Study On Biochemistry And Molecular Biology Of A Novel Fibrinolytic Protease From The Polychaete Nereis Virens

Thrombotic disease (TD) is a lethal medical complication with high incidence, especially when happen in brain and heart. Thromboembolism is the lending cause of human death in western affluent countries, which mainly damages the blood systems of heart, brainand lung, resulting in human death and disability in adult population. Thrombolytic agents have been extensively used as the therapeutic treatment of thromboembolism complication. At present, there are all three generations of thrombolytic agent, but more or less side effects are present in each of them.Many organisms are important sources of thrombolytic agents. The resource of marine biology is a resourceful warehouse of the novel medicinal exploitation in the future. The novel structure and unique function of marine biology are active domain of new medicinal exploitation. At 20th century, with a trend of blue medicine,"For Ocean Medicine"has become a novel mark. The exploitation of anti-thrombolytic activity of marine biology has been in the ascendant.The polychaete Nereis virens of the Bohai Sea of China is a common marine annelid with abundant protease, its activity needs to be further explored. For the reason, our laboratory has investigated and identified a novel marine biology thrombolytic agent, with a strong protelytic activity such as earthworm autolyzing. Therefore, in this paper the main achievements in our investigations are now showed as follows.1. We successfully identified a novel oceanic fibrinolytic activity component----- The clamworm fibrinolytic protease from the coelomic fluid and bowels. Isolation of this fibrinolytic enzyme was carried out by the combination of various column chromatographic steps: anion-exchange chromatography DEAE-Sepharose Fast Flow,canion-exchange chromatography CM-Sepharose Fast Flow,gel permeation chromatography Sephacryl S-200 High Resolution and Sephadex G-25 Superfine. Every litre of clamworm sample can get 0.2mg activity protein, yield is 28.9%. The clamworm fibrinolytic protease and its purified method have applied for invention patent (The clamworm fibrinolytic protease, its purified method and application, the number of application: 02144828.0, the open number: CN 1500873A).2. The clamworm fibrinolytic protease showed a protein band in the SDS-PAGE and PAGE, which indicated the purified produce has achieved electrophoretic standard, and the protease is a single chain protein. The protease was analyzed by 2-DE and MALDI-TOF MS, and the estimated molecular mass is 29,000Da, its pI was about 4.5.3. Peptide Mass Fingerprinting (PMF) of the clamworm fibrinolytic protease was obtained by MALDI-TOF mass spectrometry, showed 33 peaks. This result was supported by the fact that the clamworm fibrinolytic protease was digested by trypsin to 33 peptides. In further studies, we have obtained 10 N-amino-acid sequences of these peptides, as follows: 1. NVVAVK; 2. INL; 3. QAPNYSTASY; 4. FLSTNNK; 5. LYIHDTGVR; 6. AVYLAGMK; 7. NFPNYYINLY; 8. VYLAANPTASS; 9. QTFNSDTL; 10. VYILDTGI, and input the sequences into the Swiss-Prot annotated protein sequence database (N-V proteinase, No. P83433). We searched the NCBI nonredundant protein sequence database using the MS Fit algorithm to look for proteins that match the peptide mass spectra of the clamworm fibrinolytic protease, but retrieved no sequence. Therefore we conclude that the clamworm fibrinolytic protease is a novel and special protein in Nereis virens.4. The fibrinolytic activity of the clamworm fibrinolytic protease and its effect of dissolving thrombus in vitro were obvious. Its fibrinolytic activity was more strong than Urokinase (UK), especially when concentration decreasing and reactive time prolonging, its specific activity was about 1.16×105U /mg.5. The fibrinolytic activity of the clamworm fibrinolytic protease was stable in the range of pH 4～9, at 30～55℃, and was optimal at pH 7.8, 45℃. These results suggest that the clamworm fibrinolytic protease is a heat, pH-resist and stable protease.6. The clamworm fibrinolytic protease was sensitive to serine protease inhibitors, especially DFP (I50 = 5.8×10-4 M) and PMSF (I50 = 5.5×10-2 M), and also TLCK (I50 = 7.7×10-1 M). However, other serine protease inhibitors, including Aprotinin, Elastinal, SBTI and Benzamidine had no obvious effect on proteolytic activity. Cysteine protease inhibitors (Iodoacetate and E64) and the metalloprotease inhibitors (EDTA and EGTA) did not affect the activity of the clamworm fibrinolytic protease, either. These results suggest that the clamworm fibrinolytic protease is a trypsin-like serine protease.7. In substrates specificity assays, four substrates (S-2444; pyro-Glu-Gly-Arg- pNA;S-2252:H-D-Val-Leu-Lys-pNA·HCl;Benzoyl-Phe-Val-Arg-pNA;Tosyl-Gly-Pro-Arg-pNA) were used to detect its enzymatic parameters. The Michaelis-Menten constant (Km) of the clamworm fibrinolytic protease is much lower measured by S-2444 than other substrates. It indicates that the active site in the clamworm fibrinolytic protease is more similar to substrate of Urokinase.8. The clamworm fibrinolytic protease has been showed by fibrin plate that it could dissolve clotting through two pathways: (1) directly via hydrolyzing fibrin and fibrinogen; and (2) indirectly via activating plasminogen to plasmin, then has the similar procedure to Lumbrinonase.9. Analyzing the hydrolysis of the clamworm fibrinolytic protease showed that it completely and rapidly digested theα-chain, and slowly hydrolyzes theβ- andγ-γchains of fibrin (α>β>γ-γ), and hydrolyzed the Aα-chain of fibrinogen with a high efficiency, and the Bβ- andγ-chains with a lower efficiency. Moreover, the proteolytic activity is associated with 29,000Da protein band (the clamworm fibrinolytic protease) by an in-gel protease activity test.10. The cDNA expression library of Nereis virens has been constructed integrally. The unamplified cDNA library consists of 2.0×107 independent clones in which the percentage of recombinant clones is about 75.2%. The average size of inserts is 1.0kb, and the titer of amplified cDNA library is 1.0×1011 pfu/ml.In summary, the clamworm fibrinolytic protease is a novel fibrinolytic protease, and trypsin-like serine protease, and has the similar procedure to Urokinase (UK). The clamworm fibrinolytic protease can be used clinically in the future due to its obvious advantages such as strong fibrinolytic activity, high stable, long half-life et al. The cDNA expression library has an excellent quality and lays solid foundation for further screening its code gene by antibody.