Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus)

Comparative Biochemistry and Physiology, Part B 161 (2012) 247–254

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Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus) Long-Zhen Zhou a, Mi-Mi Ruan a, b, Qiu-Feng Cai a, Guang-Ming Liu a, Le-Chang Sun a, Wen-Jin Su a, Min-Jie Cao a,⁎ a b

College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Xiamen, 361021, China College of Food Science and Technology, Shanghai Ocean University, Shanghai, 209306, China

a r t i c l e

i n f o

Article history: Received 18 October 2011 Received in revised form 27 November 2011 Accepted 27 November 2011 Available online 2 December 2011 Keywords: Snakehead Trypsin Purification Molecular cloning

a b s t r a c t A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5–9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M− 1. Apparent Km was 1.02 μM and kcat was 148 S− 1 for fluorogenic substrate Boc-Phe-Ser-ArgMCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Hydrolysis of proteins is a crucial step during the process of food digestion in animals. The digestion process is catalyzed by proteinases from digestive gland, including pepsin, chymotrypsin and trypsin. Among these proteinases, trypsin (EC 3.4.21.4) is a well-known and important serine proteinase. Trypsins possess a catalytic triad consisting of His, Asp and Ser residues, which specifically hydrolyzes proteins and peptides at the carboxyl side of arginine and lysine residues (Muhlia-Almazan et al., 2008). Trypsins are synthesized in pancreas as proenzyme and activate themselves by cleaving a short propeptide from the N-terminus of the inactive zymogen. They are also responsible for activating other enzymes in pancreas (Rypniewski et al., 1994). As a well studied digestive proteinase, purification and characterization of trypsins and trypsin-like serine proteinases have been carried out in many fish species in the last decade, such as common carp (Cyprinus carpio) (Cao et al., 2000), Japanese anchovy (Engraulis ⁎ Corresponding author at: College of Biological Engineering, Jimei University, Jimei, Xiamen, 361021, China. Tel.: + 86 592 6180378; fax: + 86 592 6180470. E-mail address: [email protected] (M.-J. Cao). 1096-4959/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2011.11.012

japonicus) (Ahsan et al., 2001), Monterey sardine (Sardinops sagax caerulea) (Castillo-Yanez et al., 2004), tongol tuna (Thunnus tonggol) (Klomklao et al., 2006), true sardine (Sardinops melanostictus) (Kishimura et al., 2006), skipjack tuna (Katsuwonus pelamis) (Klomklao et al., 2007a), Atlantic bonito (Sarda sarda) (Klomklao et al., 2007b), walleye Pollock (Theragra chalcogramma) (Kishimura et al., 2008), hybrid tilapia (Oreochromis niloticus ×O. aureus) (Wang et al., 2010) and Japanese sea bass (Lateolabrax japonicus) (Cai et al., 2010). In higher vertebrates, the molecular structure accompanying with the evolution of trypsins from primitive organisms to the diverse and more complex functions has made this enzyme an excellent model for studying structure and functional relationships (Ahsan et al., 2001; Kanno et al., 2010; Kanno et al., 2011). Till now, cDNA sequences of trypsins from a few fish species were reported, including Atlantic cod (Gadus morhua L) (Gudmundsdottir et al., 1993), Atlantic salmon (Salmo salar) (Male et al., 1995), anchovy (Engraulis mordax) (Ahsan and Watabe, 2001), and Japanese flounder (Paralichthys olivaceus) (Suzuki et al., 2002). cDNA cloning is therefore important for elucidating the structure and function relationship of fish trypsins. Freshwater fish snakehead (Channa argus) is native to the Yangtze River, China and belongs to the family Channidae. Because of its

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delicious taste and even curative functions in traditional Chinese medicine, snakehead is an important economic fish and widely cultured in different provinces of China with the production of 358,000 tonnes in 2009 (Anonymous, 2010). Mature snakeheads feed on other fish, crustaceans, amphibians, small reptiles and even small mammals. They have great capacity to withstand poor water quality, low temperature and anoxic condition, so snakeheads are considered a threat to local biodiversity in many countries. A detailed study of digestive proteinases is thus important and may be helpful to the manufactures of fish feed and the aquaculture of snakehead. In our previous research, we have purified pepsinogens from snakehead and clarified the properties of these pepsingens (Chen et al., 2009). However, molecular and biochemical characterization of another major digestive proteinase trypsin from the fish have not been performed. In this study, we described the purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead. 2. Materials and methods 2.1. Fish Cultured snakeheads (C. argus) with body mass of about 2000 g were purchased alive from the market of Jimei, Xiamen, China. The fish were sacrificed and hepatopancreas was collected instantly and washed with ice water for experiment. 2.2. Chemicals DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q column were purchased from GE Healthcare (Piscataway, NJ, USA). t-Butyloxy-carbonyl-Phe-Ser-Arg-4-methyl-coumaryl-7-amide (BocPhe-Ser-Arg-MCA) and other synthetic fluorogenic peptide substrates (MCA-substrates) were obtained from Peptide Institute (Osaka, Japan). Lima bean trypsin inhibitor (LBTI) was from Worthington Biochemical Corporation (Lakewood, CO, USA), pepstatin and Pefabloc SC were purchased from Roche (Mannheim, Germany); L-3-carboxy-trans-2,3-epoxy-propionyl-L-Leu-4-guanidinobutylamide (E-64) was a product of Amresco (Solon, OH, USA); phenylmethanesulfonyl fluoride (PMSF) and benzamidine were products from SigmaAldrich (St. Louis, MO, USA). Protein marker for SDS-PAGE was from Bio-Rad (Richmond, VA, USA). Mung bean trypsin inhibitor (MBTI) was purified in our laboratory as described (Sun et al., 2010). All other chemicals used were of analytical grade. 2.3. Purification of trypsin Protein purification was done at 0–4 °C. Snakehead hepatopancreas (80 g) was minced and homogenized in 5 vol. of 20 mM Tris–HCl buffer (pH 7.5) using a tissue homogenizer (PT-2100, Kinematica, Littau, Switzerland). The extract was centrifuged at 12,000 g for 20 min (Avanti J-25, Beckman Coulter, USA). The supernatant was collected as crude enzyme. The crude enzyme was subjected to ammonium sulfate fractionation from 30% to 80% saturation. After centrifugation at 12,000 g for 30 min, the precipitate was dissolved in a minimum volume of 20 mM Tris–HCl buffer (pH 7.5) and dialyzed extensively against the same buffer. The dialyzed sample was applied to DEAESepharose (2.5 × 15 cm) pre-equilibrated with dialysis buffer. The column was washed with starting buffer until the absorbance at 280 nm reached baseline, and the bound proteins were eluted with a linear gradient of NaCl from 0 to 0.5 M in 20 mM Tris–HCl buffer (pH 7.5) at a flow rate of 1 mL/min. The unadsorbed active fraction from DEAE-Sepharose column was concentrated by ultrafiltration (YM-10 membrane, Millipore Corporation, Billerica, MA, USA) and then applied to a gel-filtration column (Sephacryl S-200 HR; 1.5 × 98 cm), pre-equilibrated with 20 mM

Tris–HCl buffer (pH 7.5) containing 0.2 M NaCl. The column was eluted with the same buffer at a flow rate of 0.5 mL/min. The active fraction was pooled and dialyzed against 20 mM Tris–HCl buffer (pH 9.0) for further experiment. The dialyzed fraction was applied to Hi-Trap Capto-Q column (5 mL) connected to AKTA protein purifier (GE Healthcare), preequilibrated with 20 mM Tris–HCl buffer (pH 9.0) and washed with the same buffer. The binding protein was eluted with a linear gradient of 0–0.5 M NaCl in 20 mM Tris–HCl buffer (pH 9.0) in a total volume of 100 mL at a flow rate of 0.5 mL/min. Purified trypsin was immediately used for enzymatic characterization or stored at − 30 °C. 2.4. Assay for trypsin activity Trypsin activity was measured as described by Lu et al. (2008), using Boc-Phe-Ser-Arg-MCA as substrate. The reaction was initiated by adding 50 μL of appropriately diluted enzyme to the incubation mixture containing 900 μL of 0.1 M Tris–HCl buffer (pH 8.0) and 50 μL of 10 μM substrate. After incubation for 10 min at 37 °C, 1.5 mL of the stopping solution (methyl alcohol:isopropyl alcohol: distilled water = 35:30:35, v/v) were added to terminate the reaction. The fluorescence intensity of liberated 7-amino-4-methylcoumarin (AMC) was measured by a fluorescence spectrophotometer (JASCO, FP-6200, Tokyo, Japan) at the excitation wavelength of 380 nm and the emission wavelength of 450 nm. Control test was conducted in the same manner without addition of enzyme. One unit of trypsin activity was defined as the amount of the enzyme to release 1 nmol of AMC per minute. 2.5. Polyacrylamide gel electrophoresis Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE) was carried out to determine the purity and molecular mass of the purified trypsin, according to the method of Laemmli (1970), using 12% separating gel and 4% stacking gel. 2.6. Determination of the protein concentration Protein concentration was determined by measuring the absorbance at 280 nm during column chromatographies or according to the method of Lowry et al. (1951) using bovine serum albumin (BSA) as standard. 2.7. MALDI-TOF/TOF-MS/MS analysis Purified protein was run on 12% SDS-PAGE and stained with Coomassie brilliant blue R-250. The single protein band obtained was identified using 4800 Plus MALDI-TOF/TOF-MS/MS Analyzer (Applied Biosystems, Carlsbad, CA, USA) by Shanghai Applied Protein Technology Company, Ltd. 2.8. Effect of pH and pH stability The effect of pH on trypsin activity was carried out in the pH range of 5.0–11.0 at 37 °C, using the following solutions (50 mM): sodium citrate buffer (pH 5.0–5.5), sodium phosphate buffer (pH 6.0–7.5), Tris–HCl buffer (pH 8.0–9.0) and Na2CO3–NaHCO3 buffer (pH 9.0– 11.0). For the effect of pH on stability, the enzyme was incubated at room temperature (20–25 °C) for 30 min in different buffers, and then the enzyme activity was measured at pH 8.0 by the method as described above. 2.9. Effect of temperature The effect of temperature on the trypsin activity was studied at 20 to 65 °C using 0.1 M Tris–HCl, pH 8.0 as reaction buffer. For thermal

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2.14. cDNA cloning of trypsinogen

Table 1 Primers used in the experiment. Name

Sequence (5′-3′)

Try-F1 Try-R1 Try-R2 GSP5-1 GSP5-2 GSP3-1 GSP3-2

GATTGTCGGAGGCTATGAGTGC GGTGTAGACGCCGGGCTTGTT GTAACCCCAGGACACCACACCC AGTTGGGATGACGGATGACCTTA CATGTTCTGTGCTGGATTCCTCG AGACCGTGTCCCTGCCCTCC GTAGCCACCGTTCAGAGAGACCT

stability assay, the purified enzyme was pre-incubated at different temperatures (20–65 °C) for 30 min. After that, the heated samples were immediately cooled in ice, and the residual activity was determined at 37 °C as described above.

Based on the amino acid sequence obtained from peptide mass fingerprinting, a sense primer (Try-F1) was designed. Two antisense primers (Try-R1 and Try-R2) were designed on the basis of the well-conversed regions of trypsin for PCR and nested-PCR. The sequences of these primers were listed in Table 1. The PCR program was performed in a thermal cycler, GeneAmp 9700 (Applied Biosystems, Carlsbad, CA, USA) as follows: 5 min at 94 °C followed by 30 cycles of 30 s at 94 °C, 45 s at 53 °C, 45 s at 72 °C, and a final extension of 7 min at 72 °C. Target DNA fragment was excised from agarose gel, purified with the Universal DNA Purification Kit (TIANGEN, Beijing, China), and cloned into pGEM-T Easy vector (Promega, Madison, WI, USA) followed by DNA sequence analysis. 2.15. 5′ and 3′-rapid amplification of cDNA ends (5′ and 3′-RACE)

2.10. Effect of proteinase inhibitors The effects of different proteinase inhibitors on trypsin were determined. The purified enzyme was pre-incubated with different inhibitors for 30 min at 25 °C, the remaining activity was determined by the method as described above. Control test was performed without addition of any inhibitor. 2.11. Substrate specificity and kinetic studies To characterize the substrate specificity of the purified trypsin, various synthetic fluorescence substrates were incubated with the trypsin and the amount of AMC released was determined. In order to analyze the kinetic constants of the trypsin, the enzyme with final concentration of 1 μg/mL was allowed to react with different substrates in the concentration range from 0.1 to 1 μM at 25 °C for 5 min. Kinetic parameters including νmax and Km were evaluated based on Lineweaver–Burk double-reciprocal plots. The turnover number (kcat) was calculated according to the following equation: kcat = νmax/[E], where [E] refers to the enzyme concentration.

From the sequence information obtained above, gene specific primers (GSP5-1, GSP5-2, GSP3-1, GSP3-2) were designed for 5′RACE and 3′-RACE, as shown in Table 1. RACE and RACE-PCR were conducted using the SMART RACE Amplification Kit (Clontech, CA, USA). Nested-PCR was adopted to improve the specificity of SMARTer RACE amplification and the program used was the same as mentioned above except the annealing temperature for 3′-RACE was 55 °C. The PCR product was then purified, cloned and sequenced. DNA sequencings were analyzed with the DNA sequencer ABI PRISM 3730 (CA, USA). 3. Results and discussion 3.1. Purification of snakehead trypsin

The enzyme activation energy (Ea) was measured by the method of Liu et al. (2007) with some modification. The Ea of trypsin was determined by measuring maximum reaction velocity (vmax) and kcat at different temperatures between 20 °C and 40 °C for 2 min using BocPhe-Ser-Arg-MCA as substrate. The activation energy was calculated by making a plot of lgkcat against 1/T (slope= −Ea/2.303R), according to the Arrhenius formula. The slope of the line was the Ea to catalyze the hydrolysis of Boc-Phe-Ser-Arg-MCA and the fit of the data to the Arrhenius equation was evaluated by least-squares regression analysis.

The purification runs had been performed for more than three times in different seasons from October to June, and similar results were obtained. In the present study, a cationic trypsin was purified from the hepatopancreas of snakehead by ammonium sulfate fractionation, DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap CaptoQ columns with results of 218-fold increase in specific activity and activity yield of 12.6% (Table 2). Ammonium sulfate precipitation with 30–80% saturation was effective in separating trypsin from most other proteins. After DEAE-Sepharose anion exchange chromatography, crude enzymes were divided into unadsorbed and adsorbed portion. The unadsorbed fraction was then collected for gel-filtration on Sephacryl S-200 HR (Fig. 1A). This column effectively separated trypsin from other proteins with little enzyme activity loss. After column chromatography on Hi-Trap Capto-Q column, the enzyme was purified to homogeneity and revealed a single band on SDS-PAGE with a molecular mass of approximately 22 kDa (Fig. 1B).

2.13. RNA preparation and cDNA synthesis

3.2. Identification of trypsin using MALDI-TOF/TOF-MS/MS

Total RNA was prepared from the hepatopancreas of snakehead using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First stranded cDNA was synthesized with TIANScript RT Kit (TIANGEN, Beijing, China) according to the manufacture's instruction.

To further confirm the purified protein, MALDI-TOF/TOF mass spectrometry was performed. The result of peptide mass fingerprinting (PMF) of the purified enzyme was shown in Fig. 2. A quantity of peptide fragments were observed in the m/z range of 800–4000 Da,

2.12. Determination of Arrhenius activation energy (Ea)

Table 2 Purification of trypsin from the hepatopancreas of snakehead. Purification steps

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Purification (fold)

Yield (%)

Crude enzyme Ammonium sulfate DEAE-Sepharose Sephacryl S-200 HR Hi-Trap Capto-Q

3520.5 1058.2 156.9 28.1 2.1

7945.5 7465.3 6467.7 5615.0 1004.5

2.3 7.0 40.5 200.5 502.0

1.0 3.0 17.6 87.2 218.3

100.0 94.0 81.4 70.7 12.6

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A 600

2.0

500 400

1.5 300 1.0 200 0.5 0

100

100 0

50

100

150

Relative activity (%)

2.5

Activity (U/ mL)

Absorbance at 280 nm

A

60 40 20

0

200

250

Fraction No. (2 mL/tube)

0

6

7

8

9

10

11

12

pH

B

0.5

1.0

500

B 100

0.4

M

1

300 200

25.0

0.2 0

0

10

20

30

100

0

18.4 14.4

40

50

0

Relative activity (%)

kDa 116 66.2 45.0 35.0

0.6

Activity (U/ mL)

400

0.8

NaCl (M)

Absorbance at 280 nm

80

80 60 40 20

Fraction No. (2 mL/tube) 0 Fig. 1. Chromatographic purification of trypsin from snakehead. (A) Sephacryl S-200 HR chromatography for unadsorbed trypsin fraction from DEAE-Sepharose; (B) Hi-Trap Capto-Q chromatography for trypsin from Sephacryl S-200 HR. (—) Absorbance of 280 nm; (●) trypsin activity for hydrolyzing Boc-Phe-Ser-Arg-MCA. Fractions under the bars were pooled. The SDS-PAGE of purified trypsin is shown in the inset of panel B. Lane 1, protein marker; lane 2, purified trypsin.

6

7

8

9

10

11

12

pH Fig. 3. The effect of pH on trypsin. A, pH profile; B, pH stability.

identity to a trypsinogen (gi|971196) from pufferfish (T. rubripes) (Fig. 2) suggesting the purified enzyme is trypsin. which was searched against the NCBI non-redundant protein sequence database. Subsequently, two signal-noise ratio peptide fragments of trypsin were subjected to MALDI-TOF/TOF-MS/MS analysis. Two peptide fragments of 25 amino acid residues revealed 100%

3.3. Effect of pH and temperature The effect of pH on trypsin was revealed in Fig. 3. Purified trypsin showed maximal activity at pH 9.0 (Fig. 3A), and the enzymatic

Takifugu rubripes LIAAAYAAPIDEDDKIVGGYECRKASVAYQ IVGGYECR Channa argus Takifugu rubripes VSLNSGYHFCGGSLVNENWSVVSAAHCYKS Takifugu rubripes Channa argus Takifugu rubripes Channa argus Takifugu rubripes

RVVVRLGEHNIRANEGTEQFISSSRVIRHP HP NYSSYNIDNDIMLIKLSKPNTLNQYVQPVA NYSSYNIDNDIMLIK LPSSCAAAGTMCKVSGWGNTMSSTADRNKL

Takifugu rubripes QCLNIPILSDRDCENSYPGMITDAMFCAGY Takifugu rubripes LEGGKDSCQGDSGGPVVCNNELQGVVSWGY Takifugu rubripes GCAERDHPGVYAKVCLFNDWLESTMASY

Fig. 2. Peptide mass fingerprinting (PMF) of trypsin. A great quantity of peptide fragments are observed in the m/z range of 800–4000 Da. Two peptide sequences of snakehead trypsin were compared to trypsinogen (gi971196) of puffer fish (Takifugu rubripes). Identical amino acid residues are shown in bold letters.

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activity decreased obviously below pH 7.0 or above 10.0 (Fig. 3B), which was in accordance with the fact that trypsins are generally alkaline proteinases. The effect of pH on snakehead trypsin was similar to these from common carp (Cao et al., 2000), tongol tuna (Klomklao et al., 2006), skipjack tuna (Klomklao et al., 2007a), sardine (Bougatef et al., 2007), bluefish (Klomklao et al., 2007c), Atlantic cod (Stefansson et al., 2010), hybrid tilapia (Wang et al., 2010) and Japanese sea bass (Cai et al., 2010). The effect of temperature on snakehead trypsin was illustrated in Fig. 4. The enzyme revealed maximal activity at 40 °C (Fig. 4A), and it was stable below 45 °C. However, the activity decreased obviously above 50 °C (Fig. 4B). These results, together with the data from common carp (Cao et al., 2000), grass carp (Ctenopharyngodon idellus) (Liu et al., 2007) and mandarin fish (Siniperca chuatsi) (Lu et al., 2008), revealed that the optimum temperature and thermal stability of trypsins from fresh water fish are generally lower than those of marine fish, such as Chinook salmon (Oncorhynchus tshawytscha, 60 °C) (Kurtovic et al., 2006), true sardine (S. melanostictus, 60 °C) and arabesque greenling (Pleuroprammus azonus, 50 °C) (Kishimura

A Relative activity (%)

100 80 60 40 20 0 20

30

40

50

Temperature (

60

70

)

B

Relative activity (%)

100

et al., 2006), skipjack tuna (60 °C) (Klomklao et al., 2007a), and sardine (60 °C) (Bougatef et al., 2007). A linear Arrhenius plot of lgkcat versus 1/T was obtained using BocPhe-Ser-Arg-MCA as substrate in the temperature range of 20–40 °C (Fig. 4C). The Ea value represents the energy barrier for trypsin to catalyze the substrate. From the slope of the line, Ea of the enzyme was determined as 24.65 kJ·M − 1. 3.4. Effects of proteinase inhibitors The effects of different proteinase inhibitors on the purified trypsin were shown in Table 3. The enzymatic activity was strongly inhibited by serine proteinase inhibitors such as PMSF, LBTI, MBTI, Pefabloc SC and benzamidine. Metalloproteinase inhibitors 1, 10-phenanthroline and EDTA, and asparatic proteinase inhibitor pepstatin partially inhibited the enzymatic activity. These results implied the possible requirement of metal ion(s) for its activity. Furthermore, cysteine proteinase inhibitor E-64, suppressed trypsin activity significantly. E-64 is an irreversible, potent, and highly selective cysteine protease inhibitor. However, E-64 is also one of the most effective low-molecular-weight inhibitors of trypsin-catalyzed hydrolysis (Sreedharan et al., 1996). Trypsins from vertebrate species commonly have six disulfide bridges (Muhlia-Almazan et al., 2008). In our present study, 80.5% of trypsin activity was restrained by E-64 at the concentration of 0.02 mM, implying the existence of Cys residue(s) near its active site, which affect the affinity of trypsin to substrate during cleavage. The result was similar to trypsin from Japanese sea bass (L. japonicus) (Cai et al., 2010). This proposal was confirmed in our present molecular cloning study as shown in Fig. 5 and will be discussed below. The enzyme activity can be slightly activated by low concentration of Ca 2 + (1 mM) (114%), which was similar to trypsins from common carp (Cao et al., 2000), hybrid catfish (Clarias macrocephalus × Clarias gariepinus) (Klomklao et al., 2011), true sardine and arabesque greenling (Kishimura et al., 2006) and mandarin fish (Lu et al., 2008). Ca 2 + could not only protect trypsin against autolysis and thus maintain its stability, but also increase its proteolytic activity (Bode and Schwager, 1975).

80

3.5. Substrate specificity and kinetic parameters 60

For substrate specificity analysis, various fluorescent MCAsubstrates were used. Trypsins strongly prefer to cleave fluorogenic substrates having Arg or Lys at P1 positions. As shown in Table 4, snakehead trypsin hydrolyzed Boc-Phe-Ser-Arg-MCA most effectively. Substrates for cathepsins (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA), and aminopeptidases (Lys-MCA and Arg-MCA) were not hydrolyzed. No hydrolysis to chymotrypsin substrate (Suc-Leu-Leu-Val-Tyr-MCA) was detected, indicating no contamination of chymotrypsin in the purified trypsin. It was reported that the preference of trypsin for cleavage Arg over Lys is 2- to 10-fold (Craik et al., 1985). This was confirmed in our present study as the relative activities to two

40 20 0 20

30

40

50

Temperature (

C

251

60

70

)

1.5

Lgkcat

1.4 Table 3 Effects of proteinase inhibitors on snakehead trypsin activity.

1.3

1.2

1.1 3.1

3.2

3.3

3.4

3.5

Temperature (K-1*1000) Fig. 4. The effect of temperature on trypsin. A, temperature profile; B, thermal stability; C, Arrhenius plot of trypsin.

Inhibitors

Concentration (mM)

Relative activity (%)

Control MBTI Pefabloc SC PMSF LBTI Benzamidine E-64 EDTA 1,10-phenanthroline Pepstatin

0 0.003 1 10 0.002 5 0.02 5 5 0.15

100 3.6 4.5 4.5 7.9 17.9 19.5 54.4 64.4 72.1

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Fig. 5. Nucleic acid and deduced amino acid sequences of snakehead trypsin. Residue numbers for nucleic acid and amino acid are indicated in the left of each row. Two cleavage sites for the signal peptide (closed triangle) and activation peptide (open triangle) are shown. The signal peptide sequence is lined and the dot-lined is the trypsinogen activation peptide. The italics are the peptide fragment observed by PMF. Two different amino acid residues are framed.

substrates Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA having Lys at P1 are 3.7–6.8 folds lower than that to Boc-Phe-Ser-Arg-MCA. Kinetic constants, Km and kcat of the trypsin for hydrolysis of specific MCA-substrates were determined according to the Lineweaver– Burk plots (Table 4). Using Boc-Phe-Ser-Arg-MCA as substrate, Km and kcat values of trypsin were 0.663 μM and 116 S − 1, respectively, and kcat/Km was 174.96 s − 1 μM − 1. The Km of the trypsin was smaller than that of trypsins from mandarin fish (Lu et al., 2008), suggesting the affinity of snakehead trypsin to substrates was higher than trypsins from mandarin fish. 3.6. cDNA cloning of snakehead trypsinogen The full-length sequence of snakehead trypsinogen was obtained by overlapping three DNA fragments, namely, conserved region, 5′RACE and 3′-RACE. The nucleic acid and deduced amino acid Table 4 Substrate specificity and kinetic parameters of snakehead trypsin on synthetic fluorogenic substrates. Substrates (10 μM)

Relative activity (%)

Km (μM)

kcat (S− 1)

kcat/Km (S− 1 μM− 1)

Boc-Phe-Ser-Arg-MCA Boc-Gln-Arg-Arg-MCA Boc-Glu-Arg-Arg-MCA Boc-Leu-Arg-Arg-MCA Boc-Leu-Lys-Arg-MCA Boc-Val-Pro-Arg-MCA Boc-Val-Leu-Lys-MCA Boc-Glu-Lys-Lys-MCA Z-Arg-Arg-MCA Z-Phe-Arg-MCA Suc-Leu-Val-Val-Tyr-MCA Suc-Leu-Leu-Val-Tyr-MCA Lys-MCA Arg-MCA

100 94.8 75.2 51.4 44.5 33.5 27.1 14.8 5.4 7.4 0 0 0 0

1.02 0.49 0.64 0.59 0.92

148 76 92 72 80

144.7 153.9 143.3 122.2 87.2

sequence was revealed in Fig. 5. The trypsinogen was 916 bp in length, with a 744 bp open reading frame at position 10-753, encoding 247 amino acid residues. The deduced protein sequence has a signal peptide of 15 amino acid residues, and a trypsinogen activation peptide (TAP) of 9 residues. Mature trypsin starts from Ile25. Asn99 and Asn-147 are putative N-glycosylation sites. Similar result was also reported in trypsin from earthworm (Wang et al., 2005). The nucleotide sequence data is available in the GenBank database under the accession number JN558641 for snakehead trypsinogen cDNA. The theoretical pI of the mature protein is 7.99, which is consistent with the fact that the trypsin was not adsorbed by DEAESepharose column at pH 7.5 while adsorbed when applied to HiTrap Capto-Q using 20 mM Tris–HCl buffer (pH 9.0). The theoretical molecular mass of the trypsin was 24.45 kDa, which is slightly higher than the result from SDS-PAGE (22 kDa). The difference of trypsin in molecular mass may be ascribed to the mobility of the enzyme on the gel or an autolysis at the C-terminal as the N-terminal sequence IVGGYECR of the mature trypsin was detected by PMF (Fig. 2). From sequence alignment in Fig. 5, if autolysis happened at the C-terminal, most possible cleavage site would be between R-224 and N-225 as the trypsin favors to cleave Arg than Lys (Table 4), which will delete a peptide of 23 amino acid residues and produce a trypsin with 21 kDa in molecular mass. As shown in Fig. 5, two amino acid residues difference (Ser-104, Leu-105) from the PMF of purified trypsin and the cloned sequence was noticed. As the cloned sequence was confirmed by analyzing 3 independent clones, a possible measurement mistake of PMF was proposed although the possibility of the existence of another isoform of trypsin should not be excluded. Comparison of the deduced trypsinogen sequence of snakehead with trypsinogens from other kinds of fish was shown in Fig. 6. Twelve Cys residues at positions 31, 49, 65, 133, 140, 160, 172, 186, 197, 207, 221 and 234 that may form six intramolecular disulfide bonds were observed (Ruan et al., 2010). In addition to the most important catalytic triad His64, Asp108, and Ser201, the neighboring residues are also highly conserved. Furthermore, Cys197 and Cys207

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Fig. 6. Alignment of snakehead trypsin sequence with trypsins from other fish. The sequence of snakehead trypsin is compared with that of Senegalese sole trypsinogen 2 (BAF76144), orange-spotted grouper trypsinogen (ADG29127), zebrafish trypsinogen (AAH92664), mandarin fish pancreatic trypsinogen (ACH53602), and mefugu trypsinogen 2 (ACT15362). Short lines indicate homology with consensus while dots fill the gaps for optional alignment. The His-64, Asp-108 and Ser-201 which form the triad active site of trypsin are marked with closed arrows.

exist nearby the active site Ser201 which explained why trypsin was significantly inhibited by a cysteine proteinase inhibitor E-64 (Table 3). Asp-195 is negatively charged for attracting Arg and Lys which are positively charged during cleavage reaction and thus it is required for the tryptic specificity for Arg and Lys (Rypniewski et al., 1994; Muhlia-Almazan et al., 2008) (Fig. 5). Although a trypsin has been purified and its sequence determined, fish trypsin usually has more than two isoforms (Cao et al., 2000; Lu et al., 2008). In order to elucidate these trypsins, further determination of the complete sequences of them is necessary. Acknowledgments This study was sponsored by the National Natural Scientific Foundation of China (Nos. 31071519, 20872049), the Science and Technology Bureau of Fujian Province (2010NZ0001-3) and the Foundation for Innovative Research Team of Jimei University (2010A005). References Ahsan, M.N., Watabe, S., 2001. Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus. J. Protein Chem. 20, 49–58. Ahsan, M.N., Funabara, D., Watabe, S., 2001. Molecular cloning and characterization of two isoforms of trypsinogen from anchovy pyloric ceca. Mar. Biotechnol. 3, 80–90. Anonymous, 2010. China Fisheries Yearbook. Agriculture press of China, p. 173 (in Chinese). Bode, W., Schwager, P., 1975. The refined crystal structure of bovine beta-trypsin at 1.8 A resolution. II. Crystallographic refinement, calcium binding site, benzamidine binding site and active site at pH 7.0. J. Mol. Biol. 98, 693–717. Bougatef, A., Souissi, N., Fakhfakh, N., Ellouz-Triki, Y., Nasri, M., 2007. Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus). Food Chem. 102, 343–350. Cai, Q.F., Jiang, Y.K., Zhou, L.G., Sun, L.C., Liu, G.M., Osatomi, K., Cao, M.J., 2010. Biochemical characterization of trypsins from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus). Comp. Biochem. Physiol. B. 159, 183–189.

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