Molecular cloning and characterization of a hemolymph clottable proteinfrom tiger shrimp (Penaeus monodon)

Maw-Sheng Yeh1, Chang-Jen Huang1,2, Jiann-Horng Leu2, Yuan C. Lee3 and Inn-Ho Tsai1,2

1Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan; 2Institute of Biological Chemistry, Academia Sinica,

Taipei, Taiwan; 3Department of Biology and McCollum-Pratt Institute, Johns Hopkins University, Baltimore, MD, USA

To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 kDa was

purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion exchange

chromatography. The purified protein was coagulated by the shrimp hemocyte transglutaminase in the presence

of Ca2+. The clottable protein contains 44% a helices and 26% b sheets as determined by circular dichroism

spectra. Its conformation is stable in buffer of pH 4±9. To solve its primary structure, partial sequences of the

purified polypeptides from cyanogen bromide cleavage and endopeptidase digestion were also determined. A

shrimp cDNA expression library was constructed. By combination with antibody screening, reverse transcriptase

PCR using degenerate primers from determined amino acid sequences and cDNA library screening with

digoxigenin-labeled DNA probes, the entire cDNA of 6124 bp was obtained. This cDNA encodes a protein of

1670 amino acids, including a 14-amino acid signal peptide. With four potential N-glycosylation sites, the

clottable protein was found to contain 3.8% high-mannose glycan; and Man8GlcNAc and Man9GlcNAc were

released upon endo-b-N-acetylglucosaminidase hydrolysis. Upon conducting a protein sequence database survey,

the shrimp clottable protein shows 36% identities to the crayfish clotting protein and lower similarities to

members of insect vitellogenins, apolipoprotein B and mammalian von Willebrand factor. Notably, a region rich

in Gln residues, a polyGln motif and five Ser-Lys-Thr-Ser repeats are present in the shrimp protein, suggesting

this protein might be a transglutaminase substrate. Northern blot analysis revealed that the clottable protein is

expressed in most of the shrimp tissues but not in the mature hemocytes.

Keywords: clottable protein; shrimp; cDNA cloning; carbohydrate analysis; tissue distribution; Penaeus

monodon.

Efficient immune systems and clotting reactions are of vitalimportance to both vertebrate and invertebrate animals. Theinvertebrate models of plasma coagulation are highly diverseand less studied. Investigation of them should help ourunderstanding of the general principles of hemostasis inanimals. Tait [1] described three types of hemolymphcoagulation in crustaceans as follows: type A is characterizedby rapid agglutination of hemocytes without clotting of theplasma; type B involves cell aggregation coupled with limitedclotting of the plasma, and type C shows limited cell lysisleading to plasma clotting and little cell aggregation. Thesethree types are likely variations of the basic mechanisminvolving both hemocyte and hemolymph [2±4]. Shrimp andother decapoda undergo the type C coagulation.

In crustaceans, clotting is mediated through clottableproteins (coagulogens) present in the plasma and cell

factors compartmentalized within the circulating cells. Theclottable protein is converted to covalently linked polymersby Ca2+-dependent transglutaminase which may be releasedfrom hemocytes during coagulation [5±7]. In the wound area,the clottable protein oligomerizes to prevent hemolymph lossthrough breaks in the exoskeleton and dissemination of bacteriathroughout the body. On the other hand, cell factors in thehemocytes have been reported to coagulate by serine proteaseswhich may be activated by lipopolysaccharide or b-1,3-glucans[2,8]. This is also linked to the prophenoloxidase activatingsystem [9]. Thus the coagulation is part of humoral immuneresponse in crustaceans [10,11].

The tiger shrimp, Penaeus monodon, is an economicallyimportant species cultured in Taiwan and south-eastern Asia.We have purified and characterized the clottable proteins oftiger shrimp and several crustaceans [12]. Being homodimericglycoproteins of about 380±400 kDa, the clottable proteins ofPenaid shrimps [12], lobster [13], crayfish [14], freshwatergiant prawn [12], and sand crayfish [15] appear to have similaramino acid compositions and N-terminal sequences. Thepurified clottable proteins formed stable clots by the trans-glutaminase from the shrimp hemocyte lysate in the presence ofCa2+. We report herein the complete nucleotide and proteinsequences, carbohydrate structure and tissue distribution of thetiger shrimp clottable protein. Its structural features and motifsare studied in detail and compared with those of the crayfishclotting protein whose sequence was published very recently[16].

Eur. J. Biochem. 266, 624±633 (1999) q FEBS 1999

Correspondence to I.-H. Tsai, PO Box 23-106, Taipei, Taiwan.

Fax: + 886 2 23635038, Tel.: + 886 2 23620261 ext 2011,

E-mail: bc201@gate.sinica.edu.tw

Abbreviations: CNBr, cyanogen bromide; HPAEC, high-performance

anion exchange chromatography; DIG, digoxigenin.

Note: the nucleotide sequence reported in this paper has been submitted

to GenBank under the accession number AF 089867

(Received 10 June 1999, revised 17 September 1999, accepted

28 September 1999)

q FEBS 1999 Shrimp hemolymph clottable protein (Eur. J. Biochem. 266) 625

M A T E R I A L S A N D M E T H O D S

Purification of clottable protein from hemolymph

Collection of hemolymph and hemocytes from tiger shrimps(P. monodon), and purification of the clottable protein were asdescribed [12]. The molecular mass of the protein wascharacterized by matrix-assisted laser desorption/ionizationtime-of-flight MS and SDS/PAGE [12]. Protein concentrationswere determined by the Bradford method [17], using BSA asstandard.

Circular dichroism

The clottable protein stock solution was mixed with buffers toprepare samples of constant protein concentration but withvaried salt concentrations or pH. CD measurements werecarried out on a J720 spectropolarimeter (Jasco) under constantflushing of nitrogen at 25 8C. Each sample was scanned from200 nm to 250 nm, and two independent experiments for eachsample were performed and found to give identical spectra. Themean residue ellipticity [u] was calculated from the meanresidue weight. The spectra were used for analyses of secondarystructure of the protein with a computer program provided byJasco and based on the method of Yang et al. [18].

Fragmentation and partial amino acid sequencing

The purified protein (2 mg) was dissolved in 100 mL of 6 mguanidine hydrochloride, 250 mm Tris/HCl, pH 8.5 andreduced by dithiothreitol (0.09 m) at 50 8C for 1 h. Afteradding 30 mL of 1 m iodoacetic acid, the mixture wasincubated at room temperature for 30 min. The desalted andlyophilized S-carboxymethyl protein was re-dissolved in100 mL of 70% formic acid with 0.2 mg of cyanogen bromide(CNBr) at room temperature for 24 h. On the other hand, thenative clottable protein (4 mg´mL21) was digested at 37 8C for18±24 h, either with Lys-C endopeptidase (Promega) in 50 mmTris, pH 8.0, or with V8 protease (i.e. Glu-C, Promega) and50 mm sodium phosphate buffer, pH 7.8, at an enzyme tosubstrate ratio of 1 : 50 (w/w). The reaction was stopped byadding dithiothreitol and heated at 95 8C for 5 min. Theresultant polypeptides were fractionated by reversed-phaseHPLC using a Chemosorb C18 column. The amino acidsequences of the purified peptides were determined with asequencer (Applied Biosystems, model 470A) coupled with anon-line phenylthiohydantoin-derivative analyzer.

General methods in molecular biology

Standard procedures in molecular biology were used forpreparation of plasmid DNA, restriction enzyme digestion,DNA agarose gel electrophoresis, DNA ligation, and thetransformation of bacteria [19].

RNA isolation and cDNA library construction

Total RNA was isolated from the tiger shrimp by extraction inacid guanidinium thiocyanate as described [20]. For libraryconstruction a total of approximately 1 mg of total RNA wasisolated from the whole shrimps. A cDNA library preparedfrom poly(A)-enriched RNA by unidirectional insertion ofcDNA into l-ZAP II [21] was constructed using a kit fromStratagene.

cDNA synthesis

The total RNA was purified using the RNAzol B kit (Biotex)and the mRNA was purified using QuickPrepR Micro mRNApurification kit with oligo(dT)-cellulose chromatography(Pharmacia). The first strand cDNA synthesis was primedwith a hybrid oligo(dT) linker-primer and random primers, andwas transcribed using moloney murine leukemia virus reversetranscriptase (Gibco BRL). The synthesized cDNA was used asa template in subsequent PCR [22].

PCR and screening of cDNA library

Degenerate primers were designed from N-terminal sequenceQPGLEYQY (forward primer) and some known peptidesequences of the clottable protein (reverse primers). A PCRproduct of 500 bp was obtained from degenerate primers, thedesign being based on the sequences QPGLEYQY andAEEVNVQ. Amplified DNA fragments were then purifiedand ligated into pGEM-T (Promega). Each clone wassequenced by the dideoxy chain termination method [23] withSequenase (US Biochemical) according to the manufacturer'sinstructions. Among the seven clones sequenced, threecontained other determined peptide sequences. Thus this500-bp DNA was used as a probe to screen the shrimp cDNAlibrary by using a digoxigenin (DIG) DNA Labeling kit(Boehringer Mannheim). Approximately 1 � 106 amplifiedclones were plated at a density of 5 � 105 plaque-forming unitsper 150-mm petri dish. Hybridization and washing were carriedout as previously describe [19]. In brief, nitrocellulose (Drassel)lifts of the phage plates were hybridized at 42 8C overnight in50% (v/v) formamide containing 5 � NaCl/Cit (NaCl/Cit =150 mm NaCl, 15 mm sodium citrate, pH 7.5), 0.1% (w/v)SDS, 0.5% (w/v) N-laurylsarcosine, 2% blocking reagent(Boehringer Mannheim), and a DIG-labeled probe. Followinghybridization, filters were washed in 2 � NaCl/Cit, 0.1% SDSat 25 8C for 30 min, then in 0.1 � NaCl/Cit, 0.1% SDS at65 8C for 30 min. Signals were detected using the DIGluminescent detection kit for nucleic acids (BoehringerMannheim). Clones of interest were further purified by threemore screen cycles. The clones were subcloned into the EcoRIsite of pBluescript II KS(+) phagemid (Stratagene).

Immunoscreening of the shrimp cDNA library

The shrimp expression cDNA library was screened by anti-clottable protein antibodies [19]. The l-ZAP phages wereplated at a density of 5 � 105 plaques per agar plate. Afterincubation for 3.5 h at 42 8C, the plates were overlaid withnitrocellulose filters (0.45 mm; Micron) that had been impreg-nated with 10 mm isopropyl-1-thio-d-galactopyranoside. Incu-bation was continued for 5 h at 37 8C. The filters were thenremoved, washed with NaCl/Pi (0.12 m NaCl, 10 mm phos-phate) at room temperature, and blocked with 1% polyvinyl-pyrrolidone in NaCl/Pi for 16 h at 4 8C. Following blocking,filters were probed with a polyclonal antiserum specific forshrimp clottable protein [12] at a 1 : 500 dilution in NaCl/Pi

containing 1% poly(vinyl pyrrolidone), 1 mm EDTA, and 0.4%Triton X-100 at 25 8C for 1 h. The filters were then washedthree times with NaCl/Pi and incubated with horseradishperoxidase-conjugated anti-rabbit IgG (Sigma) for 1 h atroom temperature. The immune complexes were then incubatedin 10 mL of NaCl/Pi containing 0.2 mg´mL21 diaminobenza-midine and viewed after adding 10 mL of 30% H2O2. Phagesdisplaying strong signals were isolated for secondary and third

626 M.-S. Yeh et al. (Eur. J. Biochem. 266) q FEBS 1999

screening. One positive clone (Cp 5) was isolated afterscreening 3 � 106 plaques, and the clone was subclonedinto the EcoRI site of pBluescript II KS(+) phagemid(Stratagene).

Sequence homology and hydropathic profile

A search for related sequences using data from GenBank, andSWISS-PROT was carried out [24]. Amino acid sequencealignment of the shrimp clottable proteins with crayfish clottingprotein [16] was accomplished with the clustal w multiplealignment program (version 1.64B) [25]. The hydropathicprofile of the shrimp clottable protein sequence was plottedfollowing the method of Kyte and Doolittle [26]. Windows ofseven amino acid residues were used to calculate the averagehydrophobicity of the central residue.

Northern blot analysis

Total RNA was isolated from various shrimp tissues usingthe RNAzol B kit (Biotex). Total RNA (20 mg) from eachtissue was fractionated on 1% formaldehyde/agarose gel in4-morpholinepropanesulfonic acid buffer and transferred onto aHybond-N membrane (Amersham). Following prehybridizationfor 3 h in 50% formamide, 5 � NaCl/Cit, 2% blocking reagent,0.1% N-laurylsarcosine and 0.02% SDS at 42 8C, the blots werehybridized with a PCR-generated DIG-labeled cDNA probe for20 h under identical conditions. The blots were washed twicefor 5 min with 2 � NaCl/Cit containing 0.1% SDS at roomtemperature and twice for 15 min at 65 8C with 0.1 � NaCl/Citcontaining 0.1% SDS. Detection of DIG signals was accom-plished using the DIG luminescent detection kit. The cDNAprobe used for Northern blotting was synthesized by a PCRDIG probe synthesis kit with two primers derived fromclottable protein sequence, namely GAGGGCATGTGAGAAC-CATC (i.e. forward primer, nucleotides 3915±3934) andCTTCCCAGACAACCTGAAGA (i.e. reverse primer, nucleo-tides 4384±4403).

Cloning of tiger shrimp b-actin gene

Degenerate primers were designed according to the amino acidsequences that are highly conserved in b-actin. The amino acidsequences for designing the two opposing primers areQIMFETF and MKCDVDI. Using these primers and theshrimp cDNA as template, a PCR product of 500 bp wasobtained (data not shown). Among the 18 clones sequenced, 16clones contained three types of the b-actin sequences. Thededuced amino acid sequence showed a high level of identity tohuman actin (91%), bovine actin (91%) and brine shrimp actin(90%). One of the three types of the b-actin cDNA wasamplified by the PCR DIG probe synthesis kit (BoehringerMannheim) with two opposing primers. It was used in theNorthern blotting experiments as an internal control of b-actinexpression.

Carbohydrate analysis

The lyophilized clottable protein (20 mg) was dissolved in100 ml of water and added to an equal volume of either 4 mtrifluoroacetic acid or 8 m hydrochloric acid in a 1.5-mL screw-capped vial; each vial was heated at 100 8C for 4 h or 6 h [27],respectively. The hydrolyzates were evaporated to drynessunder vacuum using a Speedvac evaporator. Monosaccharidecontents were analyzed with high-performance anion exchange

chromatography (HPAEC) consisting of a Bio-LC system(Dionex) equipped with a CarboPac PA-1 column(9 � 250 mm) and a pulsed amperometric detector (PAD-II)[27]. The chromatographic data were managed with AI-450chromatography software (Dionex). Seven monosaccharidesincluding glucose, mannose, galactose, 6-methylgalactose,fucose, N-acetylglucosamine and N-galactosamine were usedas standards. On the other hand, the clottable protein (40 mg)dissolved in 50 ml of water was denatured by heating at 100 8Cfor 5 min. After cooling down it was added 5 mU (5 mL) ofendo-b-N-acetylglucosaminidase and 145 mL of sodium acet-ate buffer (pH 6.5) and incubated at 37 8C for 18 h. Thereleased oligosaccharides were analyzed by a Dionex HPAEC[28], using the established conditions [29]. Ribonuclease B(Sigma) was hydrolyzed by the same enzyme to giveMan5GlcNAc,Man9GlcNAc standards.

Binding test of biotinylated clottable protein

Following the protocol of an immunoprobe biotinylation kit(Sigma), 0.75 mg of purified clottable protein in 200 mL of0.1 m sodium phosphate buffer (pH 7.4) was mixed with10 mL of biotinamidocaproate-N-hydroxy-sulfosuccinimideester (10 mg´mL21) and incubated with gentle stirring for30 min at room temperature. The biotinylated protein waspurified by a Sephadex G-25 column (1 � 10 cm) equilibratedwith NaCl/Pi and stored at 220 8C until use; it was confirmedby Western blot with ExtraAvidin conjugated with alkalinephosphatase (Sigma). The hemolymph was freshly withdrawnfrom the shrimp and fixed with 10% formalin in 2.25 � NaCl/Pi for 15 min at room temperature. The hemocytes were spundown at 500 g for 10 min washed twice with NaCl/Pi, and thensmeared on slides. The slides were overlaid with biotinylatedclottable protein (50 mg´mL21) for 1 h at room temperature andthen washed with NaCl/Pi. Finally, the slides were incubatedwith 100 mL of 50 times-diluted fluorescein isothiocyanate-conjugated UltraAvadin (Leinco) for 1 h. After washing withNaCl/Pi, the slides were mounted in 50% glycerol in NaCl/Pi for observation under a fluorescence microscope (OlympusAHBS3).

R E S U LT S

Partial amino acid sequence

The N-terminal amino acid sequence of the native protein wasdetermined up to the 30th residue [12]. The clottable protein

Fig. 1. Restriction enzyme map (A) and sequencing strategy (B) of the

clottable protein cDNA. The clone Cp 1 was obtained by PCR (see

Materials and methods) whereas Cp 5 was isolated by immunoscreening.

Other clones, Cp 2, Cp 3, Cp 4, were isolated by using both Cp 1 and Cp 5

as probes. Abbreviations of restriction enzyme sites are denoted as: B,

BglII; E, EcoRI; H, HindIII; X, XhoI.

q FEBS 1999 Shrimp hemolymph clottable protein (Eur. J. Biochem. 266) 627

was also digested directly with Lys-C and Glu-C endopepti-dases since its solubility was greatly decreased when theclottable protein was reduced by dithiothreitol. We obtained atotal of ten purified polypeptides from reversed-phase HPLC,including two from CNBr cleavage, one from Lys-C digest, andseven from Glu-C digest. Table 1 shows their amino acidsequences.

cDNA cloning of the clottable protein

Degenerate primers were designed based on the partial aminoacid sequences obtained from Table 1. After PCR amplifi-cation, we isolated a 500-bp clone (Cp 1, see Fig. 1) whichencodes the N-terminal sequence of the clottable protein. Onthe other hand, we got the Cp 5 clone by immunoscreening.The cDNA library was re-screened using both Cp 1 and Cp 5 asprobes, and Cp 2, Cp 3 and Cp 4 were thus cloned. These fiveclones were isolated and sequenced to complete the entirenucleotide sequence of the clottable protein. The deducedamino acid sequence is shown in Fig. 2B. The cDNA has a totallength of 6124 bp, including 502 bp of the 5 0-untranslatedregion, an open reading frame of 5010 bp, and 612 bp ofthe 3 0-untranslated region. The putative initiating ATG codon,which agrees with Kozak's rule [30], is at nucleotide 503. Theopen reading frame is predicted to encode a protein of 1670amino acids, including a 14-amino acid signal peptide, twoRGD (Arg-Gly-Asp) motifs and four potential N-glycosylationsites (Fig. 2B). The authenticity of the cDNA sequence isconfirmed by its accordance with the amino acid sequences ofthe fragments obtained from CNBr cleavage and proteolyticdigests of the clottable protein. The results are consistent withits molecular mass determined by matrix-assisted laserdesorption/ionization time-of-flight MS [12].

Table 1. Partial amino acid sequences of tiger shrimp clottable protein.

X denotes unidentified residues.

Cleaving

agent

Fragment Amino acid sequence

CNBr CN1 AADYSVVVQFSNIEVGDLNKVDL

CNBr CN2 KLPVNLAEEVNVQREH

Glu-C E1 QAQKQTQQQVQGTQWEEXFP

Lys-C K1 GFVNIFRVPLFWSTEL

Lys-C K2 LNYGAVEETLVGRXQ

Lys-C K3 TSPGQLANVPHIEDTLWSVR

Lys-C K4 IMHSLINGEEGL

Lys-C K5 HLXKPAAPASILYSTNFFLH

Lys-C K6 RPQSSAQEIVSGMWEELKE

Lys-C K7 EAIEKGXDYSTYAPGIDIIT

Fig. 2. Deduced amino acid sequence and 5 0 and 3 0 untranslated regions of the shrimp clottable protein. (A) Nucleotides 1±600 of the shrimp clottable

protein cDNA, containing the 5 0 untranslated region (1±502) and the beginning of the coding region (503±600). The deduced amino acid sequence up to

residue 33 is also shown. Repeated nucleotide sequences are underlined. (B) Deduced amino acid sequence of the clottable protein. The sequences confirmed

by protein sequencing are underlined. The putative signal sequence, N-glycosylation sites, and RGD motifs are doubly underlined. (C) Nucleotides 5401±

6124 of the shrimp clottable protein cDNA, containing the stop codon (5512±5514) and the 3 0 untranslated region (5515±6124). Three types of repeated

sequences are underlined. The two polyadenylation signals are doubly underlined.

628 M.-S. Yeh et al. (Eur. J. Biochem. 266) q FEBS 1999

Circular dichroism and fluorescence spectra

Figure 3A shows the CD spectra of the purified clottableprotein in neutral buffer. The clottable protein is predicted tocontain 44% a helix, 26% b sheet, 16% b turn. The tigershrimps are normally grown in seawater of 0.5±4.5% salinity(i.e. 0.09±0.77 m NaCl), but its CD spectra were not affectedby the addition of 0.1±0.5 m NaCl salt (Fig. 3A) or divalentmetal ions (e.g. 0.1±5 mm CaCl2, 0.1±5 mm MgCl2 or 0.1 mmZnCl2) to the protein solution. The protein was stable in buffersof pH between 4 and 9 but denatured at pH $ 10 (Fig. 3B).

The intrinsic fluorescence spectrum of the clottable protein hasan emission maximum at 336 nm when excited at 295 nm,typical of tryptophan fluorescence (data not shown). Theaddition of 5 mm CaCl2, 5 mm MgCl2, 0.1 mm ZnCl2 or 5 mmEDTA did not change the fluorescence spectra. Moreover, theabsence of Ca2+ or Zn2+ in the protein was confirmed by atomicabsorption analysis.

Glycan analyses

Sugar composition analyses confirmed that the tiger shrimpclottable protein contains mannose (2.6%) and N-acetylgluco-samine (1.2%) (Fig. 4). Although glucose was also found, itsvalue varied widely from analysis to analysis, and thus it wasregarded as a contaminant and not an integral sugar constituent.The sugar composition was also analyzed by a precolumnlabeling method [31] with the same result. After heatdenaturation and endo-b-N-acetylglucosaminidase digestion,the glycans released from the clottable protein were subjectedto HPAEC analysis. The high-mannose oligosaccharides ofribonuclease B released by endo-b-N-acetylglucosaminidasewere used as standards (Fig. 5A). It was found that theoligosaccharides released from the clottable protein weremainly Man8GlcNAc and Man9GlcNAc (Fig. 5B).

Northern blot

The presence of mRNA of the clottable protein in differentshrimp tissues was examined by Northern blotting using itspartial cDNA as the probe (Fig. 6A). The same tissue blot wasalso examined with a tiger shrimp b-actin probe (Fig. 6B) as aninternal control. The DIG-labeled cDNA probe for the clottableprotein hybridized with a single 6.2-kb band in all the samplesexcept the hemocytes, and the highest levels were found in gilland heart (Fig. 6A). The size of the mRNA identified agreeswith that expected for the clottable protein clone (6124 bp).The size of the b-actin mRNA identified is about 2 kb, alsosimilar to that of brine shrimp actin [32].

Hemocytes binding test

The clottable protein sequence contains two Arg-Gly-Aspmotifs that are potential sites for cell adhesion or binding [33].The binding of the biotinylated clottable protein to fixed

Fig. 3. Circular dichroism spectra of the clottable protein. Instruments

and analysis program used were described in Materials and methods.

Cuvettes of 0.2 mL and 0.1 cm light-path were used. The protein

concentration was 0.2 mg´mL21. (A) In (±±) 50 mm Tris/HCl buffer

(pH 8.0) only; or plus NaCl at: (´ ´ ´) 0.1 m; (- ´ -) 0.25 m; (- - -) 0.5 m.

(B) In (- - -) acetate buffers of pH 4.0; Tris buffers of: (±±) pH 7.5;

(´ ´ ´) pH 8.8; (- ´ -) pH 10.0.

Fig. 4. Carbohydrate composition of the clottable protein. The glyco-

protein (20 mg) was hydrolyzed in acid at 100 8C and dried (see Materials

and methods). The HPAEC sugar peaks were identified using a standard

containing seven monosaccharides. PAD, pulsed amperometric detector.

q FEBS 1999 Shrimp hemolymph clottable protein (Eur. J. Biochem. 266) 629

hemocytes was examined by immunofluorescence but nosignificant response was detected (data not shown).

D I S C U S S I O N

Among invertebrate coagulation, the clotting system in horse-shoe crab has been well-studied as a model [34,35]. Fromsequence homology search, the 380 kDa tiger shrimp clottableprotein is most similar to the 420 kDa clotting protein of afreshwater crayfish recently published [16]. So far only thesetwo homologous clotting proteins from crustacean have beencompletely sequenced (Fig. 7); they are 36% identical or about57% similar. Their low sequence identity is consistent with thefact that the anti-(tiger shrimp clottable protein) serum cannotrecognize the crayfish (Procambarus clarki) hemolymphclotting protein [12]. They also show even lower sequencesimilarities to insect vitellogenins [36], apolipoprotein B [37]and to the D domain of mammalian von Willebrand factor [38].Thus crustacean clottable proteins are evolutionarily related tovitellogenins but apparently play different functions. They donot resemble any protein in the coagulation cascade ofhorseshoe crab [39] or the vertebrate fibrinogens [13]. Theshrimp as well as crayfish clotting proteins are plasma proteinsin contrast to the horseshoe crab coagulogen which is muchsmaller and localized in the blood cells [39]. Apparently,coagulation in crustaceans is rather different from that inhorseshoe crab and many types of clotting mechanism existamong the vast number of existing invertebrate species.

The hydrophobic profile (Fig. 8) reveals that the shrimpclottable protein contains a relatively hydrophobic region

between amino acid residues 4 and 14, which fits the knownfeature of signal sequence. No large hydrophobic regions ormembrane spanning domain can be identified. Major hydro-philic regions in the protein are located at regions 179±234,306±323, 771±786, 1077±1090, 1585±1604, and 1625±1656.Three potential N-glycosylation sites at positions 106, 319, and1301 are located at hydrophilic regions (Fig. 8).

In contrast to other members of the vitellogenin family, themost prominent structural features of the shrimp protein includea Lys-rich domain containing five Ser-Lys-Thr-Ser repeats atpositions 203±231, another Ser/Thr-rich region at 1038±1292,a Gln-rich region at 1088±1132, and a polyglutamine run(QQQAQQQQQQQVQGTQ) at the C-terminal region. Exceptfor the polyQ, these features are also conserved in the crayfishclotting protein (Fig. 7), but crayfish clotting protein containsless SKTS repeats but more TKTTG repeats [16] than theshrimp counterparts. A potential clue to the functional roles ofthe polyQ and Ser-Lys-Thr-Ser repeats of tiger shrimp is thatthe protein is a substrate for the hemocyte transglutaminase.Structural homology of arthropod transglutaminase to that ofthe mammalian enzymes was reported [40] and previous study

Fig. 5. Oligosaccharide analysis of the clottable protein. (A) Standard of

oligosaccharides derived from digest of ribonuclease B with endo-b-N-

acetylglucosaminidase. The peaks shown are Man5Ä9GlcNAc, denoted as

M5, M6, M7, M8 and M9, respectively. (B) Oligosaccharides derived from

the clottable protein after digestion with endo-b-N-acetylglucosaminidase.

PAD, pulsed amperometric detector.

Fig. 6. Tissue distribution of the clottable protein as revealed by

Northern blotting. Total RNA (20 mg) from different tissues were

fractioned on a 1% formaldehyde-agarose gel by electrophoresis and

blotted onto a Hybond-N membrane. The blot was hybridized with (A) a

DIG-labeled clottable protein cDNA probe and (B) the shrimp b-actin

cDNA probe, and visualized with luminescent detection.

Fig. 7. Alignment of clottable proteins from tiger shrimp (ts) and crayfish (cr) [16]. Identical residues are shown in white on a black ground. The Lys-rich

and Gln-rich regions are boxed. Arrowheads indicate Lys-containing repeated sequences. Asterisks above the residues denote the conserved N-glycosylation

sites. Gaps (±) are added to improve alignment.

630 M.-S. Yeh et al. (Eur. J. Biochem. 266) q FEBS 1999

q FEBS 1999 Shrimp hemolymph clottable protein (Eur. J. Biochem. 266) 631

revealed that the shrimp enzyme has similar properties to thevertebrate platelet transglutaminase or the a-subunit ofcoagulation Factor XIII [41]. The transglutaminase-catalyzedreaction involves a Ca2+-dependent acyl transfer [6,42]. When aGln-containing acyl donor polypeptide binds to the enzyme, thenucleophilic attack of the enzyme active site on the g-carboxamide group of a Gln residue leads to formation of anacyl-enzyme intermediate with release of ammonia. The Lys-containing polypeptide then binds, and the acyl group istransferred to its 1-amino group, resulting in formation of anisopeptide bond. The shrimp clottable protein presumably alsocontains specific donor (Gln) and acceptor (Lys) residues tobind to the enzyme active site.

According to previous results on the substrate specificitiesof mammalian transglutaminase [43±45], only certain aminoacid residues frequently precede the acceptor lysine residues,e.g. Ser, Val, Gln, or Thr. We notice that there are in totalten Ser-Lys, five Val-Lys, five Gln-Lys, and eight Thr-Lysarrangements in the clottable protein sequence. For example,KSK (residues 205±207) and KQK (residues 772±774) of theshrimp protein bear identical sequences to those of the activeacceptors in another transglutaminase substrate [44]. The C-terminal polyQ and the Gln-rich region also have high potentialfor isopeptide formation [46,47]. As the hydropathic profile ofthe protein (Fig. 8) revealed, these Lys-rich or Gln-rich regionsand polyQ are located in major hydrophilic region and easilyaccessible surface of the clottable protein. Thus some of these

Gln and Lys residues of the protein are probably utilized in theclotting process catalyzed by the hemocyte derived transgluta-minase.

Ten cysteine residues and a GICG or GLCG motif (atposition 1511±1514 of the shrimp protein) are conservedin the C-terminal regions of insect vitellogenins and thecrustacean clottable proteins (Fig. 7). Previously, a similarmotif TCGLCG was found to be conserved in variousinvertebrate and vertebrate vitellogenins, and in the domainsD1 and D2 of human von Willebrand factor and the domain D3

of human mucin 2 [36,48]. The D domains play essential rolesin the polymerization of mucin 2 and von Willebrand factorinto large polymers, and are involved in the formation ofdisulfide bonds between subunits [49,50]. Therefore thecysteine-rich C-terminal domain in the clottable proteinprobably also involves intersubunit interaction.

Functional RGD-recognizing receptors have been found inthe granular hemocytes of crayfish [51] but the crayfish clottingprotein does not contain any RGD motif [16]. Although twoRGD motifs exist in the shrimp clottable protein, both are notflanked by disulfide bonds or other types of conformationalconstraint [52] and their binding to the hemocytes was notdetected. Whether the RGD motif of this clottable protein isfunctional toward other cell types is not known.

The carbohydrate content (3.8%) of the shrimp clottableprotein (Fig. 4) is lower than that reported for the crayfishclotting protein (about 20%, w/w) [53]. The crayfish protein

Fig. 8. Hydropathic profile of the clottable protein based on its amino acid sequence. The average hydrophobicities over seven residues were calculated.

Major hydrophilic regions are underlined. Stars indicate the positions of potential glycosylation sites.

632 M.-S. Yeh et al. (Eur. J. Biochem. 266) q FEBS 1999

contains six potential N-glycosylation sites [16] in contrast tofour in the shrimp protein; two of the sites are at identicalpositions in both proteins (Fig. 7). The N-glycan structureof the shrimp clottable protein belongs to the high mannosetype very often found in glycoproteins of invertebrates (Fig. 5).However, results of the monosaccharide composition analysisof the shrimp protein gave a ratio of GlcNAc/Man = 2.0 : 5.4(Fig. 4). It is likely that other side chains, e.g. GlcNAc-GlcNAc-Asn or GlcNAc-Asn which could not be cleaved byendo-b-N-acetylglucosaminidase or O-linked GlcNAc [54], arealso present. Alternatively, there could have been dispro-portionate decomposition of Man over GlcN, which is oftenobserved during acid hydrolysis of glycoproteins of lowcarbohydrate contents.

Northern analyses (Fig. 6A) revealed that the clottableproteins are expressed in most of the shrimp tissues. Similarto that found in the crayfish [16], the clottable protein is alsoexpressed in hepatopancreas of the shrimp (Fig. 6A, lane 4).The expression of the clottable protein is at its highest levels ingill and heart of the shrimp (Fig. 6A), with a lower level inhepatopancreas, lymphoid organ and muscle, but not in themature hemocytes (Fig. 6A, lane 3). In order to confirm theNorthern results, the blot is also examined with a tiger shrimpb-actin probe (Fig. 6B). In mature hemocytes, the actin genetranscript we obtained was intact and the corresponding bandon the gel was as strong as those from the other tissues(Fig. 6B). Therefore the degradation of the clottable proteinmessenger during total RNA isolation from the maturehemocytes should not occur (Fig. 6B, lane 3). Using anisotonic anti-coagulant improved since previous experiments[59], we could prepare washed and intact shrimp hemocytes,but could not detect the protein in the hemocyte lysate inimmunochemical experiments (data not shown). From thisevidence, we conclude that the mature shrimp hemocytesapparently do not express, synthesize or contain the clottableprotein. Previous studies also showed that clottable protein wasnot found in the hemocytes of other decapods, e.g. Sicyoniaingentis [55] and Penaeus japonicus [56]. This seems logicalsince active transglutaminase causing polymerization of theprotein is localized in the hemocytes [41]. However, theclottable protein is expressed in the shrimp lymphoid organ(Fig. 6A, lane 5) which is part of the hematopoietic tissuescapable of generating mature hemocytes [57,58]. Whether ornot the hemocytes express the clottable protein before theybecome mature and released is not clear.

We have previously used the rocket immunoelectrophoreticanalyses to quantitate the hemolymph clottable protein of tigershrimp [59]. The basal level of tiger shrimp clottable protein isabout 3 mg´ml21 of hemolymph in normal intermolt tigershrimp; it increased by twofold before molting and decreasedafter molting to the normal level. It could be elevatedabout 4-fold by repetitive bleeding or surgical ablation of tail oreyestalks of the shrimp [59]. Results of Northern analysisindicate that the highest levels of expression of the clottableprotein are at gill and heart of the shrimp (Fig. 6A). These areorgans playing a major role in the hemolymph circulation.Presumably these organs are sensitive to bleeding conditions,and may express clottable protein rapidly to compensate for itsloss and to help wound healing. By analogy, in vertebrate bloodthe expression of fibrinogen increases many fold under acute-phase condition.

In the cDNA of crayfish clotting protein, repeated sequencesare found in the 3 0 and 5 0 untranslated regions and aresuggested to be involved in regulating translation and/orstabilizing the mRNA [16]. Similar repeats in the 3 0 and 5 0

untranslated regions were also identified in the shrimp gene(Fig. 2). The genomic structure, especially the promoter regionof the clottable protein gene, should be investigated inmore detail to understand the transcriptional regulation ofcoagulation in crustacea.

A C K N O W L E D G M E N T S

We thank Shun-Wen Chen and Chen-Sien Chang for amino acid

sequencing, Chih-Ming Chou and The-Li Su for DNA sequencing, Mei

Tang for carbohydrate analyses, Kuan-Fu Liu and Wen-Ten Cheng for

collecting shrimp hemolymph, and Kay-Hooi Khoo for reading of the

manuscript. Materials in this paper form part of the dissertation submitted

by M.-S. Y. in partial fulfillment for the requirement of the degree of Doctor

of Science at the National Taiwan University.

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