The Proteomic Analysis Of Earthworm And Cleavage Specificity Analysis Of Haematopoietic Serine Proteases

The whole thesis is mainly on the proteomic analysis. Part I is based on comparative proteomics to analyse the influence of two different drying process on the thrombolytic effects of chinese traditional medicine, the earthworm. The dried earthworm is a traditional thrombolytic medicine in East Asia. Its thrombolytic mechanism has been extensively studied. A group of fibrinolytic enzymes isolated from earthworm were reported to confer fibrinolytic and fibrinogenolytic properties, reduce platelet aggregation, lower blood viscosity and promote thrombus degradation. The activity components, fibrinolytic enzymes are highly possible to be degraded by the high temperature in the drying process. However, the effects of drying process on thrombolysis were rarely investigated. Herein, we compared the thrombolytic activity of the sun-and freeze-dried earthworms, and then revealed the underlying mechanisms for different thrombolytic effects by two-dimensional gel electrophoresis (2-DE).The part 1 focuses on the proteomics analysis, while the part II mainly analyses the characteristics of certain proteases. Cells from several of the hematopoietic cell lineages including mast cells, basophils, neutrophils, cytotoxic T cells and natural killer cells, store proteases at very high levels within their cytoplasmic granules. In mast cells, these proteases can accout for up to 35% of the total cellular protein, and the absolute majority of these belong to the chymotrypsin related serine protease family. A number of very diverse functions have been identified for these proteases, including apoptosis induction, blood pressure regulation, inactivation of insect and snake toxins, killing of bacteria, degradation of cytokines. Currently the difficulty in defining physiological roles for these proteases is their ability to cleave a vast array of substrates in vitro and to substantiate targets in vivo is challenging. Therefore, in the part Ⅱ we focused on the extended cleavage specificities of several serine proteases and their potential in vivo substrates, which may shed light on their physiological roles or the appearance of proteases during verebrate evolution.In this thesis, the main contents and innovative work are as following.(1) Comparative proteomic analysis of the sun- and freeze-dried earthworm with differentially thrombolytic activities. In the second chapter we compared the thrombolytic activity of earthworm Eisenia fetida processed by sun-drying to that by freeze-drying. Fibrin plate and blood clot lysis assays showed that freeze-dried earthworms gave dramatically higher fibrinolytic and thrombolytic activities than the sun-dried earthworms. To address the thrombolytic difference, comparative proteomic analysis was carried out using fibrin zymography and two-dimensional gel electrophoresis. The freeze-dried and sun-dried earthworms generated remarkably different 2-DE protein spot patterns. A total of 126 differential protein spots were detected,83 of them were identified by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry and database searching with 13 quantitative changes and 70 qualitative changes. Five of these differential proteins were identified as fibrinolytic proteases (lumbrokinases), responsible for dissolving fibrin, the main protein component of thrombus. The total abundance of these fibrinolytic proteases in the freeze-dried earthworms was significantly higher, consistent with the results of fibrin zymography. Therefore, the higher concentration of fibrinolytic enzymes along with their broad substrate specificity explained the stronger fibrinolytic and thrombolytic activities of the freeze-dried earthworms. This study suggest that freeze-drying represents an improved processing method for earthworm as the thrombolytic therapy in the future.(2) An analysis of rMCP-2 extended cleavage specificity and its potential role in regulationg intestinal permeability and parasite clearance. Mast cells of the rat intestinal mucosa express three chymotryptic enzymes named rMCP-2,-3 and 4. In the forth chapter we presents the extended cleavage specificity of rMCP-2, the most abundant of these three enzymes. Phage display analysis shows that it is a true chymase with a specificity similar to the corresponding enzyme in the mouse, mMCP-1, with a preference for Phe or Tyr in the PI position and a general preference for aliphatic amino acids both upstream and downstream of the cleavage site. The consensus sequence obtained from the phage display analysis was used to screen the rat genome for potential targets. A few of the most interesting candidate substrates were cell adhesion and cell junctional molecules. To see if these proteins also are susceptible to cleavage when being presented in their native conformation we let the active enzyme cleave 5 different recombinant cell adhesion and cell junctional proteins. Three potential targets were identified, the loop 1 of occludin, protocadherin a-4 and cadherin 17, which indicate that these proteins are at least partly responsible for the previously observed prominent role of rMCP-2 in mucosal permeability and in parasite clearance.(3) The cleavage specificity of human mast cell chymase and neutrophil cathepsin G and their highly selective cleavage of cytokines. hCG and HC are two closely-related chymotyrpin-like proteases that are released predominantly from cytoplasmic granules of activated neutrophils and mast cells. Both of hCG and HC are encoding on mast cell chymase locus, and they have a number of common substrates as cytokines, chemokines and ECMs. To understand the distinctive roles in inflammatory events involving both neutrophils and mast cells, the fifth chapter in the thesis compared the extended cleavage specificity of HC and hCG. The phage-display and a panel of recombinant substrate proteins indicate that HC and hCG are two very similar serine proteases, but the chymase-like activity of HC is more restrictive in comparison with hCG. HC shows a strong preference for aromatic amino acids in P1 position indicating its restrict chymase-like primary cleavage specificity. In contrast, a relatively broad selection of amino acids are tolerated in the P1 position of hCG including aromatic amino acids and Leu. In order to obtain a better view of the selectivity for various potential substrates for these two enzymes we have also performed cleavage of a panel of 50 recombinant cytokines and chemokines. Our results show a surprisingly high selectivity for HC and slightly more broad range for hCG. Only 3 out of 50 were efficiently cleaved by HC and 10-11 by hCG, which suggests the high selecity of cytokines by HC and hCG. All these cytokines may be the potential targets for enzymes; therefore, the results of different selective cleavage of cytokines by HC and hCG suggest their distinctive role in inflammatory events.(4) Asp-ase activity of the opossum grathepsodenase supports the role of granzyme B as part of anti-viral immunity already during early mammalian evolution. Granzyme B is one of the key effector molecules in our defense against viruses and intracellular bacteria. This serine protease induces, together with the pore forming protein peforin, caspase dependent apoptosis in target cells. In sixth chapter we present the first characterization of a granzyme B homologue in a non-placental mammal, the American opossum, Monodelphis domestica. The enzyme was produced in a human cell line and after purification and activation used to study its primary and extended cleavage specificity by a panel of chromogenic substrates and recombinant protein substrates. The opossum granzyme B was found to have a specificity similar to human granzyme B. It has a primary specificity for negatively charged amino acids as aspartic and glutamic acid. However, compared to human granzyme B the opossum GzmB was found to be less restrictive in both its primary and its extended specificity. The identification of a granzyme B homologue with asp-ase specificity in a non-placental mammal gives strong indications for that caspase dependent apoptosis by a serine protease with primary specificity for negatively charged amino acids has been part of anti viral immunity since early mammalian evolution. This finding also indicates that a chymase and an asp-ase were the first two serine protease genes to appear in the mammalian chymase locus.