Abu-Elheiga L, Almarza-Ortega D B, Baldini A, Wakil S J
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 1997 Apr 18;272(16):10669-77. doi: 10.1074/jbc.272.16.10669.
cDNA encoding the 280-kDa acetyl-CoA carboxylase 2 (ACC2) isoform was isolated from human liver using the polymerase chain reaction. Sequencing the cDNA revealed an open reading frame of 7,449 base pairs (bp) that encode 2,483 amino acids (Mr 279,380). Using 5-kilobase pair cDNA clones as probes, we localized the gene encoding the 280-kDa human carboxylase to chromosome 12q23. When the cDNA of ACC2 was compared with that of ACC1, the nucleotide sequences and the predicted amino acid sequences had about 60 and 80% identity, respectively. Ser77 and Ser79, which were found to be critical for the phosphorylation and subsequent inactivation of rat ACC1 (Ser78 and Ser80 of human ACC1), are conserved in ACC2 and are represented as Ser219 and Ser221, respectively. On the other hand, Ser1200, which is also a phosphorylation site in rat ACC1 (Ser1201 of human ACC1), is not conserved in ACC2. The homology between the amino acid sequences of the two human carboxylases, however, is primarily found downstream of residues Ser78 and Ser81 in human ACC1 and their equivalents, that is Ser219 and Ser221 in ACC2, suggesting that the sequence of the first 218 amino acids at the N terminus of ACC2 represents a unique peptide that accounts, in part, for the variance between the two carboxylases. Using a cDNA probe (400 bp) that encodes the N-terminal amino acid residues of ACC2 in Northern blot analyses of different human and mouse tissues showed that ACC2 is predominantly expressed in liver, heart, and the skeletal muscles. Polyclonal antibodies raised against the N-terminal peptide (amino acid residues 1-220) reacted specifically and equally with human and rat ACC2 carboxylases, confirming the uniqueness of this N-terminal peptide and its conservation in animal ACC2. In addition, we present evidence for the presence of an isoform of ACC2 (Mr 270,000) in human liver that differs from the 280-kDa ACC2 by the absence of 303 nucleotides that encode 101 amino acids in the region between Arg1114 and Asp1215. The regulation and physiological significance of the two ACC2 isoforms remain to be determined.
利用聚合酶链反应从人肝脏中分离出编码280 kDa乙酰辅酶A羧化酶2（ACC2）同工型的cDNA。对该cDNA进行测序后发现其开放阅读框为7449个碱基对（bp），编码2483个氨基酸（分子量279380）。使用5千碱基对的cDNA克隆作为探针，我们将编码280 kDa人羧化酶的基因定位到染色体12q23上。当将ACC2的cDNA与ACC1的cDNA进行比较时，核苷酸序列和预测的氨基酸序列的同一性分别约为60%和80%。已发现对大鼠ACC1（人ACC1的Ser78和Ser80）的磷酸化及随后的失活至关重要的Ser77和Ser79在ACC2中保守，分别表示为Ser219和Ser221。另一方面，也是大鼠ACC1（人ACC1的Ser1201）磷酸化位点的Ser1200在ACC2中不保守。然而，两种人羧化酶氨基酸序列之间的同源性主要存在于人ACC1中Ser78和Ser81及其等同物（即ACC2中的Ser219和Ser221）下游的区域，这表明ACC2 N端前218个氨基酸的序列代表一种独特的肽段，这在一定程度上解释了两种羧化酶之间的差异。在对不同人和小鼠组织进行的Northern印迹分析中，使用编码ACC2 N端氨基酸残基的cDNA探针（400 bp）表明，ACC2主要在肝脏、心脏和骨骼肌中表达。针对N端肽段（氨基酸残基1 - 220）产生的多克隆抗体与人及大鼠的ACC2羧化酶发生特异性且同等程度的反应，证实了该N端肽段的独特性及其在动物ACC2中的保守性。此外，我们提供证据表明人肝脏中存在一种ACC2同工型（分子量270000），它与280 kDa的ACC2不同，在Arg1114和Asp1215之间的区域缺少编码101个氨基酸的303个核苷酸。两种ACC2同工型的调节作用和生理意义仍有待确定。
