1972, 1976). However, to date, only one proteo- lytic digestive enzyme has been isolated in the mollusc (Groppe and Morse, 1993). Otherwise, in the arthropod ...
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Comp. Biochem. Physiol. Vol. 110B, No. 4, pp. 777-784, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/95 $9.50 + 0.00
Pergamon
0305-0491(94)00211-8
Purification and partial characterization of chymotrypsin-like proteases from the digestive gland of the scallop Pecten maximus P. Le Chevalier, D. Sellos and A. Van Wormhoudt Marine Biology Laboratory, Coll6ge de France, BP 225, 29182 Concarneau, Cedex, France Three variants of a chymotrypsin-like protease were purified from scallop digestive glands successively by ion-exchange, gel filtration and high-performance liquid chromatographies. Enzyme activity was detected using succinyl-Ala-Aia-Pro-Phe-p-nitroanilide as a specific synthetic substrate for chymotrypsin. This proteinase was inhibited by chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. Estimated molecular mass of the purified enzyme is around 32 kDa. These isoenzymes exhibit very low activities in hydrolyzing small synthetic specific substrates used for trypsic, elastolytic and collagenolytic measurements and referred mainly to a chymotrypsin-like proteinase. Very few differences were measured concerning pH profiles among the three isoenzymes. Stability is higher at low temperature for two variants. An N-terminal analysis was performed on one variant (B) among the three isoenzymes. The alignment of the N-terminal amino acid sequence indicates some homologies with abalone chymotrypsin-like protein and arthropod chymotrypsin proteases as well as with vertebrate serine protease counterparts (trypsin, chymotrypsin and elastase). Key words: Invertebrate; Molluscan; Scallop; Chymotrypsin-like serine protease; Purification; Characterization; Isoenzyme; N-terminal sequence.
Comp. Biochem. Physiol. llOB, 777-784, 1995.
Introduction Lamellibranch bivalves feed by filtering particular organic matter. Selected nutritive particles are picked up and passed via an oesophagus to a complex digestive system composed of a stomach including a digestive gland. Digestion is partially extracellular and occurs in the lumen of the stomach. In this system, the bivalve crystalline style plays a role in the grinding and hydrolysis of ingested nutritive material by rotating against a gastric shield (a triturating mechanism causing abrasion) and also by secreting digestive enzymes (Purchon, 1971; Reid and Sweeny, 1979). Intracellular digestion occurs in the digestive gland by absorption and Correspondence to: P. Le Chevalier, Marine Biology Laboratory, Coll~ge de France, BP 225, 29182 Concarneau, Cedex, France. Tel. 33-989-70659; Fax 33-989-78124. Received 11 April 1994; revised 1 September 1994; accepted 15 September 1994. 777
assimilation of digested food by digestive diverticulata (Robinson and Langton, 1980). Most of the data concerning enzymes involved in digestion have a qualitative basis and are often based on methods that are not very specific (Morton, 1983). Histochemical surveys of enzymes associated with the process of digestion located areas of glucanolytic, lipolytic and proteolytic activities only in the stomach and digestive gland (Palmer, 1979). Generally, bivalves feed an unicellular algae containing cellulose except in the Septibranch group where animals are carnivorous (Yonge, 1928). In bivalves, a series of digestive enzymatic activities has already been reported and many specific activities of carbohydrases have been specifically measured (Wojtowicz, 1972; Theo and Sabapathy, 1990). Some bivalves have been shown to contain a wide range of carbohydratedegrading enzymes and can thus potentially
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utilize a wide variety of carbohydrate food (Stark and Walk, 1983). Protease activities, including an alkaline endopeptidase harboring trypsic activity and an acid endopeptidase with similar activity to vertebrate cathepsin B and D, have also been detected in Bivalvia (Reid and Rauchert, 1970, 1972, 1976). However, to date, only one proteolytic digestive enzyme has been isolated in the mollusc (Groppe and Morse, 1993). Otherwise, in the arthropod group, some proteases such as trypsin, chymotrypsin and collagenolytic proteinase have been sequenced (Jany et al., 1978; Sellos and Van Wormhoudt, 1992) and characterized relative to enzyme catalytic properties (Zwilling and Neurath, 1981; Van Wormhoudt et al., 1992). In this work, we report for the first time the presence in Pecten maximus of a chymotrypsinlike enzyme in the digestive gland. The enzyme has been purified and partially characterized with respect to molecular mass and enzymatic activities. The N-terminal sequence was compared with that of other serine proteases from various invertebrates and vertebrates.
Materials and Methods Scallops (Pecten maximus) were collected by trawling in the bay of Concarneau (Brittany, France). Chemicals T F A (Trifluoroacetic acid), EDTA (ethylenediaminetetraacetic acid), SANA (succinyl-(Ala)3p-nitroanilide), T A M E (p-tosyl-L- arginine methyl ester), SAAPFNa (succinyl-(Ala)2-ProPhe-p-nitroanilide), F A L G P A (2 - furanacryloyl - leucyl - glycyl - phenyl - alanine ), D F P (diisopropylfluorophosphate), PMSF (phenylmethylsulfonyl fluoride) and chymostatin were purchased from Sigma Chemical Co. (St Louis, MO). All other chemicals were of analytical grade. Extraction of scallop digestive glands Frozen scallop digestive glands (30 g) were homogenized for 5rain in a Sorvall blender at 4~'C in 200ml ammonium acetate buffer (10mM, pH 8.3), containing EDTA 10-4M. After centrifugation (10,000g for 60rain), the supernatant was dialyzed and lyophylized. Enzyme and protein measurement Chymotrypsic activity was detected using succinyl-(Ala)2-Pro-Phe-p-nitroanilide as specific substrate by the method of Delmar et al. (1979). One modification was made to the method; the concentration of the SAAPFNa was four times less concentrated as was previously described.
At each step of the chromatographies, 5 or 10/~1 were stored for the estimation of protease activity by the above-mentioned method. Trypsic activity was measured using T A M E as substrate according to Hummel (1959), elastolytic activity using SANA as substrate according to the method of Bieth et al. (1974) and coUagenolytic activity using F A L G P A according to Van Wart and Steinbrink (1981). Protein concentration was determined by the method of Bradford (1976) using reagents from Bio-Rad (Richmond, CA) or by the method of Lowry (1951). Bovine serum albumin (BSA) was used as standard. Purification chromatography Lyophylized powder (1.2 g) of scallop digestive gland was dissolved in 5 ml ammonium acetate buffer (10 mM, pH 8.3). The extract was loaded on a DEAE-52 cellulose column (20 x 3 cm) equilibrated with extraction buffer. The column was washed with the same buffer until absorbance at 280 nm was zero. Elution was performed with a linear gradient from 0 to 0.5 M NaC1 at a flow rate of 40 ml/hr and 5ml fractions were collected. DEAE fractions hydrolyzing SAAPFNa were applied to a Sephadex G-75 column (160 x 1.2cm) and eluted with an ammonium acetate buffer (10raM, pHS.3) at a flow rate of 15ml/hr. Fractions of 10 ml were collected. Absorbance was monitored at 280 nm. M o n o - Q H R anionexchange column (7.5 x 0.75 cm) from Pharmacia (Uppala, Sweden) was equilibrated in Tris-HC1 buffer (10 mM, pH 8.2). Proteins were eluted from the gel using NaCI gradients (0-1 M) and detected at 226 nm. Two high-performance liquid chromatographies were necessary to achieve purification. Firstly, a nucleosil C~8 column (C 12-Cluseau, France) 300A (25 x 0.46 cm) was equilibrated in phosphate buffer (10raM, pH 6.2). Proteins were eluted using an acetonitrile (ACN) gradient from 0 to 60%, Then, a Vydac column (218 TP 54) was equilibrated in H 2 0 / T F A 0.1% acetonitrile 35%. The elution was performed with acetonitrile/H20 TFA 0.1% gradient from 35 to 38%. Polyacrylamide gel electrophoresis Extract was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) according to the method of Weber and Osborne (1969) or under native conditions (Davis, 1964). The gel was stained with 0.2% Coomassie Blue and protein bands were visualized after destaining in methanol: acetic acid: water (4.5 : 1 : 4.5) or proteins were revealed with silver-stain (Merril et al.,
Proteases from scallop digestiveglands 1984). The apparent molecular mass of scallop chymotrypsin-like protease was determined using low molecular mass markers from Pharmacia. Glycoprotein detection After migration of proteic fractions in SDS-PAGE, proteins are blotted into a PVDF membrane. Glycoproteins are visualized according to the method of detection of sugars in glycoconjugates by an enzyme immunoassay (DIG--Glycan Detection Kit from BoehringerMannheim Biochemica, Mannheim, Germany) Ref No. 1142372). N-terminal sequence determination The N-terminal sequence analysis was performed with Applied Biosystems Model 470 gas-phase sequencer in the chemical analysis laboratory of CNRS at Vernaison (Hunkapiiler et al., 1986). Effect of inhibitors The enzyme was preincubated in the presence of inhibitors in ammonium acetate buffer 10mM, pH8, and at 30°C for 30min. The residual enzymatic activity was then measured as described in Enzyme and protein measurement. The enzyme, preincubated without an inhibitor, was used as reference. Biochemical studies The effect of pH on the activity of chymotrypsin-like protease towards SAAPFNa was determined using different buffers with specific pH range. These buffers were: sodium acetate 100 mM (pH 4--6), phosphate 100 mM (pH6-7), Tris 100mM (pH7-9) and ammonium acetate 100mM (pH9-11). The activity was measured as described in Enzyme and protein measurement. The effect of temperature on the activity of chymotrypsin-like enzyme, using SAAPFNa, was estimated by measuring the hydrolysis of this specific substrate in different temperature incubations (20-70°C). Experimental conditions of assays were described in Enzyme and protein measurement.
Results and Discussion Purification A chymotrypsin-like protease, detected by the measure of activity using SAAPFNa as specific substrate, has been isolated from the digestive gland of the scallop, Pecten maximus. The extract obtained by homogenization of digestive glands in an alkaline buffer was very viscous. This may be due to the presence of a large amount of mucopolysaccharides in
779
this tissue. To eliminate this viscosity, sample was first applied to an ion-exchange column (DEAE-cellulose 52). The fraction, eluted at 0.2 M NaCI, was subjected to chromatography on a Sephadex G-75 column. The gel filtration fraction, recovered just after the void volume, was submitted to a Mono-Q column. A major peak, which eluted at 0.2 M NaC1, contained chymotrypsic activity and was submitted to HPLC chromatography on a Nucleosil column under saline conditions. The recovered Nucleosii fraction (N) contained a mixture of chymotrypsin-like enzymes consisting solely of three variants that were separated on a Vydac column using acetonitrile (ACN) gradient (Fig. la). The HPLC pattern shows three separated peaks (A, B and C) corresponding to three different subfractions (Fig. l b). The results of this purification procedure are summarized in Table 1. The recovery after the Nucleosil step was around 2%. At the last step of purification (Vydac step), the decrease of specific activity is important (Table 2) and is caused by the TFA/acetonitrile solvant remaining at a very low pH. Van Wormhoudt et al. (1992) collected immediately after Vydac column, used in acetonitrile/TFA conditions, fractions of interest in ammonium acetate buffer to avoid the loss of shrimp chymotrypsic activity. Groppe and Morse (1993) have recently isolated a protease related to chymotrypsin from a gastropod (abalone, Haliotis rufescens). This enzyme consisted of only a single form and, was extracted from the lumenal fluid of the intestine. In the scallop, chymotrypsic activity in this tissue was much lower than that measured in the digestive gland. Reid and Rauchert (1970) suggested that protein digestion is mainly intracellular and occurs in digestive gland diverticulata of bivalvia. However, it is clear that this process occurs, at least partially, in the gastric juice. Substrate specificity Substrate specificity was studied in the recovered fraction (N) obtained from Nucleosil chromatography and in the three purified variants (A, B and C) obtained after the Vydac step, using different synthetic substrates. These are active towards SAAPFNa but fraction N, consisting of the mixture of the three chymotrypsin-like isoenzymes, also exhibits the ability to degrade some synthetic substrates used to measure tryptic (TAME), elastolytic (SANA) and collagenolytic (FALGPA) activities (Table 2). There is higher activity using SAAPFNa than that measured with other small synthetic substrates (TAME-SANAFALGPA). No activity towards these substrates was detected for any isoform of chymotrypsin-
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like protease except when using T A M E . Some tryptic residual hydrolysis, considered as esterase activity, was observed for the variants A, B a n d C, b u t was n o t quantified. A n y w a y , the lack o f response towards the tested synthetic
substrates does n o t necessarily exclude elastolyric or collagenolytic activity (Tsai" e t al., 1991; Chen e t al., 1991). It seems that this disappearance of activities for the A, B a n d C variants after Vydac c h r o m a t o g r a p h y is connected to the
Table 1. Purification of the chymotrypsin-like protease from scallop digestive gland Purification step Crude extract DEAE-52 Filtration G75 Mono-QHR Nucleosil (N)
Total protein (mg)
Total activity (units)
6000 180 13.4 5.4 0.1
2900 720 480 291 66
Specific activity (units/mg) 0.48 4 36 54 660
Y i e l d Enrichment (%) (factor) 100 25 16.5 10 2.2
1 8.3 75 112.5 1375
Proteases from scallop digestive glands
781
Table 2. Activity (micromol/min/mg) of the different fractions with small synthetic specific substrates Purification s t e p S A A P F N a T A M E S A N A F A L G P A BTEE Nucleosil (N) 660 0.1 0.05 0.025 nd Vydac (A) 50 nm nd nd nd Vydac (B) 65 nm nd nd nd Vydac (C) 75 nm nd nd nd No activity was detected with BTEE. (nd: not detected; nm: detected but not measured). great loss of S A A P F N a activity, and that the detection limit of the other substrates is also connected to a certain amount of S A A P F N a activity. Scallop chymotrypsin-like protease exhibit limited specific activities towards the tested substrates. Grant et al. (1981) isolated from a crustacean fiddler crab a collagenolytic protease, related to the serine protease family: this enzyme is capable of degrading a wider variety of synthetic substrates (at least among those used) than chymotrypsin-like proteinase of scallop. Dendiger and O ' C o n n o r (1990) reported preliminary results concerning the presence of a protease exhibiting elastase and chymotrypsic activities in the blue crab. N o activity has been detected using BTEE to search chymotrypsic protease in digestive gland of scallop. In the same way, Tsai et al. (1986) reported similar data for shrimp digestive extracts while this enzyme is now wellcharacterized in this animal (Sellos and Van Wormhoudt, 1992). Therefore, the use of a specific substrate if very important in detecting discret activity of the enzyme of interest (Van W o r m h o u d t et al., 1992; Reid and Rauchert, 1972).
Molecular weight The apparent molecular mass of scallop protease was estimated by S D S - P A G E and was found to be around 32 kDa (Fig. 2) for each variant. We demonstrated, using a digoxygenin glycan detection system, that the scallop chymotrypsin-like protease is a glycoprotein (data not shown). From known amino acid sequences of chymotrypsin and trypsin from mammals, crustaceans and insects, the molecular mass has been estimated to be around 25 kDa, though, Heidtman and Travis (1993) isolated, from a human lung, a chymotrypsinlike proteinase with an apparent molecular mass of 30 kDa and suggested that this enzyme is glycosylated. Honjo et al. (1990) reported a molecular mass of 36 kDa for a shrimp trypsinlike protease: they presumed that this higher
Inhibitory study In order to confirm the identity of the scallop protease, some inhibitors were used (Table 3). Chymostatin was a stronger inhibitor than D F P or PMSF, so the scallop enzyme was classified as a serine proteinase and, furthermore, this result confirms that this purified protease is closed to the chymotrypsin group.
Table 3. Effects of inhibitors on the activity of chymotrypsin-like protease from digestive gland of scallop Residual relative Inhibitor I/E* activity (%) None -I00 Chymostatin 2 0 DFP 100 10 PMSF 100 15 *Molecular ratio of inhibitor to enzyme. The enzymatic extract is obtained from the Nucleosil chromatography. This fraction (N) is only composed of the three variants of scallop chymotrypsin-like protease.
Fig. 2. SDS gel electrophoretic pattern of the Nucleosil fraction (N). Proteins are revealed by use of Coomassie Blue. (M: weight markers in kDa).
782
P. Le Chevalier et al. 120 --a- Variant A Variant B -.e,- Variant C
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mass was due to the presence of oligosaccharides linked to the protein (Kimoto et al., 1983). G r o p p e and Morse (1993) compared the mass of abalone chymotrypsin-like protease obtained using S D S - P A G E with that deduced from primary structure analysis and found a 3 k D a difference, reflecting the possibility of glycosylation. Hajjou and Legal (1994) reported that partially deglycosylated tuna aminopeptidase was more sensitive to the denaturing effects of SDS and ionic strength. Further enzymatic investigations must be undertaken to estimate the importance of the glycosylation of scallop chymotrypsin-like protease. p H and temperature effect Effects of p H and temperature on scallop chymotrypsin activity were studied. Relative enzymatic activities of the three variants were measured at different temperatures (20-70°C) (Fig. 3a). A maximum of activity was obtained at 50 and 55°C for both variants A and B, and for variant C, respectively. Isoenzymes A and B hydrolyze S A A P F N a in a wider
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