Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency.

Abstract

Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA thiolase (T2) (EC 2.3.1.9) were cloned and sequenced. The cDNA inserts in these clones were 1,518 bases in length when overlapped, and encoded the 427-amino acid precursor of this enzyme (45,199 mol wt). This amino acid sequence included a 33-residue leader peptide moiety and a 394-amino acid subunit of the mature enzyme (41,385 mol wt). The T2 gene expression in fibroblasts from four patients with 3-ketothiolase deficiency was analyzed by Northern blotting. The T2 mRNA in all four cell lines had the same 1.7 kb as that of the control. However, the amounts of T2 mRNA differed: the content was reduced in two cell lines (cases 1 and 3), whereas it was within a normal range in others (cases 2 and 4). Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the fibroblasts from these patients are present in the mitochondria. These results suggest that different mechanisms are involved in the enzyme defects in the four patients. (J. Clin.

Clinical and Mutational Characterizations of Ten Indian Patients with Beta-Ketothiolase Deficiency

“…It spans approximately 27 kb and contains 12 exons and 11 introns. T2 cDNA is about 1.5 kb long and encodes a precursor protein of 427 amino acids, including a 33-amino-acid leader polypeptide (Fukao et al 1990). Mutations in the ACAT1 gene are highly heterogeneous; to date, more than 70 mutations have been identified (Fukao et al 2010;unpublished data).…”

Molecular basis of zellweger syndrome,β-ketothiolase deficiency and mucopolysaccharidoses

“…No large deletion or insertion has been observed .in Southern blot analysis. Seyenteen mutations were identified in 13 T2-deficient patients: nine missense (Song et al, 1994), one nonsense , and five splice-site mutations , and two small deletions (Fukao et al, 1990). Two polymorphic base substitutions were also detected (Kuwahara et aL, 1992).…”

“…They differ in substrate specificity and/or intracellular localization. Human cDNAs for these thiolases have been cloned (T2: Fukao et al, 1990; TI: Abe et al, 1993;PT: Fairbairn and Tanner, 1989;CT: Song et al, 1994;TFP: Kamijo et al, 1994). There is 35-46% homology among the deduced amino acid sequences of these thiolases.…”

“…Human T2 is a homotetramer of the 41-kDa subunit (Middleton, 1973;Fukao et al, 1990). An anti-[rat T2] antibody can immunoprecipitate human T2 without cross-reactivity with other thiolases (Yamaguchi et aL, 1988).…”

“…An anti-[rat T2] antibody can immunoprecipitate human T2 without cross-reactivity with other thiolases (Yamaguchi et aL, 1988). Rat and human T2 cDNAs were cloned and sequence d (Fukao et al, 1989(Fukao et al, , 1990. Human T2 cDNA is about 1.5 kb long and is encoded for a precursor of 427 amino acids, including a 33-amino acid leader polypeptide.…”