Baumgart E, Vanhooren J C, Fransen M, Marynen P, Puype M, Vandekerckhove J, Leunissen J A, Fahimi H D, Mannaerts G P, van Veldhoven P P
Katholieke Universiteit Leuven, Faculteit Geneeskunde-Campus Gasthuisberg, Departement Moleculaire Celbiologie, Belgium.
Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13748-53. doi: 10.1073/pnas.93.24.13748.
Peroxisomes in human liver contain two distinct acyl-CoA oxidases with different substrate specificities: (i) palmitoyl-CoA oxidase, oxidizing very long straight-chain fatty acids and eicosanoids, and (ii) a branched-chain acyl-CoA oxidase (hBRCACox), involved in the degradation of long branched fatty acids and bile acid intermediates. The accumulation of branched fatty acids and bile acid intermediates leads to severe mental retardation and death of the diseased children. In this study, we report the molecular characterization of the hBRCACox, a prerequisite for studying mutations in patients with a single enzyme deficiency. The composite cDNA sequence of hBRCACox, derived from overlapping clones isolated via immunoscreening and hybridization of human liver cDNA expression libraries, consisted of 2225 bases and contained an open reading frame of 2046 bases, encoding a protein of 681 amino acids with a calculated molecular mass of 76,739 Da. The C-terminal tripeptide of the protein is SKL, a known peroxisome targeting signal. Sequence comparison with the other acyl-CoA oxidases and evolutionary analysis revealed that, despite its broader substrate specificity, the hBRCACox is the human homolog of rat trihydroxycoprostanoyl-CoA oxidase (rTHCCox) and that separate gene duplication events led to the occurrence in mammals of acyl-CoA oxidases with different substrate specificities. Northern blot analysis demonstrated that--in contrast to the rTHCCox gene--the hBRCACox gene is transcribed also in extrahepatic tissues such as heart, kidney, skeletal muscle, and pancreas. The highest levels of the 2.6-kb mRNA were found in heart, followed by liver. The enzyme is encoded by a single-copy gene, which was assigned to chromosome 3p14.3 by fluorescent in situ hybridization. It was absent from livers of Zellweger patients as shown by immunoblot analysis and immunocytochemistry.
人类肝脏中的过氧化物酶体含有两种具有不同底物特异性的独特酰基辅酶A氧化酶：（i）棕榈酰辅酶A氧化酶，氧化极长的直链脂肪酸和类二十烷酸；（ii）一种支链酰基辅酶A氧化酶（hBRCACox），参与长链支链脂肪酸和胆汁酸中间体的降解。支链脂肪酸和胆汁酸中间体的积累会导致患病儿童严重智力发育迟缓甚至死亡。在本研究中，我们报告了hBRCACox的分子特征，这是研究单酶缺乏症患者突变的先决条件。hBRCACox的复合cDNA序列来自通过免疫筛选和人类肝脏cDNA表达文库杂交分离的重叠克隆，由2225个碱基组成，包含一个2046个碱基的开放阅读框，编码一个681个氨基酸的蛋白质，计算分子量为76739道尔顿。该蛋白质的C末端三肽是SKL，这是一种已知的过氧化物酶体靶向信号。与其他酰基辅酶A氧化酶的序列比较和进化分析表明，尽管hBRCACox具有更广泛的底物特异性，但它是大鼠三羟基胆甾烷酰辅酶A氧化酶（rTHCCox）的人类同源物，并且不同的基因复制事件导致了哺乳动物中具有不同底物特异性酰基辅酶A氧化酶的出现。Northern印迹分析表明，与rTHCCox基因不同，hBRCACox基因也在心脏、肾脏、骨骼肌和胰腺等肝外组织中转录。在心脏中发现2.6 kb mRNA的水平最高，其次是肝脏。该酶由单拷贝基因编码，通过荧光原位杂交将其定位到染色体3p14.3。免疫印迹分析和免疫细胞化学显示，Zellweger患者的肝脏中不存在该酶。
