Detection of the interaction between Complement 3 and DNase I using the proteomic approach – Biological significance of the interaction

• Main Authors: Chao-Hsuan Yu, 余兆軒
• Other Authors: Ta-Hsiu Liao
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/95400623082854831068

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 94 === Bovine pancreatic deoxyribonuclease I (bpDNase I) is the best-characterized DNase. Previous studies in this laboratory have shown that there was a DNase I-binding proteins (DBP) in bovine small intestine and this DBP is not the DNase I inhibitor, actin. Until today, actin is the only known DNase I binding protein The covalent chromatography was utilized for isolation of the DNase I-binding protein in the extract from bovine small intestine. To achieve this goal, first, the mutant brDNase I C101A , contained a free sulfhydryl group on Cys104, was constructed and expressed in BL21 (DE3) pLysE . The expressed protein was subsequently purified to homogeneity through a SOURCE 15Q anion and a S-hyperD cation-exchange columns. CD spectra analysis revealed the mutation did not alter secondary structures and the presence of 0.5 mM β-MSH in the buffer could avoid the brDNase I C101A dimer formation. The purified brDNase I C101A was then covalently linked to Thiopropyl Sepharose 6B beads. When H293T cell lysates were added to brDNase I C101A conjugated beads, the result showed that the DNase I-Sepharose beads could bind actin indicating this method is feasible for isolation of the DNase I-binding proteins. Crude extracts from bovine small intestine were than collected and subjected to a gel filtration Sephadex G-100 column. Fractions with DNase I activities were pooled and used for pull down assay. The interacted DBPs were eluted from the beads. The DBPs were identified from the SDS-PAGE using the tandem mass spectroscopy approach. Two proteins identified were complement 3 (C3) and α-macroglobulin and further confirmed by Western blot analysis. Since C3 is distributed in serum, we assumed the interaction of C3 and DNase I existed in serum. Therefore, bovine serum was used for pull down assay and the result showed, C3 in serum could interact with DNase I. Finally, we performed computational algorithm to simulate molecular docking between C3 and DNase I. The result provided the predicted interface and possible interacting amino acid residues. Here, we proposed that when infection occurred and necrotic cells were needed for clearance, C3 could be activated and bring serum DNase I to surface of dying cells. Activated C3 could cause downstream effectors assembled and form membrane attack complex which could lyse cell membrane. DNase I then enter the cell and cleave disposal DNA to avoid releasing undigested DNA, which may stimulate immune response in blood.