Conceived and designed the experiments: YC SZ. Performed the experiments: YC. Analyzed the data: YC BG. Contributed reagents/materials/analysis tools: CF YL. Wrote the paper: YC SZ.
The authors have declared that no competing interests exist.
Serine protease inhibitors, or serpins, are a group of widely distributed proteins with similar structures that use conformational change to inhibit proteases. Antithrombin (AT) is a member of the serine protease inhibitor superfamily and a major coagulation inhibitor in all vertebrates, but its evolutionary origin remains elusive. In this study we isolated for the first time a cDNA encoding an antithrombin homolog,
Blood coagulation, or clotting, is of vital importance for the survival of both vertebrates and invertebrates-by preventing the leakage of blood from the sites of injury and impeding infection by the microbial invaders, although the coagulation system of invertebrates is distinct from that of vertebrates
Serine protease inhibitors, or serpins, are a group of widely distributed proteins with similar structures that use conformational change to inhibit proteases. The first members of the serpin superfamily studied extensively were the human plasma proteins antithrombin and antitrypsin
All animal experiments were carried out in accordance with the guidelines of the Laboratory Animal Administration Law of the People's Republic of China, with the permit number SD2007695 apporved by the ethics committee of the Laboratory Animal Administration of Shandong province.
Total RNAs were extracted with Trizol (Invitrogen) from
Primer | Sequence (5′-3′) | Sequence information |
S1 (sense) |
|
BjATl cDNA fragment primer |
A1 (antisense) |
|
BjATl cDNA fragment primer |
S2 (sense) |
|
3′RACE primer |
A2 (antisense) |
|
5′RACE primer |
S3 (sense) |
|
Recombinant primer |
A3 (antisense) |
|
Recombinant primer |
A4 (antisense) |
|
Recombinant primer |
The deduced amino acid sequence was analyzed with the BLAST algorithm at NCBI web site and SWISS-MODEL server at the Expert Protein Analysis System (
The tertiary structure of BjATl was predicted with a homology-modeling method via ESyPred3D using neural networks, using human AT as template
The complete coding region of
The complete coding region of BjATl cDNA was amplified by PCR with specific primers S3 and A4 (
The constructed plasmid pPICZαA/BjATl was linearized with SacI and transformed into the competent cells of
The recombinant BjATl expressed in
The activity, if any, of the recombinant BjATl expressed in
The purified recombinant BjATl expressed in
Total RNA was extracted with Trizol (Gibco) from the adult amphioxus
The sexually-matured amphioxus were cut into 3 to 4 pieces and fixed in freshly prepared 4% paraformaldehyde in 100 mM phosphate buffered saline (PBS; pH 7.4) 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 equilibrated in double distilled H2O containing 0.1% DEPC. The digoxigenin (DIG)-labeled BjATl riboprobes of about 500 bp were synthesized
A cDNA fragment of approximately 988 bp containing the domain SERPIN was obtained from
Blastp searching at NCBI revealed that BjATl had the conserved domain SERPIN at residues 1–336, and shared 38.2%, 36.7%, 38.5%, 41.1%, 39.1%, 39.6%, 39.6%, 41.7%, 38.5% and 40.8% identity to the antithrombins from fugu, salmon, zebrafish, frog, turtle, tuatara, chicken, ostrich, cow and humans, respectively (
Mature human antithrombin numbering is used. Secondary structural elements of BjATl predicted based on the structure of human antithrombin are shown above the sequences. Solid arrows indicate β-sheet, cylinders represent α-helices, triangles show the heparin-binding sites, and star indicates potential glycosylation site. Amino acid residues that are conserved in at least 50% of sequences are shaded in dark.
α-helix residues are colored with red, β-sheet residues with yellow, and loop and unassigned residues with green. Pink spheres show the heparin-binding sites, and blue spheres indicate the potential glycosylation site. Orange sticks show the RCL (reactive center loop) region.
Sequence comparison showed that BjATl contains a reactive center loop (RCL) similar to that of ATs. The RCL forms an extended and exposed conformation above the body of AT scaffold, and is responsible for the interaction with target proteases. The 20 amino acid residues constituting the RCL are numbered Pn- … -P1-P1′- … -Pn′, where P1-P1′ is ultimately cleaved
Clade C (upper panel) and Clade B (lower panel) serpin RCLs from P15-P4′ were aligned. Residues from P2, P1 and P1′ are framed as box, and the residues absolutely conserved are shaded in dark. The strictly conserved residues at P8 and P10 are shaded in grey.
Among the 16 clade serpins, BjATl shared high sequence identity with clade B and clade C serpins. The clade B serpins lack the signal peptide, are primarily intracellularly localized, and are supposedly the ancestors to the majority of extracellular serpin proteins (including ATs)
(A) The neighbor-joining (NJ) tree constructed using the package MEGA4.0; (B) The maximum likelihood (ML) tree using the program PhyML3.0; and (C) The Bayesian inference (BI) tree using MrBayes3.04b. Branches with bootstrap value <50% are cut off. Accession numbers for the sequences used are listed in
The constructed plasmid pPICZαA/BjATl was linearized with SacI and transformed into
(A) SDS-PAGE of recombinant BjATl purified on Ni-NTA resin column. Lane 1, molecular mass standards; Lane 2, recombinant BjATl. (B) Western blotting. Lane 1, the supernant of
The inhibitory activity of BjATl was quantified by comparison to a standard curve prepared with diluted normal human plasma. By definition, AT activity of diluted normal plasma is 100%. As shown in
(A) The inhibitory activity of recombinant BjATl in the presence (+) or absence (−) of heparin. (B) The inhibitory activity of different concentrations of recombinant BjATl in the presence of heparin. The inhibitory activity of BjATl was determined for each group and values were shown as means ± SD (n = 3). Significant differences (
To detect the interaction between BjATl and thrombin, BjATl was exposed to bovine thrombin. Pilot experiments showed that anti-BjATl serum reacted with BjATl, forming a single band of ∼45 kDa, whereas it was not reactive with bovine thrombin (
Purified BjATl or amphioxus humoral fluids were incubated with bovine thrombin. After SDS-PAGE (8% gels) under reducing condition, the reaction products were immunostained with anti-BjATl antiserum. (A) Lane 1, purified BjATl; Lane 2, bovine thrombin. (B) Lane 1, purified BjATl incubated with bovine thrombin; Lane 2, amphioxus humoral fluids incubated with bovine thrombin. The positions and molecular masses of marker proteins are indicated on the right.
Northern blotting revealed the presence of an approximately 2000 bp transcript in
(A) The blot was hybridized with Dig-labeled BjATl RNA probe. The arrow indicates the position of molecular size equivalent to 2000 bp. (B) A total of 5 µg RNA was analyzed in 1.2% agarose formaldehyde-denaturing gel.
(A) A low magnification section of a male amphioxus showing the presence of
Previous studies have shown the presence of AT in jawed vertebrates
Clade B serpins lack signal peptide and reside primarily within cells, most members are normally shorter (350–400 amino acides
Several clade B serpins were found to exist in both intracellular and extracellular forms
The liver is the major synthesis site of AT in vertebrates
In summary, the present study demonstrates molecularly and functionally the presence of a novel member of serpins with AT-like activity in amphioxus
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The authors thank the anonymous reviewers for their valuable suggestions.