Molecular characterization of the human peroxisomal branchedchain acyl-CoA oxidase: cDNA cloning, chromosomal assignment, tissue distribution, and evidence for the absence of the protein in Zellweger syndrome

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.

人类肝脏过氧化物酶体（peroxisomes）含有两种底物特异性各异的酰基辅酶A氧化酶（acyl-CoA oxidases）：其一为棕榈酰辅酶A氧化酶，可催化极长链直链脂肪酸与类二十烷酸的氧化反应；其二为支链酰基辅酶A氧化酶（hBRCACox），参与长链支链脂肪酸及胆汁酸中间体的降解过程。支链脂肪酸与胆汁酸中间体的蓄积可引发严重智力发育迟缓，最终导致患病儿童死亡。本研究对hBRCACox进行了分子特征解析，这是研究单酶缺陷患者相关突变的必要前提。研究人员通过免疫筛选及人肝脏cDNA表达文库的杂交实验，获得了相互重叠的克隆，并由此得到hBRCACox的复合互补DNA（cDNA）序列：该序列全长2225个碱基，包含一段长度为2046个碱基的开放阅读框（open reading frame, ORF），可编码一个含681个氨基酸残基的蛋白质，理论分子量为76739 Da。该蛋白质的C端三肽序列为SKL，属于已知的过氧化物酶体靶向信号（peroxisome targeting signal, PTS）。通过与其他酰基辅酶A氧化酶的序列比对及进化分析可知：尽管hBRCACox具备更广泛的底物特异性，但其实为大鼠三羟基胆甾烷酰基辅酶A氧化酶（rTHCCox）的人类同源物；哺乳动物中底物特异性分化的酰基辅酶A氧化酶，源自多起独立的基因重复事件。Northern印迹分析（Northern blot）结果显示：与rTHCCox基因不同，hBRCACox基因在心脏、肾脏、骨骼肌及胰腺等肝外组织中亦可发生转录。该2.6 kb的mRNA转录本在心脏中表达水平最高，其次为肝脏。该酶由单拷贝基因编码，研究人员通过荧光原位杂交（fluorescent in situ hybridization, FISH）将其定位至3号染色体p14.3区域。免疫印迹分析与免疫细胞化学检测结果证实，泽韦格综合征（Zellweger syndrome）患者的肝脏组织中不存在该酶。