Cloning of human acetyl‐CoA carboxylase cDNA

Abstract

Acetyl-CoA carboxylase is the rate-limiting enzyme in the biogenesis of long-chain fatty acids. In order to understand the mechanisms that regulate human acetyl-CoA carboxylase at the gene level, and the relationship between its structure and function, cDNA clones for human acetyl-CoA carboxylase have been isolated and sequenced. Human acetyl-CoA-carboxylase cDNA contains 7020 nucleotides encoding a protein of 2340 amino acids with a calculated relative molecular mass of 264575. The human enzyme shows approximately 85 % identity in nucleotide sequence with previously cloned rat acetyl-CoA carboxylase, and shows 90% identity in the amino acid sequence. Two human acetyl-CoA-carboxylase mRNA species, which differ in the 5' untranslated region with the same coding sequence, have been identified. The sequence analysis reveals that type I and type I1 acetyl-CoA-carboxylase mRNA contain 31 3-and 173-base-long 5' untranslated regions, respectively. The first 240 nucleotides in the 5' untranslated region of type I acetyl-CoA-carboxylase mRNA replace the first 100 nucleotides of the (G+C)-rich region of the 5' untranslated region of the type I1 mRNA. These two species of mRNAs are the only species of human ACC mRNA which have been detected compared to at least five species in rat tissues, and they are expressed in a tissue-specific manner.Acetyl-CoA carboxylase (ACC) catalyzes the fate-limiting reaction in the biogenesis of long-chain fatty acids, namely the ATP-dependent carboxylation of acetyl-CoA into malonyl-CoA [l -31. The subunit relative molecular mass of ACC from animal sources is approximately M, 260000; thus, it represents one of the largest proteins with enzyme activity. The protomer contains two identical subunits which undergo polymerization in the presence of activator molecules, such as citrate, into aggregates with M, values of several million [4]. The extremely large size of the subunit and its property of aggregation into different polymeric states have hindered our progress toward understanding the primary structure of the enzyme as well as its structure/function relationship. Since the enzyme is readily broken down by proteolysis, the real size of the enzyme was also a matter of dispute for a long time. Recent success in cloning the rat [5] and chicken enzyme [6] has provided some information of the size, primary structure, biotin-binding site [7] and other putative functional sites of the protein. However, there has not been any similar information on the human enzyme, largely because of the lack of suitable human tissues in addition to the difficulties involved in studying the enzyme itself, as indicated above. Regulatory mechanisms for the rat liver enzyme include covalent modification of ACC protein ; phosphoryla-

Cloning and characterization of the 5′ end and promoter region of the chicken acetyl-CoA carboxylase gene

“…If we assume that this similarity exists upstream as well, the upper extended bands become compatible with the presence of an additional exon about 240 bp in length, similar to exon 1 described in the rat and human ACC genes [25,49].…”

“…Thus we may conclude that the splicings observed in different class-2 rat ACC mRNAs are not present in chicken liver or other tissues. The same situation occurs in humans [25].…”

“…These upper bands could reflect non-specific elongation or the presence of multiple species of ACC mRNA in chicken liver, transcribed upstream and coding for an additional exon. The structure of rat [22][23] and human [25] liver mRNA supports the latter hypothesis, since in these species two classes of primary transcripts are generated by two promoters PI and PII [25,49].…”

“…It is also proposed that there are several ACC mRNA species differing in their 5h-untranslated regions. Moreover, class-2 ACC mRNAs are subject to differential splicing in the rat and mouse [25]. They result from the loss of the 47 bp exon or the appearance in the rat of a new 61 bp exon [24].…”

“…In liver and mammary gland, two spatially distinct promoters (PI and PII) generate two primary transcripts, which are differently spliced to produce five mRNAs differing in the composition of their 5h-untranslated regions [22][23][24]. In humans, two mRNAs differing in their 5h-untranslated region are also presumably generated by two promoters [25]. In this species a recent study suggests the presence of two genes, giving rise to two mRNAs differing in their 3h end [26].…”