Site-Directed PEGylation Studies Reveal the MechanisticInsights into Bile Acid Transportation of ASBT

• Main Authors: Guan-Syun Li, 李冠勳
• Other Authors: Nien-Jen Hu
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/93490373834889863094

碩士 === 國立中興大學 === 生物化學研究所 === 105 === Bile acid is responsible for lipid and vitamin absorption in human’s intestine. After lipid digestion, bile acids are recycled at the ileal epithelium by Apical Sodium-dependent Bile acid Transporter (ASBT), and undergo enterohepatic circulation, by which most of bile acids are reabsorbed back to the liver. Recent studies have determined three different crystal structures of ASBT bacterial homologous proteins. The first structure of the ASBT bacterial homologous protein was ASBTNM, showing an inward-open conformation in complex with two sodium ions and one taurocholate. The others were ASBTYf, revealing both inward-open and outward-open conformations, respectively, but no sodium ions and taurocholate were observed. Although the three crystal structures demonstrated that ASBT may adopt different conformations for taurocholate translocation, the critical factor triggering such drastic conformational changes and the mechanism of transport remains unclear. In this study, we developed a non-invasive biochemical approach, PEGylation In Gel Fluorescence (PIGF) to detect the solvent accessibility of ASBTNM substrate-binding crevice in different conditions. The results indicate that the PIGF profiles are in great agreement with crystal structures and the results further provide deep insight into the mechanistic determinants for conformational alternation of ASBT. Besides, we used ASBTNM L44C mutant as the DNA template and replaced the Na+-binding residues individually with alanine. We found that L44C/N115A mutant showed the highest extent of PEGlyation, suggesting N115A substitution may lock ASBTNM in outward-facing conformation.