Schmidt H H, Remaley A T, Stonik J A, Ronan R, Wellmann A, Thomas F, Zech L A, Brewer H B, Hoeg J M
Molecular Disease Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1995 Mar 10;270(10):5469-75. doi: 10.1074/jbc.270.10.5469.
Apolipoprotein A-I (apoA-I), the major protein of high density lipoproteins, facilitates reverse cholesterol transport from peripheral tissue to liver. To determine the structural motifs important for modulating the in vivo catabolism of human apoA-I (h-apoA-I), we generated carboxyl-terminal truncation mutants at residues 201 (apoA-I201), 217 (apoA-I217), and 226 (apoA-I226) by site-directed mutagenesis. ApoA-I was expressed in Escherichia coli as a fusion protein with the maltose binding protein, which was removed by factor Xa cleavage. The in vivo kinetic analysis of the radioiodinated apoA-I in normolipemic rabbits revealed a markedly increased rate of catabolism for the truncated forms of apoA-I. The fractional catabolic rates (FCR) of 9.10 +/- 1.28/day (+/- S.D.) for apoA-I201, 6.34 +/- 0.81/day for apoA-I217, and 4.42 +/- 0.51/day for apoA-I226 were much faster than the FCR of recombinant intact apoA-I (r-apoA-I, 0.93 +/- 0.07/day) and h-apoA-I (0.91 +/- 0.34/day). All the truncated forms of apoA-I were associated with very high density lipoproteins, whereas the intact recombinant apoA-I (r-apoA-I) and h-apoA-I associated with HDL2 and HDL3. Gel filtration chromatography revealed that in contrast to r-apoA-I, the mutant apoA-I201 associated with a phospholipid-rich rabbit apoA-I containing particle. Analysis by agarose gel electrophoresis demonstrated that the same mutant migrated in the pre-beta position, but not within the alpha position as did r-apoA-I. These results indicate that the carboxyl-terminal region (residue 227-243) of apoA-I is critical in modulating the association of apoA-I with lipoproteins and in vivo metabolism of apoA-I.
载脂蛋白A-I（apoA-I）是高密度脂蛋白的主要蛋白质，可促进胆固醇从外周组织逆向转运至肝脏。为了确定对调节人apoA-I（h-apoA-I）体内分解代谢重要的结构基序，我们通过定点诱变在第201位残基（apoA-I201）、217位残基（apoA-I217）和226位残基（apoA-I226）处生成了羧基末端截短突变体。ApoA-I在大肠杆菌中作为与麦芽糖结合蛋白的融合蛋白表达，通过因子Xa切割将麦芽糖结合蛋白去除。对正常血脂兔体内放射性碘化apoA-I的动力学分析显示，apoA-I截短形式的分解代谢速率显著增加。apoA-I201的分解代谢分数率（FCR）为9.10±1.28/天（±标准差），apoA-I217为6.34±0.81/天，apoA-I226为4.42±0.51/天，远快于重组完整apoA-I（r-apoA-I，0.93±0.07/天）和h-apoA-I（0.91±0.34/天）的FCR。所有apoA-I截短形式均与极高密度脂蛋白相关，而完整的重组apoA-I（r-apoA-I）和h-apoA-I与HDL2和HDL3相关。凝胶过滤色谱显示，与r-apoA-I不同，突变体apoA-I201与富含磷脂的兔apoA-I含颗粒相关。琼脂糖凝胶电泳分析表明，相同的突变体在前β位置迁移，但不像r-apoA-I那样在α位置迁移。这些结果表明，apoA-I的羧基末端区域（第227 - 243位残基）在调节apoA-I与脂蛋白的结合以及apoA-I的体内代谢中起关键作用。
