Molecular characterization of two trypsinogens in the orange-spotted grouper, Epinephelus coioides,...

Molecular characterization of two trypsinogensin the orange-spotted grouper, Epinephelus coioides,and their expression in tissues during early development

Chun-Hung Liu • Ya-Huei Chen • Ya-Li Shiu

Received: 7 November 2011 / Accepted: 5 July 2012 / Published online: 17 July 2012

� Springer Science+Business Media B.V. 2012

Abstract In this study, we cloned two trypsinogens

of the orange-spotted grouper, Epinephelus coioides,

and analyzed their structure, expression, and activity.

Full-length trypsinogen complementary (c)DNAs,

named T1 and T2, were 900 and 875 nucleotides,

and translated 242 and 244 deduced amino acid

peptides, respectively. Both trypsinogens contained

highly conserved residues essential for serine protease

catalytic and conformational maintenance. Results

from isoelectric and phylogenetic analyses suggested

that both trypsinogens were grouped into trypsinogen

group I. Both trypsinogens had similar expression

patterns of negative relationship with body weight;

expression was first detected at 1 day post-hatching

(DPH) and exhibited steady-state expression during

early development at 1–25 DPH. Both expression and

activity levels significantly increased after 30 DPH

due to metamorphosis. Grouper larval development is

very slow with insignificant changes in total length

and body weight before 8 DPH. The contribution of

live food to an increase in the trypsin activity profile

may explain their importance in food digestion and

survival of larvae during early larval development.

Keywords Orange-spotted grouper � Trypsinogen �Trypsin activity � Digestion � Larval development

Introduction

Trypsin is an endopeptidase belonging to the serine

protease family (Rawlings and Barrett 1994). It plays a

key role in the digestive capacity of fish larvae and

other animals. Trypsins are characterized by a cata-

lytic triad composed of three essential amino acid

residues of His-75, Asp-102, and Asp-189 in the

S1-binding pocket, which confers specificity to cleav-

age at the peptide bond on the carboxyl side of basic

L-amino acids such as arginine and lysine residues

(Graf et al. 1988), and Tyr-172 participates in

substrate specificity (Hedstrom et al. 1994). Trypsin

is synthesized as a preproenzyme that is processed to

the proenzyme of trypsinogen (Mithofer et al. 1998).

The N-terminal activation peptide of trypsinogen is

removed by specific cleavage between a Lys or an Arg

residue and an Ile residue to convert it into its active

form, trypsin (Light and Janska 1989). In addition, the

Ile residue at the amino-terminal end bends inward and

makes several internal contacts when forming the

catalytically active trypsin (Bolognesi et al. 1982). In

turn, the resulting trypsins themselves activate all

other pancreatic digestive zymogens including more

trypsinogens (Chen et al. 2003).

Several isoforms of trypsin were described in both

mammals and fish (Manchado et al. 2008). On the

C.-H. Liu (&) � Y.-H. Chen � Y.-L. Shiu

Department of Aquaculture, National Pingtung University

of Science and Technology, Pingtung 91201,

Taiwan, ROC

e-mail: [email protected]

123

Fish Physiol Biochem (2013) 39:201–214

DOI 10.1007/s10695-012-9691-4

basis of their amino acid sequence identities and to a

lesser degree, their charges at physiologic pH, they are

classified into three groups: I, II, and III. Most

vertebrates possess at least one trypsinogen gene from

groups I and II. However, group III has so far only

been found in teleosts (Manchado et al. 2008). The

isoforms of trypsin have different expression patterns

in teleosts during their life cycle and in different

tissues indicating functional differences (Murray et al.

2004; Manchado et al. 2008).

Digestive enzymes affect the digestive capacity and

set physiologic limits on the growth rate and feed

conversion ratio (Perez-Casanova et al. 2006). Trypsin

activity in the pyloric cecum was shown to be

positively involved in the growth rate of Atlantic

cod, Gadus morhua (Lemieux et al. 1999), and

Atlantic salmon, Salmo salar (Rungruangsak-Torris-

sen et al. 2006). However, a contrary finding about

trypsin activity with the growth status of orange-

spotted grouper, Epinephelus coioides, was found in

our previous study (Liu et al. 2012). The different

expression patterns of trypsin are considered to be

species specific, and other proteases, like pepsin, play

more important roles in regulating growth of grouper

through protein digestion (Liu et al. 2012). However,

trypsin plays important roles in digestive function

during the early larval stage. The study of trypsin

activity and other digestive enzymes during early

development of the digestive system in marine fish is a

valuable tool to better understand the digestive

physiology of larvae, since they can be used as

indicators of the nutritional status (Eusebio et al. 2004;

Fujii et al. 2007).

In this study, two complete trypsinogen cDNAs

from orange-spotted grouper were isolated, and a

phylogenetic analysis revealed the presence of two

distinct groups. In addition, the expression and

enzymatic activity of each isoform were analyzed

during early development.

Materials and methods

Experimental rearing and sampling

Orange-spotted grouper, E. coioides (at 3 cm in total

length), were reared for 5 months and sampled from a

farm of the Department of Aquaculture, National

Pingtung University of Science and Technology,

Taiwan. Fish were reared in a cement tank

(6 9 2 9 1.5 m) with 10 tons of saltwater at 15 %salinity and continuous aeration through an air stone.

The temperature, pH, and dissolved oxygen (DO) were

maintained in ranges of 27–29 �C, 7.5–8.0, and

5.6–6.1 mg/l, respectively. Fish were fed a commer-

cial diet at a ratio of 5 % of their body weight once

daily.

For the analysis of trypsinogen expression during

larval development, grouper larvae were reared.

Orange-spotted grouper-fertilized eggs were a kind

gift from a private fish farm in Pingtung County,

Taiwan. Eggs were placed in fiberglass-reinforced

plastic (FRP) tanks with 500 L of 30 % saltwater and

continuous aeration. Grouper larvae hatched in about

24 h after fertilization at a temperature of 27 �C. After

hatching, larvae were transferred with water into two

cement tanks (6 9 2 9 1.5 m) with 10 metric tons of

30 % saltwater and continuous aeration. In addition,

larval stock in a smaller cement tank (1.8 9 1.3 9

1 m) with 2 metric tons of 30 % saltwater and

continuous aeration was used as the unfed treatment.

The water temperature, pH, and DO were maintained

in ranges of 27–29 �C, 7.7–7.9, and 5.3–6.0 mg/l,

respectively, during the experiment. The stock density

was 5000 larvae per metric ton of water. During larval

breeding, the water was not changed. Two days after

hatching, larvae were fed oyster trochophores three

times per day for 5 days. Rotifers screened through a

200-mesh phytoplankton net were fed to larvae four

times per day from 6 to 10 days post-hatching (DPH).

Thereafter, rotifers that passed through a 150-mesh

phytoplankton net were fed to larvae four times per day

until 22 DPH. After 17 DPH, copepods were also used

as live food to gradually replace the rotifers, and only

copepods were used for larval feeding four times per

day after 22 DPH until more than 95 % of the fish had

undergone metamorphosis (the pelvic fins and dorsal

fin completely shortened) (at 35 DPH). Fish were then

fed ad libitum with shrimp paste for 5 days followed by

semi-moist feed, mixed with shrimp paste and com-

mercial feed powder at a ratio of 8:2 (Table 1).

Grouper were randomly sampled from the rearing

tank using a hand net. Before sampling, fish were not

fed for 2 days in order to empty the gut and facilitate

dissection. For trypsinogen cDNA cloning, total RNA

isolated from the pyloric cecum of juvenile

(35.6 ± 3.6 g) fish was used. Fish with an average

weight of 118.5 ± 1.27 g were used for expression

202 Fish Physiol Biochem (2013) 39:201–214

123

analysis of the two trypsinogens (T1 and T2) in

different tissues, including the esophagus, stomach,

pyloric cecum, anterior intestine, middle intestine,

hind intestine, eyes, liver, gills, skin, heart, head

kidney, mid-kidney, hind kidney, spleen, brain, dorsal

muscles, and blood by a reverse-transcription poly-

merase chain reaction (RT-PCR). In addition, tissues

with abundant trypsinogen expression, including the

pyloric cecum, anterior intestine, middle intestine,

hind intestine, and spleen were used for a real-time

PCR analysis. To analyze the relationship between

size and expression of the two trypsinogen genes by a

real-time PCR, the pyloric cecum, anterior intestine,

middle intestine, and hind intestine were dissected

from grouper at a size range of 26.35–179.1 g. In order

to investigate the beginning of trypsinogen transcrip-

tion in the life cycle, an RT-PCR was performed.

Thirty fertilized eggs, 10 living larvae from the

feeding treatment at 0, 2, 4, 6, and 12 h, and 1, 2, 3,

and 4 DPH, and 10 living larvae from unfed treatment

at 2 and 3 DPH were sampled to analyze trypsinogen

expression by an RT-PCR. Samples consisting of 30

eggs or 10 larvae from each sampling were pooled.

Trypsinogen expression and trypsin activity assays

during larval development were also carried out.

Twenty larvae were pooled for each replicate at 1, 2, 3,

and 4 DPH, five larvae were pooled for each replicate

at 8 DPH, three larvae were pooled for each replicate

at 12 and 16 DPH, and one larva was used for each

replicate at 20, 25, 30, 35, 40, 45, and 50 DPH. Each

sampling consisted of six replicates. Larvae were

individually collected; the body weight and total

length were measured; then larvae were washed with

DEPC water, frozen in liquid nitrogen, and stored at

-80 �C. At 1–25 DPH, whole larvae were used for the

analysis. The digestive tract of a larva was sampled for

analysis at 30–50 DPH. Oyster trochophores were also

prepared for the analysis of trypsin activity.

Cloning of trypsinogens and phylogenetic analysis

Total RNA was extracted from the pyloric cecum of

fish and further purified using ULTRASPECTM RNA

and Total RNA Isolation Reagent (Biotecx, Houston,

TX, USA) following the manufacturer’s instructions.

First-strand cDNA synthesis in RT was accomplished

using Super-Script II RNase H- reverse transcriptase

(Promega, Madison, WI, USA) to transcribe poly (A)?

RNA with oligo-d(T)18 as the primer. Reaction

conditions recommended by the manufacturer were

followed.

Full-length trypsinogen cDNA of grouper was

obtained by an RT-PCR, and 30 and 50 rapid ampli-

fication of cDNA (RACE) methods. Degenerate

primers were designed based on the highly conserved

teleost amino acid sequence of trypsinogen in Gen-

Bank and another database (Benson et al. 1994) and

using the ClustalW program (http://align.genome.jp/).

The degenerate primer pair of CTF1 and CTR1

(Table 2) was used to amplify partial grouper tryp-

sinogen cDNA fragments. The PCR was carried out in

a 50-ll reaction volume containing 10 ng of cDNA,

1 9 ProTaq buffer (Protech, Taipei, Taiwan), 2.5 U of

Pro Taq Plus DNA polymerase (Protech), 0.25 mM of

dNTPs, and 0.25 lM of each primer. PCRs were

performed as follows: 30 cycles of denaturation at

94 �C for 1 min, annealing at 50 �C for 1 min, and

elongation at 72 �C for 2 min, followed by a 10-min

extension at 72 �C and cooling to 4 �C.

Total RNAs from the grouper pyloric cecum were

also used for the 50 and 30 RACE. The RACE cDNA

template was synthesized using the ExactSTARTTM

Eukaryotic mRNA 50-& 30-RACE Kit (cat no.

ES80910, Epicentre, Madison, WI, USA) according

to the manufacturer’s protocol and amplified by a PCR

with PRIMER 1 and PRIMER 2 supplied by the kit.

For trypsinogen 1 (T1), the 50 and 30 ends were

amplified by nested PCRs with PRIMER 1 and specific

primers (T15RACER1 and T15RACER2), and PRI-

MER 2 and specific primers (T13-GTA-F1 and T13-

GTA-F2), respectively. For trypsinogen 2 (T2),

50and 30 ends were amplified by nested PCRs with

PRIMER 1 and specific primers (T25RACER1 and

T25RACER2), and PRIMER 2 and specific primers

(T2GT3-1F and T2GT3-2F), respectively (Table 2).

Table 1 Feeding schedule for grouper larval rearing

Day post-hatching Feeding schedule

2 ? 7 Oyster trochophores 3 times per day

6 ? 10 Rotifer screened through 200 mesh

net 4 times per day

11 ? 22 Rotifer screened through 150 mesh

net 4 times per day

17 ? 35 Copepods 4 times per day

35 ? 40 Shrimp paste (Litopenaeus vannamei)

41 ? 50 Semi-moist feed (mixed with shrimp

paste and feed powder at a ratio of 8:2)

Fish Physiol Biochem (2013) 39:201–214 203

123

http://align.genome.jp/

The PCR was performed in 50-ll reactions as

described above and under the following cycling

conditions: 30 cycles of denaturation at 94 �C for

1 min, annealing at 50 �C for 1 min, and elongation at

72 �C for 2 min, followed by a 10-min extension at

72 �C and cooling to 4 �C.

The PCR fragments were subjected to electropho-

resis on a 1.5 % agarose gel for length difference, and

all PCR-amplified cDNA fragments were cloned into

the PCRII TOPO vector of the TOPO TA cloning

system (Invitrogen) and transferred into Escherichia

coli cells according to the manufacturer’s protocol.

Recombinant bacteria were identified by blue/white

screening and confirmed by a PCR. Plasmids contain-

ing the insert were purified using the Wizard� Plus

Miniprep DNA Purification System (Promega) and

used as a template for DNA sequencing.

A nucleotide sequence analysis was performed

using the dideoxynucleotide chain termination method

(Sanger et al. 1977) on a DNA sequencer (Model

373A, Applied Biosystems, Lincoln, NE, USA).

Plasmid DNA at 1 lg was used for sequencing with

a Dye Terminator Cycle Sequencing Kit (Applied

Biosystems) and was subjected to electrophoresis on

6 % denaturing gels. Clones were sequenced with the

M13 forward and reverse primers. Trypsinogen gene

sequences were analyzed and compared using the

BLASTX and BLASTP search programs (http://blast.

genome.ad.jp) with a GenBank database search.

Multiple sequence alignments of grouper trypsinogen

genes were created using the ClustalW program

(http://align.genome.jp/).

Phylogenetic trees were constructed on the basis of

the proportion of amino acid differences (p-distances)

by the neighbor-joining (NJ) method (Saitou and Nei

1987) using MEGA 4 software (Kumar et al. 1993).

For phylogenetic tree construction, indels were

removed from multiple alignments. The reliability of

the tree obtained was assessed by bootstrapping using

1000 bootstrap replications (Felsenstein 1985).

Gene expression

For expression of the two trypsinogen genes in

different tissues and expression of two trypsinogen

and trypsin activity during early development by an

RT-PCR, 3 lg of total RNA from each tissue was

transcribed with oligo (dT). Specific primer pairs,

TI-F1/TI-R1 and TII-F1/TII-R2, were, respectively,

used for T1 and T2 fragment amplification, and the

primer pair, ActinF/ActinR, was used to amplify the

b-actin fragment as the internal control. The PCR

conditions were the same as those described above

except the respective annealing temperatures for T1,

T2, and b-actin were 49, 56, and 50 �C.

For the real-time PCR analysis, a SYBR green I

real-time PCR assay for transcription of the relative

grouper trypsinogens was carried out using an ABI

PRISM 7900 Sequence Detection System (Applied

Biosystems) according to the method described by Liu

et al. (2007a). The accuracy and reproducibility of the

real-time PCR assays were determined by running

standard curves. The amplification efficiency was

obtained from given slopes of the standard curve:

Eslop = 10(-1/slope) - 1. The slopes of T1, T2, and

b-actin were -3.2153, -3.3133, and -3.2186, which

Table 2 Primers used for full-length trypsinogen cDNA

cloning, and gene RT-PCR and real-time PCR

Primers Sequence (50 ? 30)

CTF1 AAGATYGTCGGAGGSTATGAGTG

CTR1 CCGTARCCCCAGGACACMACACC

T13-GTA-F1 CCTACCCTGGCATGATCACTG

T13-GTA-F2 TCGTGTGCAACGGTGAGCTTC

T15RACER1 GTGGAGCTCATGGTGTTG

T15RACER2 AGAGCCTCCACAGAAGTG

T2GT3-1F GCCACCCTCAACCAGTAC

T2GT3-2F ATTGACTACACCATGTTCTG

T25RACER1 GTGGAGCTCATGGTGTTG

T25RACER2 AGAGCCTCCACAGAAGTG

PRIMER2 TAGACTTAGAAATTAATACGACTC

ACTATAGGCGCGCCACCG

PRIMER1 TCATACACATACGATTTAGGTGACACTA

TAGAGCGGCCGCCTGCAGGAAA

Q-TIF4 CTGCCTCTTCAACGACTG

Q-TIR4 AGATGTAGGACTTTATTGACTCA

Q-TIIF2 CTATTAACTCCACATCCAACCT

Q-TIIR2 TGATTCCAGCAAAGACAAGA

TI-F1 CCTGGCATGATCACCAATG

TI-R1 GAACATGCTCGTTCAGATC

TII-F1 GGAGGCTCTCTGATCTCCAGC

TII-R1 TGTCATTGTCCAGGTTGCGGC

Q-actinF CTATTAACTCCACATCCAACCT

Q-actinR TGATTCCAGCAAAGACAAGA

ActinF TAGGTGGTCTCGTGGATGCC

ActinR GAGACCTTCAACACCCCCGC


123

http://blast.genome.ad.jp

http://blast.genome.ad.jp

http://align.genome.jp/

correlated with respective amplification efficiencies of

104.7, 100.4, and 104.5 % (Fig. 1). All of them were

within the acceptable efficiency of 100 ± 10 %.

The respective primer pairs used for T1, T2, and

b-actin (internal control) for amplification were

Q-TIF4/-TIR4, Q-TIIF2/Q-TIIR2, and Q-actinF/Q-

actinR (Table 2). Amplifications were performed in a

96-well plate in a 25 ll reaction volume containing

12.5 ll of 29 SYBR Green Master Mix (PE Applied

Biosystems), 2.5 ll each of the forward and reverse

primers (10 mM), 1 ll of template (1 lg cDNA), and

9 ll of DEPC water. The thermal profile for the SYBR

green real-time PCR was 50 �C for 2 min and 95 �C

for 10 min followed by 40 cycles of 95 �C for 15 s and

60 �C for 1 min. Each sample was analyzed in

duplicate. Distilled water replaced the template as

the negative control. Relative mRNA expression of

target genes to the reference gene was calculated using

the 2-DDCt method (Livak and Schmittgen 2001). To

analyze trypsinogen expression during larval devel-

opment, relative expression of T1 and T2 from whole

larvae at 1–25 DPH and digestive tracts from larvae at

30–50 DPH was separately analyzed using the 2-DDCt

method. To calculate relative expression levels of T1

(A)

(C)

(B)

Y = 0.3214X - 3.2153

R2 = 0.99720

1

2

3

4

5

6

7

Ct

Lo

g (

dilu

tio

ns)

(D)

Y = 0.3326X - 3.3133

R2 = 0.99370

1

2

3

4

5

6

7

Ct

Lo

g (

dilu

tio

ns)

(E)

Y = 0.3251X - 3.2186

R2 = 0.99710

1

2

3

4

5

6

7

10 12 14 16 18 20 22 24 26 28 30

10 12 14 16 18 20 22 24 26 28 30

10 12 14 16 18 20 22 24 26 28 30

Ct

Lo

g (

dilu

tio

ns)

(F)

Fig. 1 Amplification efficiency determination via a standard

curve analysis. Amplification profiles for T1 (a), T2 (b), and b-

actin (c) generated by six quantities of cDNA, ranging from 4 to

0.00004 lg in tenfold decrements. T1 (d), T2 (e), and b-actin

(f) standard curves were generated by plotting the cycle

threshold value against the log of concentration


123

and T2, data were calibrated to whole larvae and

digestive tracts at 1 and 30 DPH, respectively.

Trypsin activity assay

The method for the trypsin activity assay was modified

from Liu et al. (2007b) using BAEE as a substrate.

Samples were individually homogenized in 250 ll of

50 mM Tris–HCl buffer (pH 8.0), and the homogenate

was centrifuged at 10,0009g for 30 min at 4 �C.

Supernatants were used to measure trypsin activity as

a crude enzyme solution. An enzyme solution at an

appropriate dilution (20 ll) was mixed with 0.6 ml of

0.5 mM BAEE in 50 mM Tris–HCl buffer (pH 8.0)

and 10 mM CaCl2, and then the increment in absor-

bance at 1 and 3 min was detected at 253 nm. One unit

of activity was defined as the amount causing an

increase of 1 in the absorbance at 253 nm per minute.

Statistical analysis

Experimental data were statistically analyzed by one-

way analysis of variance (ANOVA), and a multiple-

comparisons (Tukey’s) test was conducted to examine

significant differences among treatments using the

SAS computer software (SAS Institute, Cary, NC,

USA). Statistically significant differences required

p \ 0.05. A linear regression was used from Microsoft

Excel (Microsoft, Redmond, WA, USA) to study the

relationship between trypsinogen expression levels of

T1 and T2 and body weight.

Results

Molecular characterization of two grouper

trypsinogens

A 900-bp cDNA of T1 contained a 50-untranslated

region (UTR) of 14 bp, an open reading frame (ORF)

of 726 bp, and a 30-UTR of 160 bp with a stop codon

(TAA), a consensus polyadenylation signal

(AATAAA) 19 bp upstream from the poly A tail,

and a poly A tail of 27 bp. T2 with a full-length

sequence of 875 bp consisted of a 50-UTR of 24 bp, an

ORF of 732 bp, and a 30-UTR of 119 bp containing a

stop codon (TAA), a consensus polyadenylation signal

(AATAAA) 12 bp upstream from the poly A tail, and

a poly A tail of 20 bp. The T1 and T2 sequences were

deposited in GenBank under respective accession nos.

JN848593 and JN848594.

T1 and T2 appeared to contain all of the structural

features of eukaryotic mRNA transcripts. The nucle-

otide sequences of T1 and T2 were predicted to encode

the preproprotein of 242 and 244 amino acids (aa),

respectively, starting from the first methionine

(Fig. 2). As with all known trypsins, those of grouper

were also assumed to be synthesized as preproenzymes

Grouper T1 MKSLIFVLLIGAAFAT--EDDKIVGGYECTPHSQPHQVSLNSGYHFCGGSLVNENWVVSA Grouper T2 MKYFILLALFAAAYAAPIEDDKIVGGYECRKNSVAYQVSLNSGYHFCGGSLISSTWVVSA

Grouper T1 AHCYKSRVEVRLGEHNLRVTEGKEQFIRSSRVIRHPEYSSYNIDNDIMLIKLSEPATLNQ Grouper T2 AHCYKSRIQVRLGEHNIAVNEGTEQFINSARVIRHPSYNSRNLDNDIMLIKLSEPATLNQ

+Grouper T1 YVQPVALPTSCAPAGTMCTVSGWGNTMSSTADKNKLQCLDIPILSFEDCDNSYPGMITDA Grouper T2 YVQPVALPTSCAPAGTMCKVSGWGNTMSSTADSNRLQCLDIPILSDEDCERSYPGIIDYT

+Grouper T1 MFCAGYLEGGKDSCQGDSGGPVVCNGELQGVVSWGYGCAEKDHPGVYAKVCLFNDWLERT Grouper T2 MFCAGYLEGGKDSCQGDSGGPVVCNGELQGVVSWGYGCAEKDHPGVYSKVCVQTDWLLET L1 S1 S1 L2 S1

Grouper T1 MAKY Grouper T2 MASY

Fig. 2 Alignment of deduced amino acid sequences of the two

grouper trypsinogens. Identical residues are indicated by dots.

The T1 and T2 signal peptides are underlined. Vertical arrowsfrom the amino-terminus indicate the activation peptide

cleavage site. Residues forming the catalytic triad (His, Asp,

and Ser) are indicated by dots. Cysteine residues are marked

with stars. Two trypsin determinant residues are marked with

plus signs. Two surface loops (L1 and L2) and residues in the S1

binding pocket are, respectively, double-underlined and boxed.

Dashed lines indicate sequences that match the peptide

sequences of a purified trypsin from the pyloric cecum of

grouper in our previous study (Liu et al. 2012)


123

that contain an amino-terminal signal peptide followed

by a short activation peptide. The deduced amino acid

sequences of both grouper trypsinogens had a hydro-

phobic signal sequence of 15 residues as predicted by

SignalP. The trypsinogen activation peptides were 4

and 6 aa long in T1 and T2, respectively. The catalytic

triads of His-61, Asp-104, and Ser-198 in T1, and of

His-63, Asp-106, and Ser-200 in T2 were conserved in

both grouper trypsinogens. Also conserved in both

grouper trypsinogens were Asp-190 in T1 and Aps-192

in T2 at the bottom of the substrate-binding pocket,

Gly-213 and Gly 223 in T1, and Gly-215 and Gly 225

in T2 lining the sides of the binding pocket, Tyr-171 in

T1 and Tyr-173 in T2 involved in trypsin substrate

specificity, and 12 cysteine residues responsible for the

formation of six disulfide bonds. Two surface loops, L1

and L2, supporting the substrate-binding pocket were

both preserved in the two grouper trypsinogens. In

addition, the consensus repeat 196GDSGG200 in T1 and198GDSGG202 in T2, and a diagnostic repeat residue of

a serine protease around the active Ser site were found

in both trypsinogens.

The grouper T1 aa sequence demonstrated a

distribution of charged residues very similar to that

of grouper T2 (80.74 % identity). The calculated

molecular masses of T1 and T2 were 26.46 and

26.58 kDa, with respective estimated pI values of 5.20

and 5.06. The composition revealed a 47-aa substitu-

tion in total. The grand average hydropathicity indices

of T1 and T2, calculated using the hydropathicity scale

given by Kyte and Doolittle (1982), were estimated to

be -0.177 and -0.100, respectively. Based on the

results of pI and hydrophobicity, both grouper tryp-

sinogens appeared to be anionic.

A phylogenetic tree constructed by the NJ method

from a multiple sequence alignment of grouper

trypsinogens, and a range of other teleost counterparts

belonging to groups I, II, and III is shown in Fig. 3.

Both grouper trypsinogens were phylogenetically

related to group I of teleost anionic trypsinogens.

Tissue expression

Tissue expression of the two grouper trypsinogens as

analyzed by an RT-PCR is shown in Fig. 4. T1 was

abundantly expressed in the pyloric cecum, all intes-

tinal tissues, and spleen, but only slightly expressed in

other tested tissues except the stomach and blood. T2

was expressed in the digestive tract, including the

esophagus, stomach, pyloric cecum, anterior intestine,

middle intestine, hind intestine, and spleen.

From the RT-PCR results, both grouper trypsino-

gens were mainly synthesized in tissues of the pyloric

cecum, anterior intestine, middle intestine, hind

intestine, and spleen. Expression levels of the two

trypsinogens in these tissues were analyzed by a real-

time PCR. Among all tested tissues, relative expres-

sion of T1 in the pyloric cecum and anterior intestine

was significantly higher compared with those of other

selected tissues. Relative expression of T1 in the

pyloric cecum and anterior intestine was 165.0 ±

25.2-fold and 110.9 ± 44.8-fold higher, respectively,

than T1 expression in the spleen (Fig. 5a). T2 had a

similar expression profile to T1. Nevertheless, even if

T2 showed higher expression levels in the pyloric

cecum and anterior intestine, it did not significantly

differ from those of the middle intestine and hind

intestine. Relative expression of T2 in the pyloric

cecum and anterior intestine were, respectively,

1469.0 ± 298.4-fold and 1639.0 ± 198.9-fold higher

compared with the expression of T2 in the spleen

(Fig. 5b).

Relationship of T1 and T2 expression

with body weight

Relationship between the expression of the two

trypsinogen genes and grouper body weight are shown

in Fig. 6. Fish within a size range of 26.35–179.1 g

had increased DCt values of T1 and T2 in all tissues,

including the pyloric cecum, anterior intestine, middle

intestine, and hind intestine with an increase in body

weight. As the copy number of the target gene and Ct

values were inversely related, a sample containing a

higher number of copies of the target gene had a lower

Ct value than that of a sample with a lower number of

copies of the same target. Therefore, expression of T1

and T2 in the pyloric cecum, anterior intestine, middle

intestine, and hind intestine decreased with increased

body weight. However, the expression of trypsinogen

exhibited individual differences resulting in insignif-

icant differences among fish of different sizes.

Expression of the two trypsinogens during early

grouper development

Grouper larvae showed slow development before 8

DPH. Thereafter, exponential growths in total length


123

and body weight were found in larvae from 12 DPH

until the end of the study at 50 DPH (Fig. 7).

Expression analyses of the two trypsinogens during

early development of grouper by an RT-PCR are

shown in Fig. 8. No amplified fragment of grouper

trypsinogens by the RT-PCR was detected in fertilized

eggs or larvae before 12 h post-hatching. T2 tran-

scription was first detected in larvae at 1 DPH.

Although T1 transcription was not detected at 1

DPH by the RT-PCR, it was detected at 1 DPH by the

more sensitive method of a real-time PCR (Fig. 9).

T1 mRNA detected by the real-time PCR is

shown in Fig. 9a. A steady-state transcript level of

T1 was found in larvae during 1–25 DPH. Increased

expression of T1 was quantified in larvae at 35–50

DPH compared with larvae at 30 DPH (Fig. 9a). T2

showed a gene expression profile similar to that of

T1 with a constant steady-state expression during

1–25 DPH. T2 showed a sharp increase in expression

after 45 DPH (Fig. 9b). The expression of b-actin did

not significantly differ in larvae at 1–25 and 35–50

DPH.

Gro

up

I G

rou

p II

G

rou

p II

I

Fig. 3 Phylogenic

relationship among full-

length amino acid sequences

of trypsinogens from a wide

range of teleosts using the

neighbor-joining (NJ)

method. Taxon names are

shown as common names of

the species plus their

corresponding GenBank

accession numbers. The

scale for the branch length is

shown below the tree.

Sequences of grouper

trypsinogens are shown as

orange-spotted grouper T1

and T2


123

An interesting variation in trypsin activity was

observed during grouper larval development. Rela-

tively high trypsin activity was detected in larvae with

a yolk sac at 1 DPH, and then trypsin activity

significantly decreased at the end of the yolk sac

absorption phase at 2 DPH (Fig. 10). Trypsin activity

in larvae fed oyster trochophores (2–5 DPH) sharply

increased from 0.38 ± 0.06 U/mg at 2 DPH to

1.53 ± 0.37 U/mg at 4 DPH and then decreased

significantly to 0.14 ± 0.01 U/mg in larvae fed SS-

type rotifers at 8 DPH. After 8 DPH, trypsin activity

did not significantly change in larvae until 25 DPH. In

the metamorphosis phase, trypsin activity significantly

increased after 40 DPH.

Discussion

Trypsin plays an important role in protein digestion

and growth of marine fish larvae (Cahu and Zambon-

ino-Infante 1994; Moyano et al. 1996; Fujii et al. 2007;

Manchado et al. 2008). In this work, two full-length

trypsinogen isoforms were cloned and identified in the

pyloric cecum of orange-spotted grouper. Nucleotide

sequences of the coding regions of T1 and T2 shared

70.74 % nucleotide sequence identity, whereas the

amino acid sequence identity of the encoded proteins

was 80.74 %. In our previous study, a trypsin was

purified from the pyloric cecum of E. coioides (Liu

et al. 2012) in which two fragments of the peptide

sequence were identified (LGEHNI and NLDN-

DIML), and it was suggested that the purified trypsin

was T2 when aligned with grouper T1 and T2

sequences. Both of them had completely conserved

catalytic triad residues of His, Asp, and Ser (His-61,

Asp-104, and Ser-198 in T1, and His-63, Asp-106, and

Ser-200 in T2). The amino acids generating the S1

substrate-binding pocket were of a typical trypsin

nature in both grouper sequences with Asp (Asp-190

in T1 and Aps-192 in T2) at the bottom and two Gly

residues (Gly-213 and Gly 223 in T1, and Gly-215 and

Gly 225 in T2) lining the sides of the pocket. The

consensus repeat, GDSGG, around the active site, Ser,

is usually diagnostic of a serine protease (Krem et al.

1999), which was also well conserved in both grouper

trypsinogens (196GDSGG200 in T1 and 198GDSGG202

in T2). The Tyr residue (Tyr-171 of T1 and Tyr-173 of

T2) was shown to be a key residue in determining

substrate specificity of trypsins (Hedstrom et al. 1994),

Fig. 4 Expression of grouper trypsinogens, T1 (a) and T2 (b),

and b-actin (c) in the esophagus (lane 1), stomach (lane 2),

pyloric cecum (lane 3), anterior intestine (lane 4), middle

intestine (lane 5), hind intestine (lane 6), eye (lane 7), liver (lane8), gills (lane 9), skin (lane 10), heart (lane 11), head kidney

(lane 12), mid-kidney (lane 13), hind kidney (lane 14), spleen

(lane 15), brain (lane 16), dorsal muscle (lane 17), and blood

(lane 18) as analyzed by RT-PCR. M: 100-bp DNA ladder

marker

020406080

100120140160180200

Pyloricceca

Anteriorintestine

Middleintestine

Hindintestine

Spleen

Rel

ativ

e ex

pre

ssio

n

(A)a

bbb

a

0200400600800

100012001400160018002000

Pyloricceca

Anteriorintestine

Middleintestine

Hindintestine

Spleen

Tissues

Rel

ativ

e ex

pre

ssio

n

(B)a

ab

a

ab

b

Fig. 5 Relative expression of T1 (a) and T2 (b) in different

tissues of grouper. Data (mean ± SE) with different letters

among different tissues significantly differed (p \ 0.05)


123

and the two loops supporting the substrate-binding

pocket (Hedstrom et al. 1992) were also conserved in

grouper trypsins as in other trypsins. In addition, two

surface loops supporting the substrate-binding pocket

and 12 cysteine residues generating six disulfide

bridges were highly conserved in both grouper tryp-

sins, which suggests that both paralogous genes are

functional.

Trypsin is activated after its secretion into the gut

via the removal of a short, highly charged activation

peptide by enterokinase, which prevents trypsinogen

from being accidentally activated within the pancreas

(Gudmundsdottir et al. 1993; Manchado et al. 2008).

Different trypsinogen genes were identified in teleos-

tean fishes (Gudmundsdottir et al. 1993; Liu et al.

2007b; Manchado et al. 2008; Ruan et al. 2010). The

Y = 0.0261X - 15.26 (R2 = 0.1892)-22-20-18-16-14-12-10

-8-6-4-2

0 20 40 60 80 100 120 140 160 180 200

ΔC

T

(E)

Y = 0.0138X - 7.2498 (R2 = 0.1304)-10

-9

-8

-7

-6

-5

-4

-3

-2

0 20 40 60 80 100 120 140 160 180 200Δ

CT

(F)

Y = 0.0173X - 7.8329 (R2 = 0.1793)-12

-10

-8

-6

-4

-2

0

0 20 40 60 80 100 120 140 160 180 200

ΔC

T(G)

Y = 0.0229X - 4.6275 (R2 = 0.2532)-7-6-5-4-3-2-1012

0 20 40 60 80 100 120 140 160 180 200

Body weight (g)

ΔC

T

(H)

Y = 0.0255X - 1.4617 (R2 = 0.1721)

-8

-6

-4

-2

0

2

4

6

8

0 20 40 60 80 100 120 140 160 180 200

ΔC

T(A)

Y = 0.0168X + 5.8241 (R2 = 0.1128)0

2

4

6

8

10

12

0 20 40 60 80 100 120 140 160 180 200

ΔC

T

(B)

Y = 0.0233X + 5.7639 (R2 = 0.2177 )0

2

4

6

8

10

12

14

0 20 40 60 80 100 120 140 160 180 200

ΔC

T

(C)

Y = 0.0356X + 7.0735 (R2 = 0.2064)02468

101214161820

0 20 40 60 80 100 120 140 160 180 200

Body weight (g)

ΔC

T

(D)

Fig. 6 Analysis of relationship of body weight with T1 and T2 expression in the pyloric cecum (a or e), anterior intestine (b or f),middle intestine (c and g), and hind intestine (d or h)


123

enzyme cleaves the activation peptide in the presence

of two acidic residues at P2 and P3 and an alkaline

residue, Lys or Arg, at P1 (Chen et al. 2003). The

activation peptide usually possesses three or four

acidic resides, Asp and Glu in groups I and II of many

teleostean fish, whereas most mammalian trypsino-

gens have four continuously arranged Asp residues

before Lys-P1 (Chen et al. 2003). This seems to be the

case for grouper T1 and T2 with three acidic residues

preceding Lys-P1, which differs from the characters of

group III trypsinogen by activation peptides possess-

ing an Arg at P1 and a deletion or a substitution of Asp

at P2 (Chen et al. 2003). In addition, both grouper

trypsinogens appeared to be anionic. This is similar to

group I trypsins that are usually, but not always,

anionic at physiologic pH, whereas group II trypsins

are usually, but not always, cationic at physiologic pH,

and group III trypsins represent a rapidly evolving

group of extremely psychrophilic enzymes (Roach

et al. 1997; Roach 2002). Those data are in agreement

with the phylogenetic analysis demonstrating that both

T1 and T2 of the grouper belong to group I

trypsinogens.

The RT-PCR analysis demonstrated that trypsino-

gen was synthesized in almost all selected tissues.

Similar results of wide expression of trypsinogens in

different tissues were also found in other fishes (Braun

et al. 1990; Lilleeng et al. 2007). Different expression

in multiple tissues may involve different functions,

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 5 10 15 20 25 30 35 40 45 50

Day post hatching

Tota

l len

gth

(cm

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Total length

Body weight

Fig. 7 Mean body weight and total length of orange-spotted

grouper larvae during the experiment

Fig. 8 RT-PCR detection of T1 (a), T2 (b), and b-actin (c) gene

expression at different life stages of the egg (lane 1), and larvae

of 0 (lane 2), 2 (lane 3), 4 (lane 4), 6 (lane 5), and 12 h (lane 6),

and 1 (lane 7), 2 (fed larvae, lane 8; unfed larvae, lane 9), 3 (fed

larvae, lane 10; unfed larvae, lane 11), and 4 (lane 12) days post-

hatching. Lane M: 100-bp ladder DNA marker

05

10152025303540455055

0 5 10 15 20 25 30 35 40 45 50

Rel

ativ

e ex

pre

ssio

n Whole larva

Digestive tract only

xaa

y

y

y

y

aaa

aa a a

(A)

y

05

10152025303540455055

Day post hatchingR

elat

ive

exp

ress

ion

(B) Digestive tract only

Whole larva

x x

y

y

z

aaaaaaaaa

0 5 10 15 20 25 30 35 40 45 50

Fig. 9 Relative expression of T1 (a) and T2 (b) during grouper

larval development quantified by an SYBR Green real-time RT-

PCR. Data (mean ± S.E.) with different letters during 1–25 or

30–50 DPH (x, y) significantly differed (p \ 0.05)

0.0

0.4

0.8

1.2

1.6

2.0

2.4

0 5 10 15 20 25 30 35 40 45 50

Day post hatching

Tryp

sin

act

ivit

y (U

/mg

) Whole larva Digestive tract only

yy

xxxcdcd

ddd

c

b

b

a

Fig. 10 Enzymatic activity of trypsin during grouper larval

development. Data (mean ± S.E.) with different letters (a, b, c,

d) during 1–25 day post-hatching (DPH) and (x, y) during 30–50

DPH significantly differed (p \ 0.05)


123

such as involvement in ionic regulation in nephrons

(Nesterov et al. 2008). However, the expression of T2

was only detected in digestion-related tissues of the

esophagus, stomach, pyloric cecum, anterior intestine,

middle intestine, hind intestine, and spleen, while T1

was widely expressed in almost all sampled tissues.

Therefore, it is thought that T2 might play a more

relevant role in the digestive function of proteins. On

the other hand, the pancreas is dispersed in the

intestinal mesentery of fish (Ostaszewska et al. 2006).

Both trypsinogen genes detected in intestinal samples

may be (at least partially) pancreatic. It is considered

that in situ hybridization could be used to determine

whether there is indeed pancreatic tissue and pancre-

atic expression.

Trypsin activity was reported to be positively

correlated with the fish growth rate in previous studies

of Atlantic cod, G. morhua (Lemieux et al. 1999), and

Atlantic salmon, S. salar (Rungruangsak-Torrissen

et al. 2006). It was also used as an indicator to evaluate

the nutritional condition and is considered to be the

main proteolytic enzyme of marine fish larvae that

lack a morphological stomach (Govoni et al. 1986;

Moyano et al. 1996; Gawlicka et al. 2000; Eusebio

et al. 2004; Darias et al. 2007; Fujii et al. 2007).

However, a different result in our previous study

showed that trypsin activity in the pyloric cecum of

E. coioides was not positively related to the fish body

weight (Liu et al. 2012), which is in agreement with

the results of the relationship between trypsinogen

mRNA expression and fish body weight in the present

study. The different expression patterns of trypsin in

grouper could be caused by zymogen activation in

ingested food rather than direct digestion of food

(Oozeki and Bailey 1995), and other proteinases, such

as pepsin (Feng et al. 2008), play more important roles

than trypsin in protein digestion in the growing

grouper.

A low survival rate and slow growth during larval

development of E. coioides are major problems

encountered by fish farms culturing grouper (Pierre

et al. 2008). In this study, early larval stages had a very

small size, and larval development presented insig-

nificant increments in body weight and total length

before 8 DPH; therefore, the larval period is very long.

With the production of grouper larvae in this study,

7.68 and 10.73 % survival rates were found with the

two feeding treatments which are higher than the

average survival rates of grouper breeding in practice.

In the period of 2–30 DPH, grouper undergo the

process of mouth-opening, followed by metamorpho-

sis, which are two critical stages with high mortality,

induced by some unknown factors, such as disease or

food. Expression and activity of trypsin in Lutjanus

guttatus were detected at hatching at very low levels

and concomitantly increased with larval development

(Galaviz et al. 2012), which agreed with other findings

in previous studies (Oozeki and Bailey 1995; Moyano

et al. 1996; Murray et al. 2006; Alvarez-Gonzalez

et al. 2010). In this study, trypsin activity was detected

in larvae before the first feeding consistent with the

result shown by Zambonino-Infante and Cahu (1994),

who reported enzymatic activities of trypsin and other

digestive enzymes in Dicentrarchus labrax at 4 DPH,

while the first feeding occurred at 6 DPH. This

suggests that enzyme activity is derived from genet-

ically preprogrammed expression and not by the first

exogenous feeding (Lazo et al. 2000; Zambonion-

Infante and Cahu 2001). The opening of the mouth

began at 2–3 DPH, in grouper larvae at 2–3 mm.

Trypsin activity of larvae increased after feeding with

oyster trochophores, but expression of T1 and T2 was

low. The increased trypsin activity may be considered

to be due to the secretion of pancreatic enzymes in the

intestinal lumen during the first 3 weeks of larval life

(Zambonino-Infante and Cahu 2001). In addition, a

relatively higher trypsin activity of 3.14 ± 0.30 U/mg

was detected in oyster trochophores compared with

trypsin activity in larvae, which may be involved in

activating other pancreatic digestive zymogens

including trypsinogens (Chen et al. 2003), leading to

the increase in trypsin activity after the first feeding. In

L. guttatus larvae, the peak of trypsin expression

preceded that of specific activity and generally

coincided with changes in the food supply. According

to profiles of trypsin activity and trypsinogen expres-

sion, the pancreas was fully functional after 25 DPH

since the maximum trypsinogen expression occurred

at 25 DPH, and maximum levels of trypsin activity

occurred at 35 DPH (Galaviz et al. 2012). An increase

in trypsinogen expression preceding that of trypsin

activity was also found in grouper larvae. Expression

of T1 and T2, respectively, increased after 35 and 40

DPH, whereas increased trypsin activity was found

after 40 DPH. Larvae of E. coioides after 30 DPH

underwent metamorphosis with a developing stomach

and pyloric cecum (Eusebio et al. 2004), and changes

in the structure of the digestive system seemed to


123

result in significant increases in trypsin activity and

trypsinogen expression.

In conclusion, two trypsinogens were cloned from

E. coioides. The phylogenetic analysis classified them

as group I trypsinogens. T2 is considered to play a

more relevant role in digestive function since it was

only found in digestive tissues. Larvae are able to

synthesize trypsinogen at 1 DPH before the first

feeding, and the increased trypsin activity after the

first feeding may be required to digest food. Increased

trypsinogen expression and trypsin activity after 30

DPH were mostly related to metamorphosis with a

developing stomach and pyloric cecum.

Acknowledgments This research was supported by a grant

from the National Science Council (NSC99-2313-B-020-005-

MY3), Taiwan. The authors thank Sian-Ru Fu and Jhih-Syuan

Chen who assisted with carrying out this project.

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Molecular characterization of two trypsinogens in the orange-spotted grouper, Epinephelus coioides,...

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Transcript of Molecular characterization of two trypsinogens in the orange-spotted grouper, Epinephelus coioides,...