Greeve J, Altkemper I, Dieterich J H, Greten H, Windler E
Medizinische Kernklinik und Poliklinik, Universitäts-Krankenhaus Eppendorf, Hamburg, Germany.
J Lipid Res. 1993 Aug;34(8):1367-83.
Two different isoproteins are encoded by the apolipoprotein (apo) B gene, apoB-48 and apoB-100. ApoB-48, core component of intestinally derived chylomicrons, has an accelerated plasma turnover as compared with the full-length protein apoB-100. A posttranscriptional modification of the apoB mRNA by conversion of cytidine into uridine at nucleotide position 6666 changes the genomically encoded glutamine codon CAA at amino acid residue 2153 into a translational stop codon UAA. This mRNA editing explains the formation of the truncated isoform apoB-48. In the present investigation editing of apoB mRNA in liver and intestine from 12 different mammalian species was measured by a quantitative primer extension analysis of reverse-transcribed and polymerase chain reaction- (PCR) amplified apoB mRNA in order to determine whether i) editing of apoB mRNA is generally restricted to the intestine or may also be found in the liver of other species than rodents, and ii) hepatic expression of apoB mRNA editing influences lipoprotein concentrations in plasma. Intestinal apoB mRNA was edited at high levels in all species, 40% in sheep, 73% in horse, 82% in pig, 84% in dog, 84% in cat, 87% in guinea pig, 88% in rat, 89% in mouse, and > 90% in human, monkey, cow, and rabbit. In liver apoB mRNA was edited to 18% in dog, to 43% in horse, to 62% in rat, and to 70% in mouse. Low levels of editing below 1% were detected in liver of rabbit and guinea pig. In contrast, hepatic apoB mRNA from human, monkey, pig, cow, sheep, and cat liver was not edited. The results of the primer extension analysis were confirmed by cloning and sequencing of the PCR products from dog, horse, cat, guinea pig, sheep, and cow for all of which the apoB cDNA sequence had not been established by previous investigations. Primer extension analysis of apoB mRNA from dog intestine and dog liver indicated C/U editing at C6655 in addition to C6666. Cloning and sequencing of apoB cDNA from dog liver and intestine confirmed additional C/U editing at C6655 which changes ACA for threonine at amino acid residue 2149 into AUA for isoleucine. Synthesis and secretion of apoB-48-containing lipoproteins from liver was demonstrated by pulse labeling of freshly isolated horse hepatocytes and immunoprecipitation with apoB-specific antibodies or density gradient ultracentrifugation. The concentrations of VLDL, LDL, and HDL in all species were determined after fractionation by density gradient ultracentrifugation.(ABSTRACT TRUNCATED AT 400 WORDS)
载脂蛋白（apo）B基因编码两种不同的同型蛋白，即apoB - 48和apoB - 100。apoB - 48是肠道来源乳糜微粒的核心成分，与全长蛋白apoB - 100相比，其血浆周转率更快。apoB信使核糖核酸（mRNA）在核苷酸位置6666处通过将胞苷转化为尿苷进行转录后修饰，使得基因组编码的位于氨基酸残基2153处的谷氨酰胺密码子CAA变为翻译终止密码子UAA。这种mRNA编辑解释了截短的同型体apoB - 48的形成。在本研究中，通过对反转录并经聚合酶链反应（PCR）扩增的apoB mRNA进行定量引物延伸分析，测定了12种不同哺乳动物肝脏和肠道中apoB mRNA的编辑情况，以确定：i）apoB mRNA的编辑是否通常仅限于肠道，或者在啮齿动物以外的其他物种的肝脏中也能发现；ii）apoB mRNA编辑的肝脏表达是否会影响血浆中的脂蛋白浓度。在所有物种中，肠道apoB mRNA的编辑水平都很高，绵羊为40%，马为73%，猪为82%，狗为84%，猫为84%，豚鼠为87%，大鼠为88%，小鼠为89%，人类、猴子、牛和兔子则>90%。在狗的肝脏中，apoB mRNA的编辑率为18%，马为43%，大鼠为62%，小鼠为70%。在兔子和豚鼠的肝脏中检测到低于1%的低水平编辑。相比之下，人类、猴子、猪、牛、羊和猫肝脏中的肝脏apoB mRNA未被编辑。通过对狗、马、猫、豚鼠、绵羊和牛的PCR产物进行克隆和测序，证实了引物延伸分析的结果，此前的研究尚未确定这些物种的apoB cDNA序列。对狗肠道和肝脏中apoB mRNA的引物延伸分析表明，除了C6666处的C/U编辑外，C6655处也存在C/U编辑。对狗肝脏和肠道中apoB cDNA的克隆和测序证实了C665５处额外的C/U编辑，该编辑将氨基酸残基2149处的苏氨酸密码子ACA变为异亮氨酸密码子AUA。通过对新鲜分离的马肝细胞进行脉冲标记，并用apoB特异性抗体进行免疫沉淀或密度梯度超速离心，证明了肝脏中含apoB - 48脂蛋白的合成和分泌。通过密度梯度超速离心分级分离后，测定了所有物种中极低密度脂蛋白（VLDL）、低密度脂蛋白（LDL）和高密度脂蛋白（HDL）的浓度。（摘要截短于400字）
