Toxicon 50 (2007) 311–321
Review
Purification and partial characterization of paralytic shellfish poison-binding protein from Acanthocardia tuberculatum
Nadia Takati
a, Driss Mountassif
b, Hamid Taleb
c, Kangmin Lee
d, Mohamed Blaghen
a,aUnit of Bio-Industry and Molecular Toxicology, Laboratory of Microbiology, Biotechnology, Pharmacology and Environment, Faculty of Sciences Ai¨n Chock, University Hassan II-Ai¨n Chock, Km 8 route d’El Jadida, B.P. 5366, Maˆarif, Casablanca, Morocco
bLaboratory of Biochemistry and Molecular Biology, Faculty of Sciences Ai¨n Chock, University Hassan II-Ai¨n Chock, Km 8 route d’El Jadida, B.P. 5366, Maˆarif, Casablanca, Morocco
cLaboratory of the Marine Biotoxins, National Institute of Halieutic Research, Casablanca, Morocco
dLaboratory of Enzyme Technology, Chonbuk National University, Chonju, Republic of Korea
Received 29 November 2006; received in revised form 21 April 2007; accepted 23 April 2007 Available online 3 May 2007
Abstract
A paralytic shellfish poison-binding protein (PSPBP) was purified 16.6-fold from the foot of the Moroccan cockles
Acanthocardia tuberculatum. Using affinity chromatography, 2.5 mg of PSPBP showing homogeneity on sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was obtained from 93 mg of crude extract. The purified PSPBP exhibits a specific activity of about 2.78 mU/mg proteins and has estimated molecular weight of 181 kDa. Observation of a single band equivalent to 88 kDa on SDS–PAGE under reducing conditions suggested it to be a homodimer. The optimal temperature and pH for the purified PSPBP were respectively 30
1C and 7.0.
r
2007 Elsevier Ltd. All rights reserved.
Keywords:PSP; PSPBP;Acanthocardia tuberculatum; Foot; Affinity chromatography
Contents
1. Introduction . . . 312
2. Materials and methods. . . 313
2.1. Materials . . . 313
2.2. Extraction of paralytic shellfish poison (PSP) and mouse bioassay. . . 313
2.3. Crude extracts preparation. . . 313
2.4. Purification of PSPBP . . . 313
2.5. Thin-layer chromatography analysis . . . 314
www.elsevier.com/locate/toxicon0041-0101/$ - see front matterr2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2007.04.016
Abbreviations:EDTA, ethylenediamine tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid;
MES, 4-N-morpholinoethanesulfonic acid; PSP, paralytic shellfish poisoning; PSPBP, paralytic shellfish poison-binding protein;
PSTs, paralytic shellfish toxins; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis Corresponding author. Tel.: +212 22 23 06 80/84; fax: +212 22 23 06 74.
E-mail address:m.blaghen@fsac.ac.ma (M. Blaghen).
2.6. Polyacrylamide gel electrophoresis (PAGE) . . . 314
2.7. Protein assay . . . 314
2.8. Determination of optimal pH and temperature of purified PSPBP. . . 314
2.9. Statistical data analysis . . . 315
3. Results . . . 315
3.1. Tissue distribution of PSP in
A. tuberculatum. . . 315
3.2. Tissue distribution of PSPBP activity in
A. tuberculatum. . . 315
3.3. Purification of PSPBP from
A. tuberculatum. . . 315
3.4. Thin-layer chromatography analysis . . . 316
3.5. Molecular weight determination of PSPBP . . . 316
3.6. Influence of pH and temperature on the purified PSPBP activity . . . 317
4. Discussion . . . 318
Acknowledgment . . . 319
References . . . 320
1. Introduction
Paralytic shellfish poisoning (PSP) represents a significant public health and safety hazard concern worldwide and causes severe economic losses globally due to bans on harvesting of contaminated shellfish and the need for costly monitoring programs. It is a serious illness, with predominantly neurological symptoms and, in severe cases, re- spiratory paralysis and death (Taylor, 1988; Kao, 1993). Paralytic shellfish toxins (PSTs) block the influx of sodium ions (Na
+) through excitable membranes (Narahashi and Moore, 1968; Hille, 1968), interrupting signal transmission and causing paralysis.
Several species of dinoflagellate, such as Alexan- drium tamarense (Prakash, 1967), Pyrodinium baha- mense var. compressum (Harada et al., 1982) and Gymnodinium catenatum (Oshima et al., 1993) are known to transmit their toxins to shellfish.
Bivalve mollusks, the primary vectors of PSP in humans, show marked inter-species variation in their capacity to accumulate PSTs, and contain a mixture of several toxins, depending on the species of algae, geographic area and type of marine animal involved (Bricelj and Shumway, 1998).
The cockle (Acanthocardia tuberculatum) is known to sequester PSTs for a long time in its tissues, even when the potentially toxin-producing microalgae are not present (Vale and Sampayo, 2002). Indeed, A. tuberculatum sequesters PSTs preferably in non-visceral organs (foot, gill and mantle) (Sagou et al., 2005). However, the toxin accumulation and metabolism systems in A. tuber- culatum have not been clarified. The studies of soluble toxin-binding proteins in different organs
are of particular interest as these proteins are suspected to be implicated in these systems.
Receptors for the saxitoxin have been reported, by ulterior studies in other species, which bind this phycotoxin with different affinities and present very distinct molecular structures and physicochemical properties. On the one hand, saxiphilin, a soluble protein, that binds STX with high affinity (K
d0.2 nM) (Mahar et al., 1991) was first dis- covered in frogs (Doyle et al., 1982; Moczydlowski et al., 1988), but similar soluble STX-binding activity has been observed in diverse species of arthropods, amphibians, reptiles, and fish (Llewellyn et al., 1997; Llewellyn, 1997). Saxiphilin appears to be a monomeric (91 kDa) protein with similar structure to the transferin family of proteins (Morabito and Moczydlowski, 1994; Morabito et al., 1995) but witch binds STX instead of Fe
3+(Li and Moczydlowski, 1991). Saxiphilin does not possess any affinity for tetrodotoxin (TTX) (Llewellyn et al., 1998; Negri and Llewellyn, 1998).
Another receptor for STX and TTX, purified from plasma of the puffer fish, Fugu pardalis, was called puffer fish saxitoxin- and tetrodotoxin-binding protein (PSTBP) (Yotsu-Yamashita et al., 2001).
This glycoprotein possessed a binding capacity of 10.6 7 0.97 nmol/mg protein and a K
dof 14.6 7 0.33 nM for [
3H] saxitoxin in equilibrium binding assays. PSTBP seemed to consist of no covalently linked dimers of a 104 kDa single subunit whose protein part was esteemed to be of 42 kDa.
The predicted amino-acid sequences of PSTBP were
not homologous to that of saxiphilin, or sodium
channels, but their N-terminus sequences were
homologous to that of the reported tetrodotoxin-
binding protein from plasma of Fugu niphobles
(Matsui et al., 2000). Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish.
Comparison of toxin profiles between cockles (A. tuberculatum) and toxigenic algae (G. catena- tum) showed that the high toxicity of cockles is due to the biotransformation of C-toxins (with low specific toxicity) into decarbamoylsaxitoxin (dcSTX) with relatively higher specific toxicity (Taleb et al., 2001; Sagou et al., 2005). However, the uptake, elimination and the transfer of toxin in other organs follows a characteristic pattern in each bivalve species (Lassus et al., 1989; Bricelj and Shumway, 1998).
In Morocco, toxic algae blooms have occurred along both Atlantic and Mediterranean coasts. This situation caused economic loss for fishermen and a significant effect on the local economy due to decreased fishery revenue. A. tuberculatum is mainly exploited in the canning industry in Morocco and Spain. A Spanish team has demonstrated that after a thermal treatment of cockles at 116 1 C for at least 51 min, toxicity drops to undetectable levels by mouse bioassay (Burdaspal et al., 1998). On the basis of the later processing, an exceptional European legislation allows harvesting in Spain of cockles with PSP toxins levels less than 300 mg STXeq/100 g meat (Official Journal of the European Communities, 1996).
Our aims are to purify the protein that bind PSTs named paralytic shellfish poison-binding protein (PSPBP) from the complex environment and to clarify its physiological function in A. tuberculatum.
Here, we present the purification and characteriza- tion of a PSPBP from the Moroccan cockle A. tuberculatum.
2. Materials and methods 2.1. Materials
Specimens of the cockle (A. tuberculatum) were collected from two stations (Kaaˆ Srass and M’diq) on the Mediterranean coast of Morocco. Cockles were washed and separated into digestive gland (hepatopancreas), foot, mantle, muscle and gills.
The cockle tissues were kept at 20 1 C until use.
Affigel 10 was purchased from Bio-Rad. Dimethyl formamide and glutaraldehyde were from Amersco.
All other chemicals (analytical grade) were from Sigma.
2.2. Extraction of paralytic shellfish poison (PSP) and mouse bioassay
Toxicity analysis for all organs was carried out by mouse bioassay according to AOAC method (1990):
100 g homogenized tissues collected from toxic cockles (Kaaˆ Srass) were mixed with 100 ml 0.1 M hydrochloric acid and boiled for 5 min, pH adjusted to 2–3. The volume of mixture was brought to 200 ml with double-distilled water, stirred and centrifuged at 3000 rpm for 10 min. The PSP mouse bioassay involves acidic aqueous extraction of selected organs. One milliliter of the supernatant was injected intraperitoneally into each of three albino mice (20 7 2 g). The mice are observed for classical PSP symptoms, such as jumping in the early stages, ataxia, ophtalmia, paralysis, gasping and death by respiratory arrest. The time from initial injection to mouse death is recorded and the values of toxicity are expressed in terms of STX equivalents per 100 g of shellfish meat (mg STXeq/
100 g meat).
2.3. Crude extracts preparation
All procedures were performed at 4 1 C. Tissue extracts were prepared from non-toxic cockles (M’diq) using both: Ultra-turrax and homogenizer T25 basic (Fisher Bioblock Scientific, Illkirch, France). Samples of tissues were quickly weighted and then homogenized with a ratio of 1:3 (w/v) in 50 mM potassium phosphate buffer (pH 7.4) con- taining 1 mM EDTA and 1 mM DTT. The homo- genates were then centrifuged for 15 min at 3000g at 4 1 C. The supernatants (crude extracts) were col- lected and stored at 20 1 C until use.
2.4. Purification of PSPBP
The crude extract from the red foot prepared as described previously was subjected to PSPBP purification by affinity chromatography. Purified PSPBP was obtained by adsorption of the crude extract with foot PSTs-coupled Affigel 10 followed by elution of the column with 1 M Tris–HCl buffer (pH 10) (buffer A).
The affinity column, using as support an acti-
vated gel of agarose (Affigel 10, Bio-Rad), was
treated as follows: 5 ml of the Affigel 10 in the
isopropanol was treated beforehand by the ethylene
diamine (50 ml) dissolved in the dimethyl formamide
(250 ml) for 30 min at room temperature. The Affigel
was then washed with 50 mM phosphate buffer (pH 7.4) (buffer B). Afterwards, 5% glutaraldehyde was added and incubated for 30 min. The column was then washed with the same buffer (buffer B).
The foot PSTs (20 mg STXeq) were then added to the actived column and loaded for 4 h at room temperature. The excess of the PSTs was removed by successive washings with buffer B. The formed complex (foot PSTs-coupled Affigel 10) was estab- lished by the addition of 0.1 M NaBH
4. The crude extract of protein foot was then added and incubated overnight at 4 1 C under weak agitation.
After loading, the column was washed with buffer B. Elution of the protein adsorbed on the column was carried out by buffer A. Fractions of 1 ml were collected and neutralized immediately by adding 100 ml of 100 mM glycocolle (pH 2.5). After elution, the column was re-equilibrated with buffer B for a possible re-use. Eluted fractions were tested for PSTs binding by mousse bioassay (AOAC, 1990).
2.5. Thin-layer chromatography analysis
Thin-layer chromatography was carried out on 20 cm 20 cm dried Silica Gel plates. One hundred micrograms of purified protein PSPBP had been loaded before and after incubation with 0.75 mg STXeq at room temperature for 30 min. A small spot of solution containing the sample (PSPBP and the mixture PSPBP–PSP) was applied on a plate at about 1 cm from the base. The plate is then dipped into a solvent system of pyridine/ethyl acetate/acetic acid/water with a ratio of 75:25:15:20 (v/v/v/v) and placed in a sealed container (Harada et al., 1983). After the end of the migration, the spots were revealed with iodine.
2.6. Polyacrylamide gel electrophoresis (PAGE)
Sodium dodecyl sulfate–polyacrylamide gel elec- trophoresis (SDS–PAGE) was performed according to the method of Laemmli (1970) on one-dimen- sional 12% polyacrylamide slab gels containing 0.1% SDS. Gels were run on a miniature vertical slab gel unit (Hoefer Scientific Instruments). Protein samples for SDS–PAGE were prepared by boiling at 100 1 C for 5 min in 60 mM Tris–HCl, pH 6.8, 1%
SDS, 10% glycerol, 1% mercaptoethanol and 0.01% Bromophenol Blue. After electrophoresis, gels were stained with 0.25% (w/v) Coomassie Brilliant Blue R-250 in methanol/acetic acid/water (4:1:5, v/v/v) for 30 min at room temperature
(Diezel et al., 1972). Destained was done in methanol/acetic acid/water (4:1:5, v/v/v). The ap- parent subunit molecular weight was determined by measuring relative motilities and comparing with the pre-stained SDS–PAGE molecular weight stan- dards (Standard proteins, Sigma).
To determine the native molecular weight of purified PSPBP, non-denaturing PAGE was per- formed according to the method of Hedrick and Smith (1968). The separating gels (6%, 8%, 10%
and 12% polyacrylamide) were buffered with 1.5 M Tris–HCl (pH 8.8). The running buffer was com- posed by 25 mM Tris and 320 mM glycine (pH 8.6).
All experiments were realized at 4 1 C. The electro- phoresis running conditions, staining and distaining were as described for SDS–PAGE. The relative molecular weight of the native purified PSPBP was estimated using a native molecular weight markers (Sigma): tetrameric urease (545 kDa), dimeric urease (272 kDa), dimeric bovine serrum albumin (BSA), (132 kDa), monomeric BSA (66 kDa) and ovalbu- min (45 kDa). By constructing the Ferguson plot [(log(R
f100) versus the concentration of poly- acrylamide gels (%)], the resulting slopes versus the standard native proteins of known molecular weight, permits to determine the molecular weight of purified PSPBP.
2.7. Protein assay
Protein content was measured according to the Bradford procedure, using BSA as standard (Brad- ford, 1976).
2.8. Determination of optimal pH and temperature of purified PSPBP
A reaction mixture containing 0.75 mg STXeq and 100 mg of purified PSPBP were incubated for 30 min.
Controls containing 0.75 mg STXeq were also done.
To determine optimal pH, PSPBP activity was measured over a wide range of pH (from 4 to 10) with a mixture of different buffers with different pK
a(Tris, MES, HEPES, sodium phosphate and sodium acetate) adjusted to the same ionic strength as the standard reaction mixture. The optimal temperature was determined in 50 mM Tris–HCl buffer (pH 7.0) for 30 min at temperature ranging from 15 to 60 1 C using a thermostated cuvette holder connected with a refrigerated bath circulator.
All the mixtures were after injected at Swiss albino
mouse to evaluate the toxicity (AOAC, 1990).
PSPBP activity was determined by the relative fixation of PSTs.
2.9. Statistical data analysis
In each assay, the experimental data represent the mean of three independent assays7standard devia- tions. Means were compared using the Student’s t-test. Differences were considered significant at the level po0.05 and very significant at the level po0.01.
3. Results
3.1. Tissue distribution of PSP in A. tuberculatum
The toxicity levels for all organs collected from toxic cockles A. tuberculatum was carried out by mouse bioassay according to AOAC method (1990).
The contribution of each organ to the total toxin body burden is function of both its absolute toxicity and relative weight contribution. The toxicity percent and relative weight contribution of different organs are shown in Fig. 1. There are substantial differences in PSTs amounts of the various organs. The toxicity decreases as follows: foot4mantle4gills4hepato- pancreas4muscle. Indeed, the foot accumulates the highest toxicity (37% of total toxicity) and the majority of toxicity (85%) is concentrated in the non- visceral bodies (foot, mantle, adductor muscle and gills). Whereas, the hepatopancreas exhibited only
15% of total toxicity. This is can be explained by the fact that the foot presents the most significant contribution to the total body weight (62.36%) (Fig. 1). According the pigmentation, foot shows two parts: red and yellow. Red part accounts for most of the toxicity in foot (69%), whereas the yellow part exhibits only 31% of foot toxicity.
3.2. Tissue distribution of PSPBP activity in A. tuberculatum
Tissue extracts (1 mg) were incubated for 30 min at room temperature with 0.75 mg STXeq (pH 7.0) to determine the capacity of PSTs binding (Fig. 2).
During this incubation, A. tuberculatum tissues yielded significant decrease of PSTs toxicity that is due to the ability of extract of each tissue to bind the PSTs. The capacity of PSTs binding showed anato- mical distribution. The foot binds 70% of the total toxicity, the mantle (40%), the hepatopancreas (34%), the adductor muscle (23%) and finally the gills (19%). The foot extract exhibited the highest capacity of PSTs binding (70%). Thus, we determined the foot to be the best source of PSP-binding protein.
3.3. Purification of PSPBP from A. tuberculatum
A total amount of about 93 mg of protein, corresponding to approximately 15.48 mU of PSPBP, was obtained from foot crude extract of
0 5 10 15 20 25 30 35 40 45
Foot Mantle Hepatopancreas muscle Gills
% of total PSP
0 10 20 30 40 50 60 70
% of total weight
*
** **
*
Fig. 1. PSP toxicities and weight distributions inAcanthocardia tuberculatumtissues. PSP was extracted from all organs and the toxicity analysis was carried out by mouse bioassay. Weight percentage of all organs was also determined. The bar graph and the superimposed line represent, respectively, % of total PSP and % of total weight. Values are given as means7S.D. of three separate experiments.**Very significantly different from the control value,po0.01.*Significantly different from the control value,po0.05 (Student’st-test).
A. tuberculatum. Table 1 summarizes a representa- tive purification protocol. The purification of the PSPBP was performed by one step (affinity chro- matography) (Fig. 3). 2.5 mg of pure PSPBP was purified with a specific activity of 2.777 mU/mg proteins, a purification factor of 16.6-fold and a yield of approximately 44.8%.
PSPBP test activity was performed using different quantities of purified protein incubated with 0.75 mg STXeq at room temperature for 30 min (pH 7.0) (Fig. 4). Intraperitoneal injection of extracted toxins by a hot acid AOAC method produced classical PSP symptoms in mice and led finally to death and the survival time depends to the quantity of PSTs present in each specimens or tissues. Incubation of extracted toxins by a hot acid AOAC method with PSPBP purified protein ex- hibited after injection an increase of the survival time and decrease of toxicity, this can be explained by the ability of PSPBP to binds PSTs. In fact, incubation of extracts PSTs in presence 0.1 mg of
PSPBP showed 48% mortality (52% of PSTs binding). So, 0.2 mg of purified protein induced 0% mortality (100% of PSTs binding).
3.4. Thin-layer chromatography analysis
Silica Gel is used in chromatography as a stationary phase. Due to silica gel polarity, non- polar components tend to elute before more polar ones. Visualization of PSPBP alone and complex PSP–PSPBP on plate made it possible to detect a delay of migration of the complex compared to the PSPBP alone (Fig. 5).
3.5. Molecular weight determination of PSPBP
The molecular weight of the native PSPBP was performed using different separating gels (6%, 8%, 10% and 12% polyacrylamide) in the absence of SDS according to the Hedrick and Smith method.
The molecular weight of the native PSPBP was
010 20 30 40 50 60 70 80
Foot Mantle Hepatopancreas muscle Gills
Relative activity (%)
**
**
**
**
Fig. 2. Anatomical distribution of PSPBP activity in Acanthocardia tuberculatum. Each tissue extract (1 mg) was incubated with 0.75mg STXeq at room temperature for 30 min (pH 7.0). The toxicity analysis was carried out by mouse bioassay. PSPBP activity was expressed by the relative fixation of PSTs. Values are given as means of three separate experiments.**Very significantly different from the control value,po0.01 (Student’st-test).
Table 1
Summary of PSPBP purification from red foot ofAcanthocardia tuberculatum
Preparation Total protein
(mg)
Total activity (mU)
Specific activity (mU/mg)
Purification (fold)
Yield (%)
Crude extract 93 15.484 0.1665 1 100
Affinity chromatography 2.5 6.944 2.777 16.6 44.8
estimated to be 181 kDa (Fig. 7). The SDS–PAGE analysis of the crude extract and purified PSPBP fraction showed a single band (Fig. 3, lane 2) with molecular weight of 88 kDa (Fig. 6), indicating that A. tuberculatum PSPBP was a homodimer.
3.6. Influence of pH and temperature on the purified PSPBP activity
The pH activity profile of purified PSPBP was determined in a pH range from 4.0 to 10.0 using a mixture of different buffers (Fig. 8A). The protein had a typical bell-shaped profile covering a broad pH range and an optimal pH of 7.0. The influence
*
*
120
100
80
60
40
20
0
1 mg 0.5 mg 0.2 mg 0.1 mg
PSPBP quantity
Relative fixation (%)
0.025 mg
Fig. 4. Purified PSPBP activity at different quantities. Different quantities of purified PSPBP (1, 0.5, 0.2, 0.1 and 0.025 mg) were incubated with 0.75mg STXeq at room temperature for 30 min (pH 7.0). The toxicity analysis was carried out by mouse bioassay. PSPBP activity was expressed by the relative fixation of PSTs. Values are given as means of three separate experiments.*Very significantly different from the control value,po0.01.
97.4 KDa
45 KDa
29 KDa 205 KDa
66 KDa 116 KDa
PSPBP
Fig. 3. Sodium dodecyl sulfate–polyacrylamide gel electrophor- esis (SDS–PAGE) pattern showing the purification of the PSPBP purified from red foot of Acanthocardia tuberculatum. Lanes 1 and 2 represent, respectively, crude extract and purified PSPBP fraction. For experimental conditions, see Section 2.
1 2
Fig. 5. Thin layer chromatogram of the PSPBP alone and the mixture PSPBP–PSP on Silica Gel. One hundred micrograms of PSPBP had been loaded before and after incubation with 0.75mg STXeq at room temperature for 30 min. Solvent system was pyridine/ethyl acetate/acetic acid/water (75/25/15/20, v/v/v/v).
Components were revealed with iodine. Lanes 1 and 2 represent, respectively, PSPBP alone and the mixture PSPBP–PSP.
of temperature on PSPBP activities was determined between 15 and 60 1 C at pH 7.0 (Fig. 8B). The optimal temperature for PSPBP activity was 30 1 C and the activity decreased significantly above 40 1 C.
4. Discussion
The present study shows a large variation in PSP toxins distribution and binding ability among
different tissues of Moroccan cockles A. tubercula- tum and describes a rapid method for purification of new toxin-binding protein named PSPBP from their foot.
A. tuberculatum showed a persistent contamina- tion with high levels of toxicity, dcSTX and STX are the toxins that account for the most of this toxicity.
Relative partitioning of PSP toxins among tissues is variable and the foot is most toxic organ followed by others organs (Taleb et al., 2001; Sagou et al., 2005). Similar studies, carried out in Spain (Beren- guer et al., 1993) and Portugal (Vale and Sampayo, 2002) on the same species, revealed the conservation of PSP toxins mainly in the foot. Our work confirms also that the most toxicity is concentrated in the foot (Fig. 1).
Furthermore, A. tuberculatum is among the slowed detoxifying bivalves in which the relative toxin proportion increases gradually in non-visceral tissues and can even surpass that of the digestive gland. The ulterior study showed that the highest PSP levels in bivalve species can be attributed to their nerves insensitive to PSP toxins. Consequently, bivalves did not exhibit physiological and behavior- al mechanisms for their detoxification (Bricelj et al., 1990). The mechanism of tissue-specific retention of PSP toxins in some species remains to be elucidated.
4.2
0 0.2 0.4 0.6 0.8
4.3 4.4 4.5 4.6 4.7 4.8 4.9
Rf
Log (MW)
205000 Da
116000 Da 97400 Da
29000 Da Purified PSPBP
(88000 Da) 45000 Da
66000 Da
Fig. 6. Determination of the molecular weight of the purified PSPBP from Acanthocardia tuberculatum foot by gel electro- phoresis on denaturing conditions. Molecular weight marker proteins used: myosine (205 kDa), b-galactosidase (116 kDa), phosphorylase B (97.4 kDa), albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). The plot represents the relative mobility of proteins versus log(molecular weight).
0.25
0.2
0.15
45 KDa 66 KDa
132 KDa
272 KDa
45 KDa
272 KDa 545 KDa 66 KDa 132 KDa Purified PSPBP 545 KDa
0.1
0.05
100 200 300
5 4.5
3.5
2.5
6% 8% 10% 12%
2 3 4
400 500 600
0
-Slope
Molecular weight (kDa)
Gel concentration
Log (Rf x100)
Purified PSPBP (181 KDa)
Fig. 7. Determination of the native molecular weight of the purified PSPBP fromAcanthocardia tuberculatumfoot by non-denaturing polyacrylamide gel electrophoresis. Proteins were electrophoresed on various acrylamide concentrations gels (6%, 8%, 10% and 12%) under non-denaturing conditions. Molecular weight marker proteins used: tetrameric urease (545 kDa), dimeric urease (272 kDa), dimeric BSA (132 kDa), monomeric BSA (66 kDa) and ovalbumin (45 kDa) (Sigma). Relative mobility of proteins plotted as log(Rf100) versus acrylamide concentration is indicated on the insert. A plot of the obtained slopes versus molecular weight was linear and used to determine native PSPBP molecular weight.
Price and Lee (1971, 1972) suggested that STX was electrostatically and reversibly bound in the melanin fraction of pigmented tissues, but subsequent work does not validate this hypothesis (Beitler, 1992).
Incubations of extracted toxins by a hot acid AOAC method with tissue extracts from unconta- minated cockles (A. tuberculatum) showed a de- crease of total toxicity. This decrease can be explained by the PSTs binding ability of selected crude extracts. The foot exhibited the highest PSTs binding activity (Fig. 2). Thus, red foot is a good starting source for the purification of PSPBP.
According the pigmentation, the foot shows two parts: red and yellow. A. tuberculatum concentrates most of the toxins as dcSTX and STX in the foot and especially in their red part. However, the toxin accumulation and prolonged retention sys- tems in A. tuberculatum have not been well clarified.
The studies of soluble toxin-binding proteins in different organs are of particular interest as these proteins are expected to be implicated in these systems.
This is the first report about the isolation of a protein that binds PSP toxins in A. tuberculatum.
Since it is difficult to purify PSPBP from foot of A. tuberculatum due to the variety of proteins, the large amounts of lipophilic substances in extracts, and after several tests. We describe in this paper a relatively simple and rapid method for the purifica- tion of PSPBP, a soluble PSP-binding protein form the foot of A. tuberculatum.
Affinity chromatography is most effective than other method of purification. Since, it give pure protein with 44.8% as yield and 17 as factor of purification and an important quantity of pure protein (2.5 mg of PSPBP from 93 mg of total proteins) (Table 1). The purified PSPBP exhibited an activity of 2.777 mU/mg proteins and having estimated molecular weight of 181 kDa (Fig. 7).
Observation of single band equivalent to 88 kDa on SDS–PAGE under reducing conditions (Figs. 3 and 6) suggested it to be a homodimer. The optimal temperature and pH for the purified PSPBP were, respectively, 30 1 C and 7.0 (Fig. 8).
The specificity of PSPBP to extracted PSP from A. tuberculatum was determined by thin layer chromatography analysis on Silica Gel which shows a delay of migration of the complex PSPBP–PSTs compared to the PSPBP alone (Fig. 5). This delay is probably due to the binding of the PSP on PSPBP.
PSP alone traveled the same distance on the plate as the mixture of PSP and PSPBP.
In summary, prolonged retention (several months to years) of PSP toxins as dcSTX and STX is a characteristic of A. tuberculatum that can be ex- plained not only by the specific retention of dcSTX (Taleb et al., 2001) and differential accumulation of PSP toxins in non-visceral organs (Sagou et al., 2005) but also by the presence of soluble toxin-binding proteins (PSPBP) in A. tuberculatum mainly in foot.
Future studies aimed at the sequencing the purified PSPBP and determining its tri-dimensional structure in order to understand its interactions with the PSP toxins. Cloning of the gene encoding for this protein, to see homologies with the already receptors of saxitoxin and saxitoxin derivatives, would be also of interest.
Acknowledgment
This work was supported by the Centre National de la Recherche Scientifique et Technique.
70 60 50 40 30 20 10
0 4 5 6 7 8 9 10
pH
15 25 30 40 50 60
Temperature (°C)
relative fixation (%)
70 60 50 40 30 20 10 0
relative fixation (%)
A
B
Fig. 8. Effect of pH (A) and temperature (B) on PSPBP activity.
PSPBP activity was measured using 100mg of purified PSPBP and determined by relative fixation of PSTs. Values are given as means of three separate experiments. For experimental conditions, see Section 2. PSPBP activity was assayed: (A) using a mixture of buffers ranging from pH 4.0 to 10 and (B) in 50 mM Tris–HCl buffer (pH 7.0) at temperature ranging from 15 to 601C.
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