| Scorpions are one of the most ancient groups of terrestrial animals on earth, they are widely spread around the world with more than 430 million years of evolution history. Scorpions use a mature and efficient venom system to capture prey, defense and deterrence competitors. Scorpion venom is a complex mixture composed of various biologically active substances. The scorpion Mesobuthus martensii is the largest and most widely distributed in China, recorded by Chinese Pharmacopoeia as the only scorpion species. To a more comprehensive understanding of the composition of scorpion venom and promote the basic research and applied basic research of scorpion venom component, a comprehensive proteomic strategy was used for proteomics research of scorpion venom and antimicrobial peptides in clinical treatment. Meanwhile, further investigation for biological function of novel protein in scorpion venom was carried out in this work.First, the work on the proteomics of the scorpion M. martensii venom was carried out. The crude venom was separated into 153 fractions by ultrafiltration, SDS-PAGE,2-DE and RP-HPLC, and then all these fractions were analyzed by LC/ESI-Q-TOF MS/MS. We identified a total of 227 non-redundant protein sequences, including 93 unknown novel sequence and 134 known sequences. Of the 134 known sequences,115 were confirmed for the first time from the M. martensii venom at the protein level. Functional classification of identified sequences covered the sodium channel toxins, potassium channel toxins, chloride channel toxins, antimicrobial peptides, defensins, enzyme inhibitors, lipolysis-activating peptides, venom enzyme, cell-associated proteins, cysteine-rich secretory proteins and venom allergens. In these identified protein sequences, there were seven novel toxin sequences, including three sodium channel toxins, three potassium channel toxins and one no-annotation toxin. On the other hand, the crude venom of M. martensii was directly separated by RP-HPLC.29 fractions were eluted and then analyzed by MALDI-TOF MS to obtain a Mass fingerprint of venom components. We had detected 202 kinds of different protein molecular weights, wherein the molecular weight ranges of 1000?5000 Da and 6000-8000 Da were two large groups, which accounted for 75% and 20% of the total. These work laid the foundation for further understanding and application of venom proteins.Secondly, the recombinant expression and functional studies of lipolysis-activating peptide (LVP) from the M. martensii venom were conducted in our work. LVP is a new kind of polypeptide molecules in scorpion venom, whose gene and protein structures are different from the classical toxin polypeptides (single-stranded sequence is similar to sodium channel toxin; structure for a heterodimer), showing a function of adipocyte lipolytic activity. Based on the identification of LVP from previous proteomics and genomics of M. martensii, we expressed and purified the LVP of M. martensii for the first time by genetic engineering method. The alpha chain of LVP (LVP-a) and beta chain (LVP-(3) were constructed recombinant expression vectors, respectively, expressed and purified. By optimizing a series of conditions of expression and purification, the recombinant LVP-a and LVP-p were obtained. After renaturations of LVP-a and LVP-?, heterodimers (refolding rate of about 20%) were achieved. Furthermore, we verified lipolytic activity of recombinant LVP proteins of M. martensii, compared to the control group, the recombinant LVP increased about 60% glycerol release (lipid hydrolysates) by a 10?M concentration. These work laid the foundation for the research and application of the biological function of scorpion LVP.Finally, the proteomic studies of exudates from chronic foot ulcers of diabetics treated by scorpion antimicrobial peptide ABP-W1 were carried out. The pathological mechanism of chronic diabetic foot ulcers is still a scientific problem to be solved. ABP-W1 was an antimicrobial peptide identified from M. martensii venom in our previous studies. After clinical trials of volunteers, ABP-W1 had a good therapeutic effect for chronic diabetic foot ulcers, but the related pharmacological action mechanism remains unclear. Therefore, the exudates from chronic foot ulcers of diabetics treated by ABP-W1 in different treatment stages (early, middle and late stages) were collected, and analyzed by bacteriological methods and iTRAQ labeling quantitative proteomics. After identification, the ulcer exudates from early and middle stages were mainly infected by the bacterium Burkholderia cepacia (conditional pathogenic bacteria), and the exudate from late stage was mainly late infected by Staphylococcus epidermidis (normal bacteria). The number of bacteria had a downward trend. In quantitative proteomics analysis, a total of 1,865 proteins were qualitatively identified, and quantitative data were analyzed using a fold change of 1.3 and p-value<0.05 for screening criteria. Compared to the exudates from early stage of ABP-W1 treatment,130 differentially expressed proteins (down-regulated 29 and up-regulated 101) were identified in the exudates from middle stage,401 differential proteins (down-regulated 82 and up-regulated 19) were identified in the exudates from late stage, and 310 differential proteins (down-regulated 56 and up-regulated 254) both appeared in the exudates of middle and late stages. Furthermore, these differential distinct proteins were analyzed in the functions, canonical pathways, transcriptional regulators of upstream and protein interaction networks. The results showed that:1) differential proteins from the middle and late stages of ABP-W1 treatment were mostly high expressed, and the number of late stage was more; 2) the activating effects of differential proteins from the middle and late stages of ABP-W1 treatment were mainly involved in protein synthesis, cell proliferation, cell differentiation, cell migration, cell movement, and organizational survival, and inhibiting effects mainly associated with cell death, inflammatory disease, inflammatory response, immunological disease, bacterial growth, etc.; 3) the activation state of middle stage of ABP-W1 treated was mainly increased and of late stage was decreased; 4) the middle and late stages of ABP-W1 treatment both had common activation and inhibition effect, besides, there were large differences in biological effects.In summary, firstly, we had got a complete proteomics map of the scorpion M. martensii venom. At the protein level, our work systematically revealed the complexity and diversity of the protein composition of M. martensii venom, enriched the understanding of protein composition of scorpion venom. Secondly, we established a new method for the recombinant production of lipolysis-activating peptide, which was a novel protein molecule from scorpion venom, and preliminarily verified its lipolysis function, which laid the foundation for the research of the basic theory and development of potential medicinal value for LVP. Finally, using quantitative proteomics technique, we analyzed the differential protein expression profile in exudates from chronic foot ulcers of diabetes treated by scorpion antimicrobial peptide ABP-W1, which provided the basic data and important reference for further revealing the molecular mechanism of diabetic foot ulcer. |
PDF Full Text Request