Frias, Jorge
(CBA - Biotechnology Centre of Azores, Faculty of Sciences and Technology, University of Azores)
,
Toubarro, Duarte
(CBA - Biotechnology Centre of Azores, Faculty of Sciences and Technology, University of Azores)
,
Fraga, Alexandra
(ICVS - Life and Health Research Institute, University of Minho)
,
Botelho, Claudia
(CEB - Centre of Biological Engineering, University of Minho)
,
Teixeira, Jose
(CEB - Centre of Biological Engineering, University of Minho)
,
Pedrosa, Jorge
(ICVS - Life and Health Research Institute, University of Minho)
,
Simoes, Nelson
(CBA - Biotechnology Centre of Azores, Faculty of Sciences and Technology, University of Azores)
Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alterna...
Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6, and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48℃ and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat tail by 11.4% in 24 h compared to non-treated animals.
Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6, and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48℃ and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat tail by 11.4% in 24 h compared to non-treated animals.
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문제 정의
This work describes a new enzyme capable of degrading blood clots without impairing blood coagulation. This enzyme showed a direct-action mechanism by degrading the fibrin substrate without the need to activate other factors.
가설 설정
2B). These important findings are consistent with previous research hypothesizing that the catalytic activity can be influenced by small structural changes in the primary sequence flanking the active site residues. The substitution of amino acids flanking the conserved catalytic triad residues may contribute to forming different structural conformations that translate into different activities.
제안 방법
The amplification of the S127e subtilisin encoding gene was performed with the degenerated primers Bk-5′- GCGCAATCCGTGCCTTACGGC-3′ and reverse Bk-5′-TTATTGTGCAGCCGCCTGTACGTTG-3′, designed based on the homologous references that were predicted by mass spectrometry analysis of the purified protein. The following PCR conditions were used: 95oC for 4 min, followed by 30 cycles of 95oC for 45 sec, 55oC for 45sec, 72oC for 1.
a/F/050/2016) from Fundação para a Ciência e Tecnologia, Portugal; Jorge Frias received a doctoral grant (SFRH/BD/131698/2017). The authors are grateful to Dr. Teresa Sampaio and Nélia Arruda, from Dr. Forjaz Sampaio Medical Center, for helping with the plasma anticoagulation assays. Duarte Toubarro and Nelson Simões designed the study and helped with the paper’s redaction.
The optimal temperature was determined at 16oC, 28oC, 37oC, 48oC, and 60oC, as described above in the buffer at pH 7. The effect of ions was investigated by adding CuSO4, ZnSO4, HgCl2, MnSO4, CaCl2, CoCl2, MgSO4, and FeSO4 to the reaction mixture at a final concentration of 1 mM, performing the reaction at optimal pH and temperature.
as the template to amplify 16S rDNA and S127e subtilisin genes. To identify the isolate, the amplification of the 16S rDNA gene was carried out using the forward primer 8F (5′-AGTTGATCCTGGCTCAG-3′) and reverse primer 1492R (5′-ACCTTGTTACGACTT-3′). The PCR amplification was carried out under the following conditions: 95oC for 3 min, followed by 35 cycles of 95oC for 30 sec, 46oC for 30 sec, 72oC for 1 min and, finally, 72oC for 2min.
대상 데이터
cgi). Sequence identities were determined by BLAST against the NCBI general database (http://www.ncbi.nlm.nih.gov/BLAST/). Multi-sequence analysis of homologous genes was performed using CLUSTAL W implemented in BioEdit 7.
Six-week-old female Wistar rats weighing 180–200 g, supplied by Charles River in France, were kept in rooms with controlled temperature and humidity with a 12 h light/dark cycle. Animals were supplied with tap water and a normal pellet diet for at least three days before the experiments.
데이터처리
In both graphs, columns represent mean ± SD, n = 3. The asterisk shows the significant difference (p = 0.079) between the control and treated group (Student’s t-test).
All experiments were performed in triplicate and repeated three times with similar results. The data from in vivo thrombolytic assay were evaluated using Student’s t-test. Differences were considered statistically significant for p values < 0.
이론/모형
The cytotoxicity of the purified AprE127 was tested in Vero cells (Sigma-Aldrich No. 84113001) using the MTT assay. Then, 100 μl of Vero cells suspension, with about 105 cells, was applied per well in 96-well plates and incubated at 37oC for 24 h in a CO2 chamber.
성능/효과
In conclusion, our work demonstrates a new subtilisin, designated AprE127, that can be useful in overcoming important limitations of current agents, namely hemorrhagic complications. AprE127 was proved to be safe and to reduce thrombus extension in injected Wistar rats, with evident prevention of thrombus formation.
subtilis DC 33 [53, 54], which first hydrolyse β-chain. In this fundamental work, it was proved that AprE127 was not toxic to cells in culture, and it is not hemolytic. Furthermore, this enzyme was able to cause a slight but evident reduction in the lengths of the infarcted region of a carrageenan-induced tail thrombosis test in rat in comparison with non-treated animals.
In this triad, His takes part of the proton donor, and Asp is needed for the orientation of the imidazolium ring of His [33]. The 3D analysis of AprE127 is allowed to predict an identical fold to BPN subtilisin (PDB Acc. No. 1s01A) with a confidence score of 1.76 and an estimated model accuracy of 0.96 ± 0.05 (TM-score). This model evidenced the α/β-fold typical of the S8 subtilisin family, with a twisted central β-sheet containing seven strands surrounded by nine α-helices, with the active site residues converging to form the substrate binding loop (Fig.
Based on the halo diameter of fibrin lysis, the isolate S127e, which presented the highest activity was selected. This isolate has a 16S rDNA (NCBI Acc. No. MN069579) and subtilisin named AprE127 (NCBI Acc. No. MF281668) sequences with 99.65% and 98.53% identity with those of B. subtilis P52 and J-5 strains, respectively. Therefore, it was assigned as a new isolate of B.
후속연구
The uniqueness of AprE127 resides in a few differences in amino-acid sequences, which probably explain the differences observed in its substrate specificity and mode of action. More research should be done to completely elucidate the differences in AprE127 and to shed more light on the structure-function relationship.
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