| 其他摘要 | Thrombotic disease (TD) is a lethal medical complication with high incidence.
Thromboembolism is the leading cause of human death in China and western countries.
During the last few decades, anti-thrombosis agents have been extensively used as the
therapeutic treatment of thromboembolism complication, including anti-clotting,
anti-platelet, and thrombolytic agents, but more or less side effects are present in each of
them. As a result, the pursuit of high quality thrombolytic agent never stopped.
Molecular mechanism of interaction between blood-sucking arthropods and vertebrate
host are hot spots of international research, which have potential applications in finding
new medical leading molecules and providing arthropod control strategy. By proteomics
coupling transcriptome analysis with pharmacological testing, three families of proteins,
which exert mainly on anti-thrombosis functions, were identified and characterized from
salivary glands of the horsefly Tabanus yao Macquart (Diptera, Tabanidae). They are: (I)
one bifunctional enzyme Tablysin, which hydrolyzes both alpha and beta chains of
fibrinogen, and acts as platelet aggregation inhibitor; (II) another fibrinogenolytic enzymes
family TY6, which hydrolyze specially alpha chain of fibrinogen; (III) one apyrase Tabapy,
acting as platelet aggregation inhibitor. We studied the diversity, structure, and molecular
mechanisms of these three proteins. The extreme diversity of horsefly anti-thrombosis
components reveals the anti-thrombosis molecular mechanisms of the traditional Eastern
medicine insect material.
Tablysin is a single-chain protein, its molecular weight is 27 kDa. The activity of the
horsefly fibrinogenolytic enzyme was optimal at pH 2.4-10.0, 37℃. These results suggest
that Tablysin is useful in human physical environment. Tablysin is sensitive to serine
protease inhibitor PMSF, but the metalloprotease inhibitor EDTA does not affect the activity of Tablysin. Plasma protease inhibitors, including Aprotinin and α-macroglobulin,
can partially inhibit the effect on proteolytic activity. These results suggest that Tablysin is
a serine protease. Tablysin was subjected to N-terminal amino acid sequence analysis.
Unfortunately, its N-terminus was blocked. We obtained the interior peptide amino acid
sequences by Q-TOF mass analysis, and its encoding gene was screened from the cDNA
library of T. yao salivary glands.
Analyzing the hydrolysis of Tablysin showed that it can hydrolyze the α- and β-chains
of fibrinogen, but can not hydrolyze fibrin, laminin and fibronectin. Tablysin is either not
plasminogen activator. Platelet aggregations induced by agonists ADP were significantly
inhibited by Tablysin in dose-dependent manner. Attempts to explain the mechanism of
inhibition, we used flow cytometry to detect the effect of Tablysin on the occupancy of
platelet glycoprotein GPIIb/IIIa, and we found that tablysin could bind to GPIIb/IIIa.
We constructed recombinated Tablysin-pET plasmid successfully, and then using
IPTG to induce the His-tagged fusion protein expression. By His-bind resin, enterokinase
cleavage, and FPLC, we finally got rTablysin. The rTablysin has the same strong
fibrinogenolytic activity as the native Tablysin. The thrombolytic effect of purified
rTablysin on carrageenan induced thrombosis model in mice was investigated. The result
shows that the tail-thrombus decreased correlate with rTablysin in a dose-dependent
manner.
A group fibrinogenolytic enzymes, TY6, were purified from T. yao SGE. Complete
cDNA sequences encoding TY6 were cloned from the the cDNA library of the salivary
glands. In these ten cDNA sequences, most of them encode a proprotein composed of 254
amino acid (aa) residues including predicted signal peptides (16 aa) and mature TY6
containing the SCP domain (Sc7 family of extracellular domain) found in insect antigen 5
proteins. TY6 is a single-chain protein; its molecular weight is 27 kDa. TY6 is sensitive to
serine protease inhibitors PMSF, the metalloprotease inhibitor EDTA did not affect the
activity of TY6. TY6 was recognized as an allergen, showing IgE reactivity of horsefly
sensitized patients by immunoblot. The presence of enzymes diversity with effective
fibrinogenolytic activity reveals the mechanism of horsefly blood meal.
An Apyrase (ATP diphosphohydrolase, EC 3.6.1.5), named Tabapy, was purified as an
active enzyme from horsefly salivary glands and the interior peptides amino acid
sequences were sequenced. Its molecular weight is 63 kDa. The amino acid sequences
obtained match the conceptual translation product of a cDNA clone isolated from horsefly salivary glands cDNA library. Platelet aggregations induced by agonists ADP were
significantly inhibited by Tabapy in dose-dependent manner. Tabapy showed IgE reactivity
of horsefly sensitized patients by immunoblot. The apparent conversion of a gene encoding
an enzyme involved in a common metabolic event at the cellular level to a gene involved
in the antihemostatic response of horsefly illustrates one way that this particular insect has
adapted to the challenges of blood-feeding. |
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