Biochemical characterization and cloning of transglutaminases responsible for hemolymph clotting in Penaeus monodon and Marsupenaeus japonicus

Biochemical characterization and cloning of transglutaminases

responsible for hemolymph clotting in Penaeus monodon

and Marsupenaeus japonicus

☆

Maw-Sheng Yeh

, Ling-Rong Kao

, Chang-Jen Huang

, Inn-Ho Tsai

⁎

a

Department of Food and Nutrition, Hung Kuang University, Sha Lu, Taiwan

b

Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan

c

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

Received 29 October 2005; received in revised form 22 March 2006; accepted 7 April 2006 Available online 21 April 2006

Abstract

To investigate the shrimp blood clotting enzyme, a transglutaminase in the hemocytes of Penaeus monodon (abbreviated as TGH) was purified.

TGH is an abundant homodimeric cytosolic protein with 84.2 kDa subunits. It clotted shrimp plasma and incorporated fluorescent dansylcadaverine

into succinyl casein upon activation by CaCl

in vitro. IC

for the activation was 3 mM, which is below the shrimp plasma Ca

level. Showing

similar properties as other type II transglutaminase, TGH was particularly unstable after activation. MALDI-TOF/TOF mass-analyses of tryptic

peptides of P. monodon TGH confirmed its identity to STG I (AY074924) previously cloned. A possible allele of the other isozyme STG II

(AY771615) has also been cloned from the P. monodon cDNA and designated as PmTG. The predicted PmTG protein sequence is 58% similar to that

of STG I and 99.2% to that of STG II. Likewise, a novel enzyme Mj-TGH was purified and cloned from Marsupenaeus japonicus hemocytes. Results

of sequence alignment and phylogenetic analyses of these transglutaminases suggest that STG I and Mj-TGH are 83% identical and orthologous to

each other, while PmTG/STG II and a previously cloned M. japonicus transglutaminase (AB162767) are their paralogs. Protein of the latter two could

not be isolated, their regulated expression was discussed.

© 2006 Elsevier B.V. All rights reserved.

Keywords: Transglutaminase; Hemolymph coagulation; Regulation by Ca2+; cDNA sequence; Shrimp (Penaeus monodon and Marsupenaeus japonicus)

1. Introduction

Transglutaminases (EC 2.3.2.13) are known to play critical

roles in blood coagulation and other biochemical process

in-volving protein cross-linking or related super-structures.

En-zymes in this family catalyze calcium-dependent acyl-transfer

reactions between glutamine residues and lysine residues in

protein substrates. The role of Ca

is to induce enzyme

con-formational changes essential for substrate-binding and catalysis

[1

–4]

. The formation of intra-molecular or intermolecular 1-(

γ-glutamyl) lysine bonds lead to polymerized or insoluble

pro-teins. In addition, the enzymes mediate other post-translational

modification of proteins, e.g., deamidation and amine

incorpo-ration

[1]

.

The enzymes are widely distributed in tissues and body fluids

of animals. For example, blood coagulation factor XIIIa

cata-lyzes the cross-linking of fibrin monomer during blood

coag-ulation

[5]

, another transglutaminase toughens skin epidermis

and hair follicle epithelia

[6]

in vertebrates. The enzyme is also

involved in forming the fertilization plug in female rodents

[7]

.

Up to ten transglutaminases in various invertebrate species have

so far been reported

[8–11]

, including those responsible for

blood coagulation in Crustacean

[9,10]

. The enzymes were also

found to undergo acetylation

[1]

, phosphorylation, or

fatty-acylation

[12]

that probably regulates their subcellular

localiza-tion and catalytic activities. Some of them are involved in signal

transduction and apoptosis

[1,13]

.

Abbreviations: Mj, Marsupenaeus japonicus; Pm, Penaeus monodon; TGH, hemocyte transglutaminase; RFE, relative fluorescence enhancement

☆ _{The cDNA sequences of PmTG and Mj-TGH have been deposited in}

GenBank with accession No. AF469484 and DQ436474, respectively. ⁎ Corresponding author. Fax: +886 2 23635038.

E-mail address:bc201@gate.sinica.edu.tw(I.-H. Tsai).

1570-9639/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2006.04.005

Tiger shrimp (Penaeus monodon) is an economically

impor-tant species cultured in Taiwan and southeastern Asia.

Coagula-tion of the hemolymph is part of the innate immune response of

Crustaceans, it prevents leakage of hemolymph from sites of

injury and dissemination of invaders such as bacteria throughout

the body. Much earlier, we have purified, characterized and cloned

the clottable protein in the hemolymph of P. monodon

[14]

.

Previously, two transglutaminases of P. monodon, STG I and STG

II were cloned and expressed as the shrimp clotting enzymes

[8,10]

. In the present study, we first reported purification and

biochemical characterization of major transglutaminases in

shrimp hemocytes (abbreviated TGH) and their stability and

regulation. Another transglutaminase from P. monodon

(nated as PmTG), and a novel hemocyte transglutaminase

desig-nated as Mj-TGH from Marsupenaeus japonicus (kuruma prawn)

were herein cloned and sequenced. Results of peptide sequencing

and peptide mass fingerprinting of the purified TGHs from both

shrimps enabled us to identify them with previously reported STG

I of P. monodon

[8]

and Mj-TGH, respectively. Moreover,

trans-glutaminases from invertebrates were subjected to phylogenetic

analysis, and the regulations of their expression were discussed.

2. Materials and methods

2.1. Shrimp hemocytes and the reagents

A total of 90 live specimens of P. monodon and 50 live specimens of M. japonicus were obtained from local markets. The hemolymph was withdrawn by a syringe of 2.5 ml with a needle of 23 G and 1.25 inch long containing 1/8 volume of anticoagulant (0.1 M sodium citrate, 0.4 M NaCl. It was centrifuged at 1600×g for 5 min at 4 °C, the supernatant was harvested for clotting assay. The pellet was washed twice with a cold buffer (1.5 mM EDTA, 0.4 M NaCl, 0.25 M sucrose in 50 mM Tris–HCl at pH 7.5). Casein and monodansylcadaverin were obtained from (Sigma Chem. Co., USA). Succinyl-casein was synthesized from casein and succinyl anhydride (Pierce, USA) as previously described [15].

2.2. Transglutaminase assay

Succinyl-casein (500 μg/ml) were mixed with 50 μM monodansylcada-verine in 50 mM Tris–HCl (pH 7.6) containing 5–10 mM dithioerythreitol and 6 mM CaCl2. This substrate solution was thermostated at 23 °C in a

cuvette before adding hemocyte lysate or purified P. monodon TGH. Incorporation of dansylcadaverin to succinyl-casein by TGH was followed by a fluorospectrometer (model MPF-4, Hitachi, Japan) at the excitation/ emission wavelengths of 360/500 nm, respectively [15]. Upon adding the TGH, the reaction started immediately in the presence of CaCl2 and no lag

phase was observed. Initial enzymatic rate within 1 min was measured to minimize errors due to enzyme instability, and relative fluorescence enhancement (RFE) was calculated based on the percent fluorescence increase relative to the blank. The shrimp plasma was diluted with equal volume of 50 mM Tris–HCl (pH 7.5) with 10 mM CaCl2 and then added

purified TGH at a final concentration of 14–35 μg/ml. The clotting time was monitored at 23 °C since the shrimps live in natural marine environment at 15–35 °C.

2.3. Purification and characterization of shrimp TGH

After washing, the hemocytes in pellet were lysed by 10 mM Tris–HCl buffer containing 1.5 mM EDTA and 0.1 mM ATP[16]and centrifuged to remove cell debris. The supernatant (i.e., hemocyte lysate) was loaded on a Fractogel TSK DEAE-650(M) column (1.4 × 7.5 cm) at 4 °C, which had been equilibrated in 50 mM Tris–HCl buffer (pH 7.5) with 1.5 mM EDTA and 0.1 mM ATP. The sample was eluted with a linear gradient of 0–0.3 M NaCl, fractions of 1 ml were collected and assayed for transglutaminase activity. Active fractions were pooled and further purified by a Sephacryl S-300 column (0.9 × 60 cm) at 4 °C, which was equilibrated and eluted with the 50 mM Tris–HCl buffer containing 1.5 mM EDTA and 0.1 mM ATP.

Molecular weight and purity of P. monodon TGH were studied by SDS-PAGE[17]. Native molecular weight of TGH was also estimated by elution volume on a gel filtration column, by interpolation from a calibration plot of “ratios of the elution volume to the void volume” against log molecular weight of the markers. In addition, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometer (Model G2025, Hewlett-Packard, USA) was used to determine its accurate mass, in which 1μl of purified TGH (0.4 mg/ml) after dialysis against distilled water was added with 1μl sinapinic acid before crystallized under vacuum.

Fig. 1. Purification of TGH from P. monodon hemocyte lysate. (A) About 12 ml hemocyte lysate was separated by a Fractogel TSK DEAE-650 (M) column at 4 °C. The column was eluted with a gradient of 0–0.3 M NaCl in 50 mM Tris–HCl (pH 7.5), containing 1.5 mM EDTA and 0.1 mM ATP. Arrow indicated start of the gradient. (B) partially purified TG was subjected to gel filtration on a Sephacryl S-300 column. Fractions of 1 ml were collected at 4 °C, and transglutaminase activity (○–○) was assayed as detailed in Materials and methods.

2.4. Protein fragmentation and amino acid sequencing

Likewise, the enzyme Mj-TGH was purified from the hemocytes of another aqua-cultured species M. japonicus. Both TGHs (1 mg) of P. monodon and M. japonicus were denatured in 100μl of 6 M guanidine hydrochloride in 0.2 M Tris–HCl (pH 8.5) and reduced by dithiothreitol at 50 °C for 1 h. After adding excess iodoacetic acid and incubated at room temperature for 30 min, the S-carboxymethyl protein was desalted and lyophilized. Amino acid sequences were analyzed by an automatic sequencer (Model 477A, Applied Biosystem, USA).

For fragmentation, M. japonicus TGH was dissolved in 100μl of 70% formic acid with 0.2 mg of CNBr at room temperature for 24 h. In addition, it was digested with Lys-C endopeptidase (Promega, Madison, WI, USA) in 50 mM Tris–HCl (pH 8.0) at 37 °C for 20 h at an enzyme to substrate ratio of 1:50 (w/w). The reaction was stopped by adding dithiothreitol and heated at 95 °C for 5 min. Then the oligopeptides were purified by reversed-phase HPLC on a C18column,

then N-terminal sequences of three of the fragments.

2.5. Enzyme assay and effect of calcium and other reagents

For the fluorescence assay, CaCl2at a final concentration of 10 mM was

added to P. monodon TGH (77μg/ml) in 50 mM Tris–HCl, 1.5 mM EDTA, 0.1 mM ATP (pH 7.5) to start the reaction and initial rate (RFL/min) was

measured[15]. For clotting assay, clotting time was measured after adding the enzyme to a buffer containing 60μl supernatant of the centrifuged hemolymph and equal volume of 50 mM Tris–HCl (pH 7.5) containing 20 mM CaCl2.

To investigate its thermostability, purified TGH in the buffer (32μg/ml) were incubated at various temperature up to 50 °C. After a period of time, 30μl aliquots were withdrawn for the fluorescence assay. In addition, we have studied effects of pre-incubation of TGH with the chlorides of IIA group metal ion or iodoacetamide which modified active site CysSH of TGH. The remaining enzyme activity was assayed by the fluorescence assay.

2.6. cDNA synthesis and cloning

Standard procedures in molecular biology were used for preparation of plasmid DNA, restriction enzyme digestion, DNA agarose gel electrophoresis, DNA ligation, and the transformation of bacteria[18]. Total RNA was purified from tiger shrimp and kuruma prawn using the RNAzol B kit (Biotecx, Friendswood, TX, USA)[19]. The mRNA was purified using QuickPrepR Micro mRNA purification kit (Amersham Pharmacia, Freiburg, Germany). The first strand cDNA was synthesis using oligo(dT)-primer and random primers, and it was used as templates in subsequent polymerase chain reaction (PCR)[20].

To amplify the transglutaminase cDNAs of both species, we have used an antisense oligo(dT) and another degenerate primer designed from the conserved protein sequence (VPAHTIK) of M. japonicus TGH, i.e., sense 5′-GTNCCN

Fig. 3. (A) Correlation between clotting and amine-incorporating activities of purified TGH. After adding Tris–HCl buffer containing CaCl2 to the same

volume of shrimp plasma, the clotting was started from the addition of TGH (final TGH concentration ranged from 14 to 35μg/ml). In separate experiments, initial rates of dansylcadaverin incorporation were determined by fluorescence assay. (B) Apparent Ca2+_{dependency of P. monodon TGH activity. Purified TGH}

(18μg/ml) was used in the fluorescence assay at various CaCl2concentrations in

Tris buffer at 23 °C. Fig. 2. (A) Molecular mass of purified P. monodon TGH as determined by

MALDI-TOF mass spectrometry. (B) Estimation of the TGH molecular weight by gel filtration. Protein markers used were:β-amylase (β-A, 200 kDa), bovine serum albumin (BSA, 67 kDa) and carbonic anhydrase (CA, 29 kDa). Veand Vo

are elution-volumes for the protein marker and blue-dextran, respectively. (C) SDS-PAGE on 12% acrylamide gel. Lane 1, molecular weight markers; lane 2, hemocyte lysate; lanes 3 and 4 purified TGH with non-reducing and reducing sample buffer, respectively. Molecular weights of the markers were shown at left.

CANCA(TC)ACNAT(TCA) AA-3′. Except for the first and last cycles, each of the 40 cycles was: 94 °C for 30 s, 42 °C for 30 s, and 72 °C for 1 min. The first cycle included an extended denaturation time (2 min) during which polymerase was added while the last cycle had an extended (10 min) elongation period. The cDNAs of 578 bp and 1071 bp were thus produced from tiger shrimp and kuruma prawn, respectively. These cDNAwere purified and ligated into pGEM-T (Promega). Each clone was sequenced by using AmpliTaq DNA polymerase and fluorescent dideoxynucleotides on a DNA sequencer (Applied Biosystems model 310)[21].

2.7. Rapid amplification of cDNA ends (RACE)

Total RNA was isolated from both shrimps and poly (A)+ RNA was prepared. The first-strand cDNA was synthesized using a modified poly-T primer and 1μg of poly (A)+_{RNA. The second-strand cDNA was obtained}

using an enzyme cocktail containing RNase H, DNA polymerase, and DNA ligase. Asymmetric adaptor primers (AP primers) were then ligated to both ends of the double-stranded cDNA. An aliquot of this cDNA was diluted 1:100 and subjected to PCR. The 5′ ends of mRNAs of PmTG and Mj-TGH were obtained by RACE methods using the Marathon cDNA amplification kit (Clontech, Palo Alto, CA, USA). Adaptor primers AP1 (external) and AP2 (internal) were supplied with the kit. RACE was performed with a 27mer sense primer (AP1) and antisense primers, 5′-GTAGTAGTCACCGTACGTCACTTCC-3′ for PmTG/ TGH and 5′-TGATAAGGTCATGGTCCACCAGCTTG-3′ for Mj-TGH, respectively. The second round of PCR was carried out with a nested 25mer sense primer (AP2) and nested antisense primers, 5′-CTTCGAGAAC GATTTCTCCCTCACG-3′ for TGH and 5′-GTAGTAGTCAGAGTAGTA

CACCTCC-3′ for Mj-TGH, respectively. Except for the first and last cycles, each cycle was: 94 °C for 30 s, 63 °C for 30 s, and 72 °C for 1.5 min. The first cycle included an extended denaturation time (2 min) during which polymerase was added while the last cycle had an extended (10 min) elongation period. After 40 PCR cycles, the products were cloned into pGEM-T vector (Promega) and sequenced[20,21].

2.8. RNA isolation from P. monodon tissues and RT-PCR

Total RNA was isolated from gill, heart, hemocyte, hepatopancreas, intestine, lymphoid organ, and muscle of P. monodon using the RNAzol reagent (Biotecx, Friendswood, TX, USA). RNAase-free DNase (Promega) was added to eradicate the genomic DNA contamination. First-strand cDNA was amplified from 2μg total RNA using 10 pmole oligo-dT primer in a 25-μl reaction containing 30 U RNasin (Promega), 1 mM dNTP, 10 mM dithiothreitol, and 300 U Superscript II (Life Technologies). After being incubated at 42 °C for 1 h in 1.5 mM MgCl2, 0.25 mM dNTP and 0.5 U ExTaq (Takara Shuzo Co.,

Japan), 2μl of the solution were added to 48 μl solution of the primers (designed from PmTG; forward, 5′-TGGAAGTGACGTACGGTGAC TACTAC G-3′ and reverse, 5′-GTTGTACAGCTGGCTGGAGTTGAAGG-3′). After amplified by PCR at the annealing temperature of 60 °C for 30 s (25–40 cycles), the 390 bp PCR products were examined on a 1.2% agarose gel.

2.9. Molecular phylogenetic analysis

Sequences of invertebrate transglutaminases were retrieved by BlastP[22]. Their amino acid sequences were aligned with the program CUSTAL W[23]. Phylogenetic tree was build by the program PHYLIP (http://www. evolution. genetics.washington.edu./phylip.html)[24]. Degree of confidence of the lineage at each node was determined by the bootstrap analyses of 1,000 replicates[25].

2.10. In-gel digestion of P. monodon TGH and peptide mass

finger-printing

After manually excised from the polyacrylamide gel, the electrophoresis band of P. monodon TGH was cut into pieces, which were dehydrated with acetonitrile for 10 min, vacuum dried, re-hydrated with 100 mM dithioerythritol in 25 mM NH4HCO3 (pH 8.5) at 37 °C for 1 h. They were subsequently

Fig. 4. (A) Thermostability of P. monodon TGH. The enzyme (0.3 mg protein/ml) in 50 mM Tris–HCl, 1.5 mM EDTA (pH 7.5) was pre-incubated at different temperature (0–50 °C) for 10 min. At indicated time, aliquots were withdrawn for amine-incorporation assay. (B) Stability of P. monodon TGH at various pH. The hemocyte lysate was diluted with 0.1 M of the following buffers with 0.4 mM EDTA at 4 °C and incubated 16 h before the amine-incorporation assay at 23 °C; sodium acetate (pH 5.0–5.8) (▵–▵), imidazole–HCl (pH 5.8–7.0) (

▪

▪

Fig. 5. Effects of metal chlorides on the enzymatic activity of P. monodon TGH. The hemocyte lysate (0.4 mg protein/ml in 50 mM Tris–HCl, 0.1 mM ATP, 0.1 mM EDTA, pH 7.5) was preincubated with 5.3 mM metal chlorides at 4 °C (▵–▵, MgCl2;

▪

▪

were withdrawn at the time indicated and amine-incorporation activity of TGH was measured at 23 °C.

alkylated with 65 mM iodoacetamide in NH4HCO3at 27 °C for 1 h in the dark,

washed twice with 50% acetonitrile in NH4HCO, dehydrated with acetonitrile

for 10 min and dried, then hydrolyzed with 25 ng modified trypsin (Promega, Madison, USA) in 25 mM NH4HCO3(pH 8.5) at 37 °C for 16 h. The

oligo-peptides were extracted twice with 50% acetonitrile containing 5% formic acid for 15 min and dried under vacuum. Their masses were analyzed with MALDI-TOF/TOF and matched by Mascot Search programs.

3. Results

3.1. Purification and characterization of P. monodon TGH

Enhancement of the substrate fluorescence upon

incorporat-ing dansylcadaverine into succinyl casein was measured to assay

Fig. 6. The cDNA and deduced amino acid sequence of PmTG. Sequences homologous to those of Mj-TGH peptides purified by HPLC are underlined. Potential N-glycosylation sites and the RGD motif are doubly underlined.

TGH during purification (

Fig. 1

). The yield was high (0.6 mg

TGH from 7.6 mg of the total hemocyte lysate proteins).

Increase of the specific activities of TGH after the two

purification steps was about 4-folds. The activity was hardly

detected in the shrimp plasma. The molecular mass of the

purified TGH was determined as 84280 ± 168 Da by

MALDI-TOF mass spectrometry (

Fig. 2

A). However, the molecular

weight measured by a gel filtration column was about 170–

180 kDa (

Fig. 2

B), and it was estimated to be about 85–90 kDa

by SDS-PAGE under both reducing and non-reducing condition

(

Fig. 2

C). Thus, the TGH most likely existed as non-covalent

homodimers, which could be dissociated in the presence of SDS

during electrophoresis. We found that TGHs of both shrimps

were N-terminal blocked, thus no results could be obtained by

automatic sequencing. Presumably like many other

transgluta-minases, the N-terminus was acetylated after removal of the

initiator-methionine residue. This acetylation possibly serves as

a signal for protein secretion

[1]

.

3.2. Enzymatic properties of P. monodon TGH and the effect of

Ca

We noticed that the amine-incorporating activity of purified

TGH and its hemolymph-clotting activity were well correlated

(

Fig. 3

A). Like other member of group II transglutaminase

family, Ca

is an essential cofactor or specific activator of

shrimp TGH. The half-maximum concentration of Ca

required

for the incorporation of dansylcadaverine of P. monodon TGH

Fig. 7. The cDNA and deduced protein sequence of Mj-TGH. Sequences confirmed by protein sequencing are underlined. The polyadenylation-signal, potential N-glycosylation sites and the RGD motif are doubly underlined.

was about 3 mM (

Fig. 3

B). Similar level of Ca

was required for

clotting of hemolymph by TGH. This is consistent with the

normal Ca

requirement of other transglutaminases

[1]

. Other

metal chlorides, e.g., 10–100 mM of SrCl

, BaCl

or MgCl

,

could not substitute CaCl

in both TGH assays (data not shown).

The strict dependency on Ca

is different from that for Factor

XIII, which can be activated by other divalent metal ions

although less effectively

[26]

.

Fig. 8. Amino acid sequence alignment of transglutaminase variants of tiger shrimp and kuruma prawn. Residues identical in all the four sequences are shown in black, and those identical in any three are marked in gray. RGD motifs are doubly underlined and the catalytic triads are shown in bold italics. Dots indicate putative calcium binding sites, and sequences matching those of the purified Mj-TGH oligopeptides are underlined.

P. monodon TGH appeared to be labile at temperature

N25 °C

and pH outside the range 7–9 (

Figs. 4

A and B). Lyophilization

or frozen–thaw could inactivate the enzyme by more than 50%.

However, more than 85% of the activity could retain for several

days if purified TGH or the hemocyte lysate was stored in the

presence of metal chelator (e.g., EDTA) at pH 7–9 at 0–4 °C, or

kept in buffer in the presence of EDTA and 35% (v/v) glycerol

below

−14 °C.

Like other type II transglutaminases, P. monodon TGH was

inhibited or inactivated by certain divalent metal ion (e.g., 10

–

20

μM ZnCl

) which competed Ca

binding

[3,26,27]

,

thiol-reactive agents (e.g., organomercurials and iodoacetamide), or

2–10 mM histamine and other primary amines (data not shown).

Like other transglutaminases

[1,28]

, addition of 5 mM Ca

to

the medium greatly facilitated the inhibition of TGH by 3–

10

μM mercurial or 0.1 mM iodoacetamide (data not shown),

suggesting exposure of the TGH active site by a Ca

induced

conformational change. Notably, TGH activity was decayed in

minutes at mM concentrations of Ca

. Addition of EDTA could

stop the Ca

-induced decay immediately but could not reverse

it, and the presence of dithiothreitol in the enzyme buffer only

weakly protected this decay (data not shown). Moreover,

pre-incubation with other metal chlorides also inactivated the

enzyme although less effectively than CaCl

(

Fig. 5

).

3.3. Cloning, sequencing and phylogenic analysis

Automatic sequencing of two oligopeptides obtained by

HPLC-purification of Lys-C digest of Mj-TGH showed their

amino acid sequences as MRQEYVLSDVGTVFVGSYP and

EXLTWEYKPPEGTRAXRVSILN, respectively. The sequence

of another purified CNBr cleavage product was YYTGVPAH

TIKRHDGELHLEPSQT. Degenerate PCR primers were

de-signed based on these Mj-TGH partial sequences. In addition to

the cDNA encoding STG I (AY074924), a 578-bp cDNA

en-coding the C-terminal part of a novel P. monodon

transglutami-nase (designated as PmTG) was produced by PCR. On the other

hand, a 1071 bp cDNA encoding the C-terminal part of Mj-TGH

was also amplified. The full-length cDNA of PmTG and Mj-TGH

were then completed by 5′ RACE. PmTG had a total length of

2,387 bp, including 36 bp of the 5′-untranslated region, an open

reading frame of 2,271 bp, and 81 bp of the 3′-untranslated

Fig. 9. Phylogenetic tree for invertebrate transglutaminases based on the amino acid sequences. Human factor XIIIa (NP-000120) was used as the out-group. Besides PmTG and Mj-TGH, the species and GenBank accession Nos. are: P. monodon STG I (AY074924) and STG II (AY771615), M. japonica-TG (AB162767), crayfish TG (AAK69205), horseshoe crab TG (A45321), starfish TG (BAB20439), sea urchin (CAD28789), drosophila TG (AAF52590) and grasshopper annulin (P52183). Bootstrap values are shown at the nodes.

Fig. 10. RT-PCR analysis of tissue expression of PmTG. Total RNA was extracted from various tissues of P. monodon. Specific primers were designed to amplify a 390-bp fragment of PmTG cDNA. PCR products at various cycles were analyzed by electrophoresis on 1.2% agarose gel containing ethidium bromide.

region. The putative initiating ATG codon, according to Kozak's

rule

[29]

, is at nucleotide 33 (

Fig. 6

). The open reading frame of

PmTG is predicted to encode a protein of 757 residues (pI 5.47)

and similar to STG II

[10]

. Its ORF differs from that of STG II by

15 nucleotides and resulted in the substitution of six amino acid

residues, i.e., L158

→F, T182→N, Q212→P, V495→A,

S499

→G, and E541→G. None of them are in the catalytic core

domain of the enzymes.

We also cloned Mj-TGH cDNA which contained 2996 bp

(included 54 bp of the 5′-untranslated region, an open reading

frame of 2274 bp, and 669 bp of the 3′-untranslated region). Its

authenticity was confirmed by sequence analyses of the

fragments obtained from CNBr cleavage and protease digestion

of the purified M. japonicus TGH. The putative initiating ATG

[29]

is at nucleotide 55, and its open reading frame encodes an

enzyme of 758 residues (pI 5.76) with seven potential

N-glycosylation sites and one RGD motif (

Fig. 7

). The predicted

Mj-TGH sequence is 83% and 59% identical to those of STG I

and STG II/PmTG, respectively (

Fig. 8

). PmTG and Mj-TGH

probably are both cytosolic, since they do not contain a typical

signal-peptide or trans-membrane domains.

Amino acid sequences of all the invertebrate

transglutami-nases so far solved were aligned and a phylogenetic tree has been

constructed to study their structural relationships (

Fig. 9

). All the

Crustacean enzymes are linked together in the tree and closer to

those of other marine arthropods than to those of insects.

Apparently, STG I and Mj-TGH are orthologous to each other.

3.4. RT-PCR analyses and tissue expression profile

The expression patterns of PmTG mRNA in various tissues of

P. monodon were analyzed by RT-PCR. As shown in

Fig. 10

, a

390-bp DNA fragment could be amplified in contrast to the

negative control. The expression levels were highest in

hemo-cyte and muscle, moderate in gill, intestine and lymphoid organ,

and lowest in heart and hepatopancreas.

3.5. In-gel digestion and mass analysis of TGH

Purified P. monodon TGH band in gel was digested with

modified trypsin before analyzed by

peptide-mass-fingerprint-ing. The masses matched nicely with those of the peptides

pre-dicted from P. monodon STG I

[8]

. We found at least 34 TGH

peptides matching those of STG I (

Table 1

), representing a total

of 396 amino acids or 52% (396/757) of the entire sequence and

evenly distributed.

Table 1

Mass match between the tryptic peptides of TGH and STG I (MSeand MScare experimental mass of TGH peptides and calculated mass of STG I peptides,

respectively)

Tryptic peptides of STG I position MSe MSc

FTNDLELCR 14–22 1167.57 1167.52 EDREFEIATELNEIADALSASK 24–45 2450.13 2450.18 EFEIATELNEIADALSASK 27–45 2050.95 2050.01 EFEIATELNEIADALSASKDPK 27–48 2390.14 2390.19 IVSVDFQPMYNAVNHHCELYELV 49–73 3047.36 3048.41 RGGLLSMVVK 82–91 1058.62 1058.63 GGLLSMVVK 83–91 902.47 902.53 LNQNVALASAQELK 92–105 1497.73 1497.82 FYFSFGSRPNVEK 106–118 1576.77 1576.77 GTQAHLVVTGK 119–129 1109.60 1109.62 IEKPHDDWDIR 132–142 1422.68 1422.69 VAAEVTLR 168–175 857.49 857.50 FDTHVYILFNPWNENDGTYLPEEK 186–209 2940.31 2940.36 MVNSNDDSGLLVGK 275–288 1447.63 1447.70 WDGEYEDGR 289–297 1125.42 1125.44 WDGEYEDGRAPAK 289–301 1492.64 1492.66 WLGSIEILEMYLR 302–314 1621.82 1621.85 VVTNFASAHDTNISLSIDEYFDEDGDQIDAADR 344–376 3642.54 3642.62 YVNPAGIR 377–384 888.41 888.48 YDVPFVLAEVNADVVR 444–459 1804.91 1804.94 LATQTSSVGR 473–482 1018.53 1018.54 AGPVATGGFFK 489–499 1050.48 1050.55 SDREDITWEYKPPEGTR 500–516 2077.94 2077.97 VSILNAAR 520–527 841.45 842.50 EDVTFQIEDR 544–553 1250.56 1250.58 TINVVMTCASMYYTGAK 577–593 1908.83 1909.86 HDGELHLEPSQTK 600–612 1489.70 1489.72 LVDHDLIK 627–634 950.47 951.54 VSFDNPIPLLLK 679–690 1354.76 1354.79 LDGPGLMRPK 696–705 1081.62 1082.59 MNFEMNLYPK 718–727 1285.55 1285.58 KAVEGTLIVQFNSR 728–741 1560.84 1560.86 AVEGTLIVQFNSR 729–741 1432.74 1432.77 ELCNLTGAK 742–750 1005.52 1005.48

4. Discussion

In shrimps, coagulation is initiated by activation of hyaline

cells which release its content including the clotting enzymes

[30

–32]

. P. monodon TGH effectively polymerizes shrimp

clottable proteins to form stabilized gel (

Fig. 3

). Notably, the

purified P. monodon TGH exists as weakly associated dimmers,

like the A subunit of mammalian FXIII which is release from

megakaryocytes as homodimers and also present as dimmers

when crystallized. However, the shrimp TGH is possibly active

as monomers like FXIII

[1]

. Dependent on seawater salinity, the

Ca

concentrations in tiger shrimp hemolymph range from 6.4

to 10 mM

[28]

. Thus, when TGH is released from hemocytes it

would be fully activated to initiate the coagulation (

Fig. 3

).

However, metal ions and high chloride ion

[2]

in the

hemo-lymph possibly reduce its enzymatic activity within minutes, as

a feedback regulation to avoid excess clotting.

The X-ray crystallographic structure

[33,34]

showed that the

catalytic domain (i.e., region 200

–550) of FXIIIa contains a

hydrogen-bonded Cys

–His–Asp triad similar to that found in

enzymes of the papain family

[35]

. The catalytic triad appears to

be also conserved in PmTG (and Mj-TGH), i.e., Cys330 (325),

His397 (392), Asp420 (415). Their Ca

-binding sites

presum-ably include the conserved Asn460, Asp462, Glu512, and

Glu517 (

Fig. 8

). With highly conserved region 350–353, His

358 (353) and Glu451 (466) in shrimp TGH may be associated

as in FXIIIa

[33]

. Many conserved Trp residues in the core

domain of TGH (

Fig. 8

) possibly are involved in stabilization of

the transition state during the enzymatic reaction

[36]

. It is

known that Ca

unmasks the active site Cys and widens the

protein substrate binding site

[1,37]

and the active site possibly

undergoes irreversible conformational changes. Being 35%

similar to FXIIIA, TGH is relatively unstable after activation at

ambient temperature and in the presence of bivalent metal ions

(

Fig. 5

). Instability of other transglutaminases has been reported

before

[2,38]

, but its exact mechanism remains to be explained

by 3D structural analyses of both the active and the inactive

enzymes or by mutagenesis study

[39]

.

The RGD (or Arg–Gly–Asp) motif, which is also present in

fibrinogen, is known to bind to integrins or specific membrane

receptors

[40]

. Positions of RGD motifs are remarkably

conserved in all invertebrate transglutaminases (

Fig. 8

) and

human keratinocyte

[8]

. This motif probably is essential for the

shrimp enzyme to bind integrins and form complex with adhesion

molecules

[1,41]

, and possibly trigger certain signal pathways,

e.g., RhoA activation and cell spreading

[42]

. Interestingly, the

RGD are all flanked by Pro and/or Gly residues (i.e.,

“proline

brackets”) and structurally constrained on the protein surface

[43]

, consistent with their role for protein

–protein interaction.

Both STG I and PmTG were transcribed in P. monodon

hemocytes. However, results of the peptide mass fingerprinting

of P. monodon TGH confirmed its identity with STG I (

Table 1

)

but not with PmTG or STG II. The molecular mass of P.

monodon TGH was determined as 84280 ± 168 Da. The

pre-dicted P. monodon TGH mass would be 84713 Da (based on the

STG I sequence,

Fig. 8

) assuming no disulfide bond was formed

and N-terminal acetylation took place after removal of Met 1, as

in the case of FXIIIa

[44]

. Alternatively, the mass would be

84285 Da if four residues (Met–Pro–Thr–Val) were removed.

Thus, the determined mass suggests that TGH possibly is

processed by deleting N-terminal residues before acetylation but

not glycosylated although they contain seven potential

N-glycosylation sites (

Figs. 6 and 7

).

Differed by merely six amino acid substitutions, PmTG and

STG II probably are alleles of the same locus with natural

polymorphism. The phylogenetic tree (

Fig. 9

) shows that the

shrimps have at least two paralogous transglutaminases, i.e.,

PmTG/STG II and STG I in P. monodon, and

Mj-transglutami-nase (AB162767) and Mj-TGH in M. japonica. Paralogs are

homologous proteins resulted from gene-duplication and they

usually co-expressed in the same organism, while orthologs are

those from the same gene lineage but expressed in different

organisms

[45]

. Previous immunodiffusion experiments showed

that antibodies prepared against of P. monodon clottable protein

reacted with M. japonicus clottable protein

[32]

. The coagulation

systems for this two genetically related species

[46]

presumably

are similar. Results in

Fig. 8

confirmed a high structural

similarity between the hemocyte enzymes (STG I and Mj-TGH)

of both species, but many distinct substitutions between STG I

and PmTG at their core regions (residues 200–500) suggesting

possible different substrate specificities. Protein sequence of a

previously cloned crayfish transglutaminase

[9]

appears to be

more close to that of PmTG or STG II (62%) than that of STG I

(55%), whether it is the only isozyme or the real functional TGH

of this fresh-water Crustacean remains to be studied.

The recombinant STG I from insect-baculovirus expression

system did not show clotting activities

[8]

, and recombinant

STG II was postulated to be the clotting enzyme of P. monodon

[10]

. However, STG I was expressed in Sf21 cells cultured at

26 °C and the cell lysate were frozen before assay, the

re-combinant STG I probably had lost most of its activity under the

culture conditions or freeze-and-thaw procedure. The instability

of STG I probably lead to the wrong conclusion that STG 1 is

not responsible for the hemolymph coagulation

[8]

. Moreover,

in vitro assays of recombinant STG II may also be misleading,

since the clot generated by recombinant STG II was less

branched and instable

[10]

, not like the clot formed naturally in

crayfish

[47]

and horseshoe crab

[2]

.

Based on microscopic observation, shrimp hemocytes

com-prised of hyaline cells, semi-granular cells, and granular cells

[48–50]

. Coagulation in shrimp is mediated by the circulating

hyaline cells

[31]

. We have cloned more cDNAs encoding

PmTG than those encoding STG I although STG I was the only

transglutaminase purified from P. monodon hemocytes. By

RT-PCR, we also found PmTG gene highly expressed in hemocytes

and muscle, but less expressed in other tissues (

Fig. 10

),

consistent with the expression patterns of STG II gene

[10]

.

Thus, PmTG mRNA is relatively abundant in the hemocytes,

while STG I gene is constitutive expressed at low-level in

various tissues

[8]

. The mRNA of a crayfish transglutaminase

(possibly orthologous to those of PmTG/STG II) was also

reported to be expressed in semigranular and granular cells

[9]

.

Since shrimp have an open circulatory system and hemocytes

are likely to rest onto different tissue surfaces, the Northern blot

results of PmTG (

Fig. 10

) need to be confirmed by in situ

hybridization. Previous in situ hybridization pointed out a low

mRNA level of STG I in circulating hemocytes but a high level in

mitotic cells of hematopoietic tissue

[8]

, and the RT-PCR

experiment also showed that STG I mRNA is decreased when

hemocytes matured and released into the hemolymph. Taken

together, these results suggest that transcription and translation of

STG I in the hemocytes possibly take place in early

develop-mental stages but down-regulated in later developdevelop-mental stages.

In conclusion, this is the first biochemical study on shrimp

hemocyte transglutaminases (TGH) responsible for hemolymph

coagulation. The purified shrimp TGHs are shown to be

relatively unstable after Ca

-dependent activation. We also

cloned two novel transglutaminases, PmTG from P. monodon

and Mj-TGH from M. japonicus, and confirmed the presence of

two transglutaminase genes in both species. By peptide mass

fingerprinting (

Table 1

) or sequencing of purified TGH peptide

fragments (

Fig. 8

), both STG I and Mj-TGH have been identified

to be the functional TGH in the two shrimp species. We failed to

isolate their paralogous enzymes, PmTG/STG II and another

transglutaminase previously cloned from M. japonicus, although

their mRNAs were expressed in shrimp hemocytes. The specific

role and regulated expression of these transglutaminases in

different Crustacean remain to be investigated.

Acknowledgements

We thank Yuh-Ling Chen for supplying peptide sequence

data of M. japonica transglutaminase and Chien-Hong Lu for

preparing the shrimp cDNA. MALDI-TOF/TOF analyses were

carried out by the Core Facilities for Proteomics and Structural

Biology Research, Institute Biological Chemistry, Academia

Sinica, Taiwan. This research was supported by grants from

Academia Sinica and Hung Kuang University, Taiwan, ROC.

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