.7 35.4 40.6 35.4 32.4 29.4 32.8 32.0 33.4 29.2
0.6 2.1 0.8 0.6 1.4 ND ND ND 0.4 0.8 0.7
1.0 1.7 1.1 1.1 2.0 1.2 1.2 1.0 1.2 1.1 1.0
13.6 14.4 14.9 14.5 13.5 14.5 15.0 14.9 14.6 15.2 18.9
48.2 41.9 47.3 43.0 47.6 51.6 54.3 51.2 51.7 49.3 50.0
