Plg (plasminogen), a member of the serine protease superfamily, is a key component constituting the fibrinolytic system, and its evolutionary origin remains unknown during the course of animal evolution. In the present study, we isolated a cDNA, designated BbPlgl, encoding a kringle-containing protease with plasminogen-like activity from the basal chordate Branchiostoma belcheri. The deduced protein, BbPlgl, consisted of 430 amino acids, which is structurally characterized by the presence of an N-terminal signal peptide of 16 amino acids, 2 kringle domains with a Lys-binding site structure, a serine protease domain with the putative tPA (tissue plasminogen activator)-cleavage site (between Arg297 and Val298), the catalytic triad His237-Asp288-Ser379 expected for protease function, and a potential N-linked glycosylation site, all characteristic of Plgs. Besides, the recombinant refolded BbPlgl was readily activated by human uPA (urokinase plasminogen activator), and exhibited Plg-like activity. BbPlgl was also able to auto-activate at neutral and alkaline pH at 4°C without the addition of uPA, and the activation was accelerated by addition of human uPA. These results demonstrate that BbPlgl is a novel member of the Plg family, with a domain structure of K-K-SP (kringle-kringle-serine protease) lacking the PAN domain, pushing the evolutionary origin of Plg to the protochordate. In addition, BbPlgl displays a tissue-specific expression pattern in B. belcheri, with the most abundant expression in the hepatic caecum and hind-gut, agreeing with the notion that the hepatic caecum of amphioxus is the precursor of the vertebrate liver.

INTRODUCTION

A number of serine proteases play critical roles in the cascade of proteolytic reactions that leads to blood coagulation, or clotting, in vertebrates [1,2]. Blood coagulation follows the same fundamental pattern in all vertebrates, with the culminating event being the thrombin-catalysed conversion of fibrinogen into an insoluble thread-like protein, fibrin, preventing leakage of blood from the sites of injury and impeding infection by microbial invaders [36]. Fibrin clots are ultimately dissolved in due course in order to restore vascular potency [5]. The enzymes involved in this physiologically important process constitute the fibrinolytic system [7,8], and include Plg (plasminogen), PAs (plasminogen activators), PAIs (PA inhibitors) and PIs (plasmin inhibitors). The central component in the fibrinolytic system is the glycoprotein Plg, which is produced by the liver and circulates in plasma as a zymogen [9,10]. Following limited proteolysis by a PA, the pro-enzyme Plg is converted into the active enzyme plasmin. The proteases associated with clotting and fibrinolysis are often referred to as the plasminogen-prothrombin family, which also includes the signaling molecules MSP (macrophage-stimulating protein), HGF (hepatocyte growth factor) and APOA [apolipoprotein(a)] [11]. These molecules perform a surprisingly diverse array of functions, and some of which have lost their proteolytic function. The members of the plasminogen-prothrombin family are all structurally characterized by the presence of one or more “kringle” (K) domains in the N-terminal portion and a domain with serine protease homology in the C-terminal portion. The phylogenetic analysis of the protease domains of the vertebrate plasminogen-prothrombin family demonstrates the presence of two major subfamilies, a subfamily containing MSP, HGF, Plg and APOA and a subfamily containing prothrombin, HGF activator and PAs [11].

The genes encoding Plg have been identified in several vertebrate species such as humans [12], mouse [13], rat [14], European hedgehog [15] and zebrafish [16]. The primary structure of this protein from different species has been quite consistent, consisting of a pre-activation peptide known as the PAN domain (about 77 amino acid residues), five tandem kringle domains (about 80 residues each), an activation cleavage site (between Arg561 and Val562; numbering as human Plg) and a catalytic domain including the serine protease triad of His603, Asp646 and Ser741 [12,17,18]. However, the Plg gene has not been reported thus far in the non-vertebrate chordates or other invertebrate animals, although a plasminogen-like molecule has recently been observed in the primitive chordate amphioxus Branchiostoma belcheri [19]. Moreover, the origin of Plg remains obscure during the course of animal evolution. Therefore, the purposes of this study were aimed to isolate the Plg-like gene, if any, from the protochordate B. belcheri, and if so, to examine its expression pattern and to determine if it is functionally similar to vertebrate Plg.

MATERIALS AND METHODS

Cloning and sequencing of Plgl (Plg-like) cDNA

Amphioxus B. belcheri were collected from the “Amphioxus ground” near Shazikou in the vicinity of Qingdao, and cultured in filtered natural seawater until used. Total RNAs were extracted with TRIzol® (Invitrogen) from B. becheri, and polyA+ RNA was purified using polyA tract mRNA isolation system II (Promega) according to the manufacturer's instructions. The first-strand cDNA was synthesized with the reverse transcription system (Promega) using an oligo d(T) primer. The fragments of B. belcheri plasminogen-like gene, termed BbPlgl, were amplified by PCR with the degenerate primer pairs, S1 and A1 (Table 1), designed based on the conserved motifs of vertebrate plasminogens. The PCR amplification was carried out at 94°C for 4 min, followed by 31 cycles of 94°C for 45 s, 53°C for 45 s, 72°C for 90 s and a final extension step at 72°C for 7 min. The PCR products were gel-purified using a DNA gel extraction kit (AXYZEN), ligated into the T/A cloning vector pGEM-T easy (Promega) at 4°C overnight, and transformed into Top10 competent cells (TIANGEN). Positive clones were selected and sequenced with an ABI PRISM 3730 DNA sequencer. The sequences were searched in GenBank® with BLASTx for comparative analysis.

Table 1
Sequences of the primers used in this study
PrimerSequence (5′–3′)Sequence information
S1 (sense) 5′-AACTA(C/T)TG(C/T)CG(C/T)AA(C/T)CCG-3′ BbPlgl cDNA fragment primer 
A1 (antisense) 5′-C(C/T)TG(G/T)CCG(A/G)AACTGGTGAC-3′ BbPlgl cDNA fragment primer 
GSP3′ (sense) 5′-AGCACAACAAGGCTTCCACCGACTCC-3′ 3′RACE primer 
NGSP3′ (sense) 5′-ACACCAATGACATCGCCCTGCTGAAG-3′ 3′RACE nested primer 
Zht1 (antisense) 5′-GGACATCACAGTGTTCCCAGCGTAC-3′ Primer for cloning of 5′-end 
Zht2 (sense) 5′-AATGTGCGGTTCGGCTACTGTGACG-3′ Primer for cloning of 5′-end 
Zht3 (antisense) 5′-GAGACTGTGAGTCCCACCGCTGA-3′ Primer for cloning of 5′-end 
ORF-1 (sense) 5′-GGAATTCCATATGGGCCTTTTGGCCTTGTCAG-3′ Recombinant primer 
ORF-2 (antisense) 5′-CGGAATTCTTAGTTGTCAGCTGTGTACTTGAG-3′ Recombinant primer 
PrimerSequence (5′–3′)Sequence information
S1 (sense) 5′-AACTA(C/T)TG(C/T)CG(C/T)AA(C/T)CCG-3′ BbPlgl cDNA fragment primer 
A1 (antisense) 5′-C(C/T)TG(G/T)CCG(A/G)AACTGGTGAC-3′ BbPlgl cDNA fragment primer 
GSP3′ (sense) 5′-AGCACAACAAGGCTTCCACCGACTCC-3′ 3′RACE primer 
NGSP3′ (sense) 5′-ACACCAATGACATCGCCCTGCTGAAG-3′ 3′RACE nested primer 
Zht1 (antisense) 5′-GGACATCACAGTGTTCCCAGCGTAC-3′ Primer for cloning of 5′-end 
Zht2 (sense) 5′-AATGTGCGGTTCGGCTACTGTGACG-3′ Primer for cloning of 5′-end 
Zht3 (antisense) 5′-GAGACTGTGAGTCCCACCGCTGA-3′ Primer for cloning of 5′-end 
ORF-1 (sense) 5′-GGAATTCCATATGGGCCTTTTGGCCTTGTCAG-3′ Recombinant primer 
ORF-2 (antisense) 5′-CGGAATTCTTAGTTGTCAGCTGTGTACTTGAG-3′ Recombinant primer 

The gene-specific primers GSP3′and NGSP3′ (Table 1) were used in RACE (rapid amplification of cDNA ends) reactions for the cloning of 3′-end cDNA. The 3′-RACE-Ready cDNA was prepared by using the SMART™ RACE cDNA Amplification kit (Clontech) according to the manufacturer's instructions. The primary 3′-RACE reaction mixture (final volume 20 μl) contained 10×Advantage 2 PCR buffer, 0.2 mM (each) dNTPs, 10×Universal Primer A Mix (UPM, Clontech), 0.4 μM of the gene specific primer GSP3′, 50×Advantage 2 polymerase mix, and 2 μl of 3′-RACE-Ready cDNA as template. The thermal conditions for the primary 3′-RACE reaction were: initial denaturation at 94°C for 4 min, followed by an amplification program encompassing 7 cycles, each consisting of denaturation (at 94°C for 30 s), annealing (starting at 70°C for 30 s in the first cycle, followed by a temperature gradient, 1°C decrease per cycle) and extension (at 72°C for 3 min). Subsequently, 26 amplification cycles under constant cycling conditions (at 94°C for 30 s/63°C for 30 s/72°C for 3 min) were performed, followed by a final extension at 72°C for 7 min. The nested PCR amplification was performed in the reaction mixture of 20 μl containing 2 μl of 1:10 dilution of the primary PCR product as template, 1×PCR buffer, 0.5 unit of Ex Taq DNA polymerase, and 0.4 μM of each of the primers NGSP3′ and Short UPM. The PCR amplification was performed as follows: predenaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 45 s/64.5°C for 45 s/72°C for 2 min, and a final extension at 72°C for 7 min. The 3′-RACE product was gel-purified, sub-cloned, sequenced and assembled.

To obtain the the 5′-end of BbPlgl cDNA, the cDNA PCR Library Kit (Takara) was used. Total RNAs were extracted and purified as described above. The first-strand cDNA was synthesized from 5 μg of polyA+ RNA and linked with cassette adaptors following the the manuals of the M-MLV RTase cDNA Synthesis Kit (Takara) in combination with a cDNA PCR Library Kit (Takara), using the gene-specific primer Zht1 (Table 1) to replace the oligo d(T)-RA primer offered in the kit. The PCR was performed to construct the cDNA PCR Library, using the first-strand cDNA as template, in a total volume of 50 μl PCR mixture containing 1×PCR buffer, 0.5 unit of EX Taq DNA polymerase, 0.2 μM of the specific primer Zht1 (Table 1), and the CA primer (forward: 5′-CGTGGTACCATGGTCTAGAGT-3′) for the adaptor. The thermal conditions for the construction of the cDNA PCR Library were: 94°C for 1 min, 35 amplification cycles at 94°C for 30 s/60°C for 30 s/72°C for 3 min, and an additional extension at 72°C for 5 min. A pair of gene-specific primers (Zht1 and Zht2; Table 1) were used to ascertain the existence of the BbPlgl cDNA in the cDNA PCR library. Subsequently, the primers Zht3 and CA primer were applied to amplify the 5′-end of BbPlgl cDNA using the cDNA PCR library as the template. PCR amplification was carried out using the following parameters: denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 45 s/59°C for 45 s/72°C for 2 min, and then a final extension at 72°C for 7 min. The DNA product was gel-purified, sub-cloned and sequenced with ABI PRISM 3730 DNA sequencer. The sequences were searched in GenBank® with BLASTx for comparative analysis and assembled with the obtained fragments.

Sequence analysis

The deduced amino acid sequence was analysed with the Expert Protein Analysis System (http://www.expasy.org/) and SMART program (http://smart.embl-heidelberg.de/). The signal peptide was predicted with SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/), and the potential sites of Asn-linked glycosylation were predicted with the NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/). Multiple alignments were performed using Clustal_X (1.81), and pairwise alignments performed using the MegAlign program (DNAstar) by the CLUSTAL W method. The phylogenetic tree was constructed by the neighbour-joining method within the PHYLIP 3.67 software package using 1000 bootstrap replicates [20].

Expression of BbPlgl in Escherichia coli

The complete coding region of BbPlgl with the predicted signal peptide deleted was amplified by PCR using the upstream primer ORF-1 and the downstream primer ORF-2 (Table 1). The reaction was carried out under the following conditions: initial denaturation at 94°C for 4 min, followed by 30 cycles of denaturation at 94°C for 45 s, annealing for 30 s at 45°C, and extension at 72°C for 2 min, and an additional extension at 72°C for 7 min. The PCR product was digested with NdeI and EcoRI, and sub-cloned into the plasmid expression vector pET28a (Novagen), previously cut with the same restriction enzymes. The identity of the insert was verified by sequencing, and the plasmid was designated pET28a/BbPlgl.

The cells of E. coli BL21 were transformed with plasmid pET28a/BbPlgl and cultured overnight in LB (Luria–Bertani) broth containing kanamycin (30 μg/ml). The culture was diluted 1:100 with LB broth and subjected to further incubation at 37°C until D600 reached 1.0. The expression of BbPlgl was induced by addition of IPTG (isopropyl β-D-thiogalactoside) to the culture at a final concentration of 0.1 mM. After further incubation at 37°C for 4 h, the bacterial cells were harvested by centrifugation at 5000 g and 4°C for 10 min, resuspended in 50 ml buffer A (pH 8.0) consisting of 50 mM Tris/HCl, 100 mM NaCl, 1 mM EDTA and 10 mM DTT (dithiothreitol), and sonicated on ice. The inclusion bodies were sedimented by centrifugation at 15000 g and 4°C for 20 min. A small amount of the inclusion bodies was sampled, suspended in SDS sample buffer, boiled and loaded onto an SDS/12% PAGE gel. The total cellular extracts of E. coli BL21 containing pET28a/BbPlgl before IPTG induction was also run on the gel in parallel.

To remove additional lipids and cellular proteins, the inclusion bodies were further resuspended in 100 ml of buffer B (pH 8.0) consisting of 50 mM Tris/HCl, 100 mM NaCl, 1 mM EDTA, 1 mM DTT and 0.5% Triton X-100, stirred at 800 rev./min for 1 h followed by centrifugation at 15000 g and 4°C for 20 min. The pellets were washed three times with buffer B and then washed three times with 50 mM Tris/HCl, 1 mM EDTA and 1 mM DTT.

Protein purification and refolding

Following the wash, the pellets were solubilized in 40 ml of 100 mM Tris/HCl, pH 8.0, containing 8 M urea, 1 mM glycine, 1 mM EDTA, 100 mM 2-ME (2-mercaptoethanol), 10 mM DTT, 1 mM GSH and 0.1 mM GSSG, and incubated at 4°C for 24 h. The supernatants were pooled by centrifugation at 15000 g and 4°C for 20 min, and loaded onto a Ni-NTA (Ni2+-nitrilotriacetate) resin column (GE Healthcare). The column was washed with 100 mM Tris/HCl, pH 7.4, containing 20 mM imidazole followed by 100 mM Tris/HCl, pH 7.4, containing 40 mM imidazole and then eluted with 100 mM Tris/HCl, pH 7.4, containing 250 mM imidazole. The purity of the eluted samples was analysed by SDS/12% PAGE, and stained with Coomassie Brilliant Blue R-250.

The purified protein was dialysed against buffer C consisting of 100 mM Tris/HCl, pH 10.5, containing 8 M urea, 1 mM glycine, 1 mM EDTA, 100 mM 2-ME, 10 mM DTT, 1 mM GSH and 0.1 mM GSSG overnight at 4°C, adjusted with buffer C to a final protein concentration of approx. 0.1 mg/ml, and dialysed successively against the refolding buffers, 20 mM Tris/HCl, pH 10, 0.2 M arginine, 0.5 mM 2-ME, 0.08 mM GSSG and 0.8 mM GSH containing 6 M, 4 M, 2 M and finally 1 M urea. Each dialysis was performed at 18°C for 4 h against 1 litre of the dialysis buffer. Subsequently, the protein was dialysed against 20 mM Tris/HCl, pH 10, containing 0.4 M urea, 0.2 M arginine, 0.5 mM 2-ME, 0.08 mM GSSG and 0.8 mM GSH at 4°C for 48 h. The refolded protein was pooled by centrifugation at 15000 g and 4°C for 20 min and loaded on to a SDS/12% PAGE gel.

Protein concentrations were determined by the method of Bradford using BSA as a standard [21].

Western blotting

The purified protein and the extracts of E. coli BL21 containing pET28a/BbPlgl before IPTG induction were mixed with SDS sample buffer, boiled for 5 min and run on a SDS/12% PAGE gel with a 5% spacer gel. After electrophoresis, the gel was washed for 5 min in the transfer buffer of 15.6 mM Tris/HCl, pH 8.3, containing 120 mM glycine and 20% methanol, and proteins on the gels were blotted on nitrocellulose membrane (Hybond, Amersham Pharmacia). The blotted membranes were incubated in 20 mM PBS, pH 7.4, containing 5% non-fat dried skimmed milk powder at room temperature (25°C) for 1 h, washed three times with 20 mM PBS, and then incubated with mouse anti-human Anti-His Antibody (TIANGEN) diluted 1:1500 with 20 mM PBS and 5% non-fat skimmed milk powder at room temperature for 1.5 h. After washing in 20 mM PBS, the membranes were incubated with goat anti-mouse IgG-HRP (horseradish peroxidase) (TIANGEN) diluted 1:400 with PBS at room temperature for 45 min. Bands were visualized using 0.06% DAB (4-dimethylaminobenzene) in 50 mM Tris/HCl buffer, pH 7.6, and 0.03% H2O2.

The molecular mass standards used were E. coli β-galactosidase (116.0 kDa), bovine serum albumin (66.2 kDa), chicken egg white ovalbumin (45 kDa), porcine muscle lactate dehydrogenase (35 kDa), E. coli Rease Bsp981 (25 kDa), bovine milk β-lactoglobulin (18.4 kDa) and chicken egg white lysozyme (14.4 kDa).

Plasminogen-like activity assays

The refolded protein was dialysed against buffer D, consisting of 50 mM Tris/HCl, pH 8.0 with 50 mM NaCl and 0.01% Tween-80, and then concentrated by PEG-mediated reverse osmosis. The protein solution obtained was approx. 300 μg/ml as determined by the method of Bradford [21]. The recombinant BbPlgl activity was detected by a chromogenic assay using plasminogen chromogenic activity kit (Beijing Bio-lab Materials Institute). The human uPA (urokinase PA) and chromogenic substrate S-2251 were both dissolved in buffer D, with final concentrations of 2000 units/ml and 3 mM respectively. The plasminogen activities were measured, in triplicate, by the chromogenic assays described by Fajardo-Lira and Nielsen [22]. Briefly, 50 μl of buffer D containing 100 units of human uPA was mixed with 200 μl of different concentrations of BbPlgl solutions (20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml and 100 μg/ml), following pre-incubation at 37°C in a water bath for 20 min, 50 μl of 3 mM S-2251 solution was added to the reaction mixtures and incubated at 37°C for 4 h. For the control, 200 μl buffer D was mixed with the same volumes of human uPA and S-2251 solutions, and processed similarly. After incubation, the reaction mixtures were centrifuged at 15000 g for 5 min, and aliquots of 100 μl were pipetted into a 96-well plate. Absorbance was measured at 405 nm and 490 nm (subtracted to correct for added absorbance due to turbidity) using a microplate reader (TECAN, GENios Plus). One unit of the refolded BbPlgl activity was defined as 1 μmol of the product released per min.

For the determination of auto-activation of the refolded BbPlgl, 200 μl of the refolded protein solutions (100 μg/ml) was mixed with 50 μl buffer D with or without 100 units of human uPA added; after pre-incubation at 37°C in a water bath for 20 min, 50 μl of 3 mM S-2251 solution was added to the reaction mixtures and incubated at 37°C for 4 h. The control was prepared similarly except that the refolded protein was withdrawn. After incubation, the reaction mixtures were processed and measured as above.

The cleavage of human uPA on the refolded BbPlgl and auto-activation of the refolded protein were further ascertained by SDS/PAGE. A total of 200 μl of buffer D containing different quantities of human uPA (0, 200, 300 or 400 units) were mixed with 100 μl of the refolded protein solutions (300 μg/ml) and incubated at 37°C in a water bath for 4 h. Similarly, 200 μl of buffer D containing 0 units and 400 units of human uPA were each mixed with 100 μl of the unfolded protein solutions (100 μg/ml). After incubation, aliquots of 50 μl of the reaction mixtures were each sampled, mixed with an equal volume of 2× sample buffer containing 2-ME, boiled and electrophoresed on a SDS/12% PAGE gel. For the auto-activation assay, the refolded protein solution (100 μg/ml), stored at 4°C for 2 weeks, was also electrophoresed under the same conditions [23].

Northern blotting and in situ hybridization histochemistry

Total RNA was extracted with TRIzol® (Gibco) from the adult amphioxus B. belcheri ground in liquid nitrogen. Aliquot containing 5 μg of each of the RNAs were each electrophoresed and blotted onto a Nylon membrane (Roche). The DIG (digoxigenin)-labelled BbPlgl riboprobes of approx. 1000 bp were synthesized in vitro from linearized plasmid DNA following the DIG-UTP supplier's instructions (Roche). Northern blot analysis was carried out as described previously [24].

Sexually-matured amphioxus were cut into 3–4 pieces and fixed in freshly prepared 4% paraformaldehyde in 100 mM PBS at 4°C for 8 h. The samples were dehydrated, embedded in paraffin, and sectioned at 6 μm. The sections were mounted onto poly-L-lysine coated slides, dried at 42°C for 36 h and de-paraffinized in xylene for 20 min (two changes for 10 min each), followed by immersion in absolute ethanol for 10 min (two changes for 5 min each). They were re-hydrated, and finally equilibrated in double distilled H2O containing 0.1% DEPC (diethyl pyrocarbonate). The DIG-labelled BbPlgl riboprobes of approx. 1000 bp were synthesized in vitro from linearized plasmid DNA following the DIG-UTP supplier's instructions (Roche). In situ hybridization histochemistry was carried out as described by Fan et al. [24].

RESULTS

Sequence and phylogeny of BbPlgl

A cDNA fragment of approx. 800 bp was obtained by RT–PCR (reverse transcription–PCR) using the primers S1 and A1. Based on this partial sequence, a 1000 bp fragment was cloned using 3′ RACE. Through the construction of the cDNA PCR library, a 5′ fragment of approx. 450 bp was obtained. The full-length cDNA of BbPlgl was assembled from the overlapped cDNA fragments, and deposited in GenBank® (accession number: EU734810). It was 1949 bp long with an ORF (open reading frame) of 1290 bp, a 5′-UTR (untranslated region) of 123 bp and a 3′-UTR of 536 bp. The initiation codon (ATG) was in accordance with the Kozak consensus sequence (A/GXXATGG), and the 3′-UTR had a polyadenylation signal AATAAA. The ORF of BbPlgl encoded a polypeptide of 430 amino acids with a calculated molecular mass of approx. 46.7 kDa (Figure 1), although this estimate does not take into consideration putative glycosylations. SignalP software analysis by the Signal IP 3.0 server [25] revealed that BbPlgl had a putative N-terminal signal peptide of 16 amino acids. There was also a potential N-linked glycosylation site in BbPlgl located at the residual position 32.

Nucleotide sequence of BbPlgl and its deduced amino acid sequence

Figure 1
Nucleotide sequence of BbPlgl and its deduced amino acid sequence

The in-frame stop codon in 5′UTR is marked by ▲. The start codon is underlined by a single line. The deduced signal peptide is doubly under-lined. The asterisk represents the stop codon. The polyadenylation signal is boxed.

Figure 1
Nucleotide sequence of BbPlgl and its deduced amino acid sequence

The in-frame stop codon in 5′UTR is marked by ▲. The start codon is underlined by a single line. The deduced signal peptide is doubly under-lined. The asterisk represents the stop codon. The polyadenylation signal is boxed.

As other Plgs, BbPlgl was similarly structurally characterized by the presence of kringle domains in the N-terminus and a serine protease domain in the C-terminus. Peculiarly, BbPlgl had two kringle domains instead of five domains, and lacked the PAN domain. Therefore, BbPlgl has a novel domain structure of K-K-SP (kringle-kringle-serine protease). A kringle domain is typically about 80 amino acids long and is characterized by a secondary structure formed by three disulfide bridges within the domain [26]. Both the kringle domains K1 and K2 in BbPlgl comprised 79 amino acids, sharing an identity of 51.9% to each other. Moreover, the K1 and K2 in BbPlgl were both closely identical to K1 (43.0–58.2% and 41.8–51.9%), K2 (41.8–51.9% and 50.6–60.9%), K3 (49.4–57.0% and 51.9–63.3%), K4 (50.6–55.7% and 46.8–55.7%) and K5 (50.6–57.0% and 44.3–50.6%) of known Plgs. Each kringle domain in BbPlgl had six cysteine residues, suggesting that they folded as expected in Plgs. In addition, the kringle LBS (lysine-binding site) structure consisting of cationic, anionic and lipophilic areas that match the electrostatic, hydrophobic and steric requirements of lysyl-type ligands [27,28], was also conserved in BbPlgl, although its K1 had only a cationic charge residue. The serine protease region of BbPlgl contained the putative tPA (tissue PA)-cleavage site (between Arg297 and Val298), which is in a position analogous to the tPA-cleavage site in human Plg. In addition, the catalytic triad His237-Asp288-Ser379 expected for protease function was also present in positions which correspond to human Plg.

A search of the recently completed draft assembly and automated annotation of B. floridae genome revealed the presence of a Florida amphioxus cDNA with the same domain structure of K-K-SP and its genomic DNA sequence (jgi|Brafl1|123738|estExt_fgenesh2_pg.C_1270052: http://genome.jgi-psf.org/cgi-bin/dispTranscript?db=Brafl1&id=123738&useCoords=1). Sequence comparison demonstrated that BbPlgl shared 92.3% identity to the deduced protein encoded by the Florida amphioxus gene at the amino acid level, implying that BbPlgl is highly conserved in intra-species (results not shown).

The kringle domains were divided into three groups indicated by I, II and III, with the I and II groups closely resembling the ancestral form [29,30]. The phylogenetic trees constructed using the amino acid sequences of the kringle domains of BbPlgl as well as their counterparts of other Plgs and plasminogen-prothrombin family members [11,31] revealed that K1 and K2 of BbPlgl were clustered together with I and II groups respectively (Figure 2A). The phylogenetic analysis of protease domains of BbPlgl, plasminogen-prothrombin family members and Plgl proteins with a structure of TSP-1-K-K-SP (where TSP-1 is thrombospondin type 1 domain) in Ciona intestinales demonstrated that both BbPlgl and Plgl in C. intestinales were located at the base of the subfamily containing Plg, HGF, MSP and APOA (Figure 2B). The above results suggest that BbPlgl may represent the archetype of the subfamily containing Plg, HGF, MSP and APOA.

Phylogenetic trees of kringle domains and serine protease regions

Figure 2
Phylogenetic trees of kringle domains and serine protease regions

The phylogenetic trees of both kringle domains (A) and serine protease regions (B) of BbPlgl as well as their counterparts of other Plgs and plasminogen-prothrombin family members constructed by the neighbour-joining method within the package PHYLIP 3.5c. Bootstrap majority consensus values on 1000 replicates are indicated at each branch point in percent. Accession numbers for sequences used are listed in Table 2.

Figure 2
Phylogenetic trees of kringle domains and serine protease regions

The phylogenetic trees of both kringle domains (A) and serine protease regions (B) of BbPlgl as well as their counterparts of other Plgs and plasminogen-prothrombin family members constructed by the neighbour-joining method within the package PHYLIP 3.5c. Bootstrap majority consensus values on 1000 replicates are indicated at each branch point in percent. Accession numbers for sequences used are listed in Table 2.

Table 2
The names and accession numbers of the plasminogen-prothrombin family

See Figure 2 for the phylogenetic trees. MST, macrophage stimulating; PT, prothrombin.

SpeciesNameAccession numbers
Homo sapiens Human serine protease 3 BC069494 
Homo sapiens Human Plg AAA36451 
B. belcheri BbPlgl EU734810 
Macaca mulatta Rhesus monkey Plg NP_001036540 
Mus musculus Mouse Plg AAA50168 
Rattus norvegicus Rat Plg NP_445943 
Bos taurus Cow Plg NP_776376 
Canis familiaris Dog Plg AAT44581 
Macropus eugenii Wallaby Plg AAB65760 
Erinaceus europaeus Hedgehog Plg AAC48717 
Gallus gallus Red jungle fowl Plg XP_419618 
Oryzias latipes Japanese medaka Plg NP_001098315 
Oncorhynchus mykiss Rainbow trout Plg CAD69012 
Danio rerio Zebrafish Plg NP_958880 
Homo sapiens Human APOA X06290 
Macaca mulatta Rhesus monkey APOA J04635 
Homo sapiens Human HGF BAA14348 
Macaca mulatta Rhesus monkey HGF XM_001108226 
Mus musculus Mouse HGF NM_010427 
Rattus norvegicus Rat HGF NM_017017 
Gallus gallus Chicken HGF X84045 
Xenopus laevis Frog HGF S77422 
Danio rerio Zebrafish HGF1 BC135005 
Danio rerio Zebrafish HGF2 XM_001921459 
Homo sapiens Human MST1 NM_020998 
Mus musculus Mouse MST1 NM_008243 
Rattus norvegicus Rat MSP X95096 
Gallus gallus Chicken MST1 NM_205213 
Xenopus laevis Frog MSPA BC044008 
Xenopus laevis Frog MSPB XLU57455 
Homo sapiens Human PT P00734 
Macaca mulatta Rhesus monkey PT EF057490 
Mus musculus Mouse PT X52308 
Rattus norvegicus Rat PT X52835 
Bos taurus Cow PT J00041 
Struthio camelus Ostrich PT BAA89046 
Xenopus (Silurana) tropicalis Frog PT BC089747 
Eptatretus stoutii Hagfish PT M81393 
Takifugu rubripes Fugu PT NP_001027864 
Oncorhynchus mykiss Rainbow trout PT NP_001117856 
Homo sapiens Human tPA A03776 
Mus musculus Mouse tPA J03520 
Rattus norvegicus Rat tPA NM_013151 
Bos taurus Cow tPA X85800 
Xenopus laevis Frog tPA BC061654 
Oryzias latipes Japanese medaka tPA NM_001104831 
Homo sapiens Human uPA X02419 
Mus musculus Mouse uPA NM_008873 
Rattus norvegicus Rat uPA X63434 
Bos taurus Cow uPA L03546 
Homo sapiens Human Factor XII NM_000505 
C. intestinales Sea squirt Plgl XP_002128167 
SpeciesNameAccession numbers
Homo sapiens Human serine protease 3 BC069494 
Homo sapiens Human Plg AAA36451 
B. belcheri BbPlgl EU734810 
Macaca mulatta Rhesus monkey Plg NP_001036540 
Mus musculus Mouse Plg AAA50168 
Rattus norvegicus Rat Plg NP_445943 
Bos taurus Cow Plg NP_776376 
Canis familiaris Dog Plg AAT44581 
Macropus eugenii Wallaby Plg AAB65760 
Erinaceus europaeus Hedgehog Plg AAC48717 
Gallus gallus Red jungle fowl Plg XP_419618 
Oryzias latipes Japanese medaka Plg NP_001098315 
Oncorhynchus mykiss Rainbow trout Plg CAD69012 
Danio rerio Zebrafish Plg NP_958880 
Homo sapiens Human APOA X06290 
Macaca mulatta Rhesus monkey APOA J04635 
Homo sapiens Human HGF BAA14348 
Macaca mulatta Rhesus monkey HGF XM_001108226 
Mus musculus Mouse HGF NM_010427 
Rattus norvegicus Rat HGF NM_017017 
Gallus gallus Chicken HGF X84045 
Xenopus laevis Frog HGF S77422 
Danio rerio Zebrafish HGF1 BC135005 
Danio rerio Zebrafish HGF2 XM_001921459 
Homo sapiens Human MST1 NM_020998 
Mus musculus Mouse MST1 NM_008243 
Rattus norvegicus Rat MSP X95096 
Gallus gallus Chicken MST1 NM_205213 
Xenopus laevis Frog MSPA BC044008 
Xenopus laevis Frog MSPB XLU57455 
Homo sapiens Human PT P00734 
Macaca mulatta Rhesus monkey PT EF057490 
Mus musculus Mouse PT X52308 
Rattus norvegicus Rat PT X52835 
Bos taurus Cow PT J00041 
Struthio camelus Ostrich PT BAA89046 
Xenopus (Silurana) tropicalis Frog PT BC089747 
Eptatretus stoutii Hagfish PT M81393 
Takifugu rubripes Fugu PT NP_001027864 
Oncorhynchus mykiss Rainbow trout PT NP_001117856 
Homo sapiens Human tPA A03776 
Mus musculus Mouse tPA J03520 
Rattus norvegicus Rat tPA NM_013151 
Bos taurus Cow tPA X85800 
Xenopus laevis Frog tPA BC061654 
Oryzias latipes Japanese medaka tPA NM_001104831 
Homo sapiens Human uPA X02419 
Mus musculus Mouse uPA NM_008873 
Rattus norvegicus Rat uPA X63434 
Bos taurus Cow uPA L03546 
Homo sapiens Human Factor XII NM_000505 
C. intestinales Sea squirt Plgl XP_002128167 

Plgl activity of recombinant BbPlgl

An expression vector including the entire ORF of BbPlgl and a 5′ additional tag of pET28a was constructed and transformed into E. coli, which resulted in the original N-terminal Met in the recombinant protein being replaced by Met-Gly-Ser-Ser-(His)6-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met. The recombinant protein BbPlgl was purified by affinity chromatography on a Ni-NTA resin column. The purified recombinant BbPlgl with the His6 tag yielded a single band of approx. 48 kDa on SDS/PAGE after Coomassie Blue staining (Figure 3A). Western blotting showed that the mouse anti-human His antibody (diluted 1:1500) reacted with the inclusion bodies of IPTG-induced E. coli BL21 with expression vector, forming a band of approx. 48 kDa on SDS/PAGE, corresponding to the molecular mass predicted for BbPlgl cDNA. In contrast, it was not reactive to the supernatant of the cell lysate of E. coli BL21 containing expression vector before induction by IPTG. This indicated that BbPlgl was correctly expressed (Figure 3B).

SDS/PAGE and Western blotting of the recombinant BbPlgl in E. coli

Figure 3
SDS/PAGE and Western blotting of the recombinant BbPlgl in E. coli

(A) SDS/PAGE. Lane M, molecular mass standards (values in kDa to the left); lane 1, inclsion bodies from IPTG-induced E. coli BL21 containing pET28a/BbPlgl; lane 2, total cellular extracts from E. coli BL21 containing pET28a/BbPlgl before induction; lane 3, diluted BbPlgl inclusion bodies; lane 4, recombinant BbPlgl inclusion bodies purified on Ni-NTA resin column. (B) Western blot analysis. Lane 1, total cellular extracts from E. coli BL21 containing pET28a/BbPlgl before induction; lane 2, recombinant BbPlgl inclusion bodies purified on Ni-NTA resin column.

Figure 3
SDS/PAGE and Western blotting of the recombinant BbPlgl in E. coli

(A) SDS/PAGE. Lane M, molecular mass standards (values in kDa to the left); lane 1, inclsion bodies from IPTG-induced E. coli BL21 containing pET28a/BbPlgl; lane 2, total cellular extracts from E. coli BL21 containing pET28a/BbPlgl before induction; lane 3, diluted BbPlgl inclusion bodies; lane 4, recombinant BbPlgl inclusion bodies purified on Ni-NTA resin column. (B) Western blot analysis. Lane 1, total cellular extracts from E. coli BL21 containing pET28a/BbPlgl before induction; lane 2, recombinant BbPlgl inclusion bodies purified on Ni-NTA resin column.

The refolded BbPlgl was readily activated by human uPA and exhibited an enzymatic activity of 8.7 m-units/mg of protein. Besides, the catalytic activity was enhanced when the amount of the recombinant protein was increased although both human uPA and chromogenic substrate were in fixed quantities, showing that BbPlgl acts in a concentration-dependent manner (Figure 4). Moreover, the refolded BbPlgl was able to auto-activate at neutral and alkaline pH at 4°C without the addition of uPA, displaying an enzymatic activity of 6.5 m-units/mg of protein toward the chromogenic substrate (P<0.01) (Figure 5). However, when human uPA was added, the activation was accelerated (P<0.05) (Figure 5), indicating that human uPA can interact with BbPlgl, resulting in its activation.

The Plgl activity of recombinant BbPlgl

Figure 4
The Plgl activity of recombinant BbPlgl

The refolded BbPlgl was readily activated by human uPA and the catalytic activity was enhanced when the amount of the refolded protein was increased, showing a concentration-dependent activity. The values of Plgl activity of BbPlgl are shown as means±S.D. (n=3). Significant differences (P<0.05) are indicated by an asterisk (*) as compared with control.

Figure 4
The Plgl activity of recombinant BbPlgl

The refolded BbPlgl was readily activated by human uPA and the catalytic activity was enhanced when the amount of the refolded protein was increased, showing a concentration-dependent activity. The values of Plgl activity of BbPlgl are shown as means±S.D. (n=3). Significant differences (P<0.05) are indicated by an asterisk (*) as compared with control.

The Plgl activity of refolded BbPlgl in the presence or absence of human uPA

Figure 5
The Plgl activity of refolded BbPlgl in the presence or absence of human uPA

The refolded BbPlgl was able to display enzymatic activity toward the chromogenic substrate without human uPA added. In the presence of human uPA, the activation was accelerated. The values are shown as means±S.D. (n=3). Significant difference (P<0.05) is indicated by an asterisk (*) as compared with the enzymatic activity in the absence of human uPA, and dramatically significant difference (P<0.01) by two asterisks (**) as compared with control.

Figure 5
The Plgl activity of refolded BbPlgl in the presence or absence of human uPA

The refolded BbPlgl was able to display enzymatic activity toward the chromogenic substrate without human uPA added. In the presence of human uPA, the activation was accelerated. The values are shown as means±S.D. (n=3). Significant difference (P<0.05) is indicated by an asterisk (*) as compared with the enzymatic activity in the absence of human uPA, and dramatically significant difference (P<0.01) by two asterisks (**) as compared with control.

The human uPA-catalysed cleavage of refolded BbPlgl was also monitored by SDS/PAGE analysis (Figure 6). The refolded BbPlgl was apparently cleaved in the presence of human uPA, yielding a cleavage product with a molecular mass of approx. 35 kDa. In contrast, non-refolded BbPlgl was not activated in the absence or presence of human uPA.

SDS/PAGE analysis of human uPA-catalysed cleavage of refolded BbPlgl

Figure 6
SDS/PAGE analysis of human uPA-catalysed cleavage of refolded BbPlgl

Lanes 1–3, refolded BbPlgl with 400, 300 or 200 units of human uPA respectively; lane 4, refolded BbPlgl without human uPA; lane M, molecular mass standards of 116.0 kDa, 66.2 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa and 14.4 kDa; lane 5, refolded BbPlgl stored at alkaline pH with no human uPA added; lane 6, refolded BbPlgl stored at alkaline pH with 400 units of human uPA added; lane 7, non-refolded BbPlgl stored at the same alkaline pH with no human uPA added; lane 8, non-refolded BbPlgl stored at the same alkaline pH with 400 units of human uPA added.

Figure 6
SDS/PAGE analysis of human uPA-catalysed cleavage of refolded BbPlgl

Lanes 1–3, refolded BbPlgl with 400, 300 or 200 units of human uPA respectively; lane 4, refolded BbPlgl without human uPA; lane M, molecular mass standards of 116.0 kDa, 66.2 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa and 14.4 kDa; lane 5, refolded BbPlgl stored at alkaline pH with no human uPA added; lane 6, refolded BbPlgl stored at alkaline pH with 400 units of human uPA added; lane 7, non-refolded BbPlgl stored at the same alkaline pH with no human uPA added; lane 8, non-refolded BbPlgl stored at the same alkaline pH with 400 units of human uPA added.

Tissue-specific expression of BbPlgl in adult amphioxus

Northern blotting revealed the presence of an approx. 2000 bp transcript in B. belcheri (Figure 7). In situ hybridization histochemistry demonstrated that BbPlgl transcript was most abundant in the hepatic caecum and hind-gut, and present in the gill and ovary at lower levels, although it was absent in the muscle, neural tube, notochord and testis (Figure 8), implicating a tissue-specific expression pattern of BbPlgl in adult B. belcheri.

Northern blotting

Figure 7
Northern blotting

(A) The blot was hybridized with the DIG-labelled BbPlgl RNA probe. The arrow indicates the position of the molecular size equivalent to 2000 bp. (B) A total of 5 μg RNA was analysed in 1.2% agarose formaldehyde-denaturing gel.

Figure 7
Northern blotting

(A) The blot was hybridized with the DIG-labelled BbPlgl RNA probe. The arrow indicates the position of the molecular size equivalent to 2000 bp. (B) A total of 5 μg RNA was analysed in 1.2% agarose formaldehyde-denaturing gel.

Localization of BbPlgl transcripts in the different tissues of adult amphioxus

Figure 8
Localization of BbPlgl transcripts in the different tissues of adult amphioxus

(A and C) Micrographs showing the presence of BbPlgl in the hepatic caecum and hind-gut. (B) Micrograph showing the presence of BbPlgl transcripts in a female amphioxus. (D) A control section. hc, hepatic caecum; hg, hind-gut; g, gill; o, ovary; t, testis; m, muscle; nt, neural tube; nc, notochord. Scale bars represent 100 μm.

Figure 8
Localization of BbPlgl transcripts in the different tissues of adult amphioxus

(A and C) Micrographs showing the presence of BbPlgl in the hepatic caecum and hind-gut. (B) Micrograph showing the presence of BbPlgl transcripts in a female amphioxus. (D) A control section. hc, hepatic caecum; hg, hind-gut; g, gill; o, ovary; t, testis; m, muscle; nt, neural tube; nc, notochord. Scale bars represent 100 μm.

DISCUSSION

Previous studies have shown the presence of Plg only in the jawed vertebrates [3,32], and a Plgl molecule has been discovered in the amphioxus B. belcheri, a basal chordate [19]. However, molecular cloning and identification of the putative Plgl in B. belcheri was lacking. In the present paper we demonstrate for the first time the presence of a kringle domain-containing protease with Plgl activity, named BbPlgl, in B. belcheri. The deduced 430-amino-acids long protein, BbPlgl, is structurally characterized by the presence of a putative N-terminal signal peptide of 16 amino acids, 2 kringle domains with the lysine-binding site structure in the N-terminus, a serine protease domain with the putative tPA-cleavage site (between Arg297 and Val298) in the C-terminus, the catalytic triad His237-Asp288-Ser379 expected for protease function, and a potential N-linked glycosylation site, which are all typical of Plgs. Moreover, the recombinant BbPlgl is readily activated by human uPA, and exhibits Plgl activity. Similar to mammalian Plgs [23], BbPlgl is also able to auto-activate at neutral and alkaline pH at 4°C without the addition of human uPA, and the activation is remarkably accelerated by addition of human uPA. Therefore, both the sequence and functional data clearly indicate that BbPlgl is a novel member of the Plg gene family, with a domain structure of K-K-SP lacking the PAN domain, pushing the evolutionary origin of Plg to the protochordate. Phylogenetic analysis shows that K1 and K2 of BbPlgl are grouped with I and II domains, respectively, and the serine protease region of BbPlgl is positioned at the base of the subfamily containing Plg, HGF, MSP and APOA, suggesting that BbPlgl may represent the archetype of this subfamily member. It is highly likely that the kringle in this ancestral kringle-containing protease gene with one kringle and one serine protease region was duplicated into two domains, one similar to group I and the other similar to group II, approx. 500 million years ago. The presence of the kringle-containing protease in B. belcherio may provide additional support to the model of a step-by-step parallel evolution, originally suggested by Jiang and Doolittle [32a], of the plasminogen-prothrombin family, that is, the members of plasminogen-prothrombin family currently present in mammals, avians and amphibians may evolve through intragenic duplications of the kringle domain, amino acid replacements of the protease domain, gene duplications, exon shuffling and deletions [11,33]. It is of note that BbPlgl has a calculated molecular mass of approx. 46.7 kDa, which is about half the size of that estimated from Western blotting analysis [19]. Although this difference may be due to the fact that the molecular mass calculated from BbPlgl cDNA has ignored the putative glycosylations, it cannot be ruled out that BbPlgl and the molecule reported by Liang and Zhang [19] are two different molecules.

The liver is the major synthesis site of Plgs in vertebrates [3439]. Amphioxus has a hepatic caecum, the pouch that protrudes forward as an outpocketing of the digestive tube and extends along the right side of the posterior part of the pharynx, which has long been considered to be the precursor of vertebrate liver [4042]. Our study reveals that BbPlgl displays a tissue-specific expression pattern in B. belcheri, with the most abundant expression in the hepatic caecum and hind-gut, confirming the observation of Liang and Zhang [19]. Generally speaking, this agrees with the hypothesis that the hepatic caecum of amphioxus is the precursor of the vertebrate liver.

In summary, the present study highlights the presence of a novel kringle-containing protease with Plgl activity, which has a domain structure of K-K-SP lacking the PAN domain, in the basal chordate B. belcheri, pushing the evolutionary origin of Plg to the non-vertebrate chordate. It also bolsters the notion that the hepatic caecum of amphioxus is equivalent to the vertebrate liver.

Abbreviations

     
  • APOA

    apolipoprotein(a)

  •  
  • BbPlgl

    Branchiostoma belcheri plasminogen-like

  •  
  • DIG

    digoxigenin

  •  
  • DTT

    dithiothreitol

  •  
  • HGF

    hepatocyte growth factor

  •  
  • IPTG

    isopropyl β-D-thiogalactoside

  •  
  • K-K-SP

    kringle-kringle-serine protease

  •  
  • LB broth

    Luria–Bertani broth

  •  
  • 2-ME

    2-mercaptoethanol

  •  
  • MSP

    macrophage-stimulating protein

  •  
  • Ni-NTA

    Ni2+-nitrilotriacetate

  •  
  • ORF

    open reading frame

  •  
  • PA

    plasminogen activator

  •  
  • Plg

    plasminogen

  •  
  • Plgl

    Plg-like

  •  
  • RACE

    rapid amplification of cDNA ends

  •  
  • tPA

    tissue PA

  •  
  • uPA

    urokinase PA

  •  
  • UTR

    untranslated region

FUNDING

This work was supported by grants [grant numbers 2006CB101805 and 30730072] from the Ministry of Science and Technology (MOST) and the Natural Science Foundation of China (NSFC).

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Author notes

The nucleotide sequence data reported for Branchiostoma belcheri plasminogen-like (BbPlgl) will appear in GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases under the accession number EU734810.