Structural and Functional Studies of Apolipoprotein H

• Main Authors: Hsueh-Hsiao Wang, 王學孝
• Other Authors: An-Na Chiang
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/18135872140985271917

碩士 === 國立陽明大學 === 生物化學研究所 === 87 === Apolipoprotein H (apoH), also known as β2—glycoprotein I, is a highly glycosylated single-chain polypeptide of 326 amino acids, containing a high proportion of lysine and cysteine residues. Some in vitro experiments have revealed several possible roles for apoH, including anticoagulant properties, lipoprotein metabolism and binding to phospholipids. A recent report also suggests that apoH may be an anti-atherogenic protein. Our previous study showed that apoH is capable of protecting low density lipoprotein (LDL) against Cu2+-mediated oxidation under physiological concentration. The aim of this study was to elucidate the structure-function relationship of apoH and investigate the regulation of the apoH gene expression. These are areas that have not been explored yet. Structural epitopes that are important for the protective function of apoH were identified by methods of chemical modification and conjugated diene formation assay. Modification of cysteine residues caused a total reverse of the antioxidative capacity of apoH, but the modification of lysine residues caused only partial reverse of the antioxidative function. Northern blot analysis showed that liver was the major site of apoH gene expression. However, detectable apoH mRNA was also observed in intestine and placenta by using reverse-transcription polymerase chain reaction (RT-PCR). To further understand the regulatory elements of the apoH gene, we have cloned and sequenced the human apoH 5'-flanking 2242 bp sequence. The transcriptional start site was determined by primer extension to be located 108 bp upstream of the translational start codon. Nucleotide sequence similarity search of this 5'-flanking region revealed a putative promoter region and numerous potential binding sites for transcription factors. By analysis of the reporter activity, the 5'-upstream DNA fragment between -9~ -677 bp of apoH gene displayed strong promoter activity. This study reveals the possible sites of apoH that might be involved in its protective function. Moreover, characterization of the cis-regulatory elements of the apoH gene will make progress in the understanding of apoH gene regulation.