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OBJECTIVE To investigate the fibrinolytic activity and protein profile change of Pheretima guillemi (Michaelsen)-derived anticoagulant proteins DPf3 after digestion in gastrointestinal tract in vitro and in vivo, and to predict the spatial structure of its active protein, DPf3 ID NO.2, by homology modeling. METHODS The in vitro digestive effects of pepsin (and trypsin) on DPf3 were investigated under simulated gastric empty state (pH 2) and semi-empty state (pH 5), and the in vivo digestion effects were evaluated via Kunming mice orally administrated DPf3. The activity and protein profile of DPf3 after hydrolysis in vivo and in vitro were analyzed by fibrin plate method combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); I-TASSER combined with SAVES v5.0 were used to homology modeling and evaluate the structure of DPf3 ID NO.2, the active protein of DPf3, and to predict its secondary and tertiary structure. RESULTS After enzymatic hydrolysis in vitro, the fibrinolytic activity of DPf3 was lost under pH 2 incubation, and the fibrinolytic activity of DPf3 incubated with heat inactivated pepsin at pH 5 was slightly higher than that of DPf3 incubated with activated pepsin. After enzymolysis in vivo, only the fibrinolytic activity of gastric perfusion fluid was significantly different from that of control group. The protein profile showed that the stable presence of DPf3 ID NO.2 might be an important reason for the fibrinolytic activity of DPf3. Spatial structure prediction showed that DPf3 ID NO.2 was a typical chymotrypsin-like serine protease, revealing its catalytic center, zymogenic center, important amino acid residues and S1 active pocket. CONCLUSION The combination of in vivo and in vitro hydrolysis provide a basis for the protein expression profile and activity changes of antithrombotic protein DPf3 after oral administration. Homologous modeling is feasible for the structural prediction of active peptides in macromolecular proteins of the traditional Chinese animal medicine.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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