Biochemical characterization of two functional human liver acyl-CoA oxidase isoforms 1a and 1b encoded by a single gene

Abstract

Human acyl-CoA oxidase 1 (ACOX1) is a rate-limiting enzyme in peroxisomal fatty acids beta-oxidation and its deficiency is associated with a lethal, autosomal recessive disease, called pseudoneonatal-adrenoleukodystrophy. Two mRNA variants, transcribed from a single gene encode ACOX1a or ACOX1b isoforms, respectively. Recently, a mutation in a splice site has been reported [H. Rosewich, H.R. Waterham, R.J. Wanders, S. Ferdinandusse, M. Henneke, D. Hunneman, J. Gartner, Pitfall in metabolic screening in a patient with fatal peroxisomal beta-oxidation defect, Neuropediatrics 37 (2006) 95-98.], which results in the defective peroxisomal fatty acids beta-oxidation. Here, we show that these mRNA splice variants are expressed differentially in human liver. We investigated the biochemical role of the two human ACOX1 isoforms by heterologous expression of the catalytically active ACOX1a and ACOX1b enzymes in Escherichia coli. ACOX1a seems to be more labile and exhibits only 50% specific activity toward palmitoyl-CoA as compared to ACOX1b.

Figure 1

A, Human ACOX1a or ACOX1b mRNAs expression analysis by real time PCR. Messenger RNAs isolated from human liver were reverse transcribed and subjected to quantitative real time PCR using specific primers. Bars represent the relative level of ACOX1a and ACOX1b mRNA using the housekeeping 18S mRNA amplification as a reference gene. The fold change in gene expression in liver was calculated and presented as the mean ± S.D. of two real-time PCR runs performed in triplicate from 3 pooled total RNA samples of human liver (Ambion). B, SDS-PAGE analysis of purified recombinant human peroxisomal ACOX1a and ACOX1b proteins. Lane 1, molecular weight marker (MM) in kDa; Lane 2, ACOX1a (5μg); Lane 3, ACOX1b (5μg).

Figure 2

A, effect of pH on the specific activities of ACOX1a and ACOX1b. Assays were carried out with 5 μg of enzyme purified and palmitoyl-CoA at 50 μM as substrate. Closed circles. B, temperature-dependent activation of ACOX1a and ACOX1b. Assays were carried out with 5 μg of purified enzyme and palmitoyl-CoA at 50 μM as substrate in 50 mM phosphate buffer, pH 7.5 at the indicated temperature. C, denaturation curves of ACOX1a and ACOX1b. Denaturation was started by incubation of purified enzyme in 50 mM phosphate buffer, pH 7.5 for 15 minutes at the indicated temperature followed by an activity assay with palmitoyl-CoA at 50 μM in 50 mM phosphate buffer, pH 7.5 at 30°C using 5 μg of the enzyme. Closed circles, ACOX1a; open circles, ACOX1b.

Figure 3

Straight chain acyl-CoA specificity of recombinant human ACOX1a and ACOX1b. Specific activities of ACOX1a (closed circles) and ACOX1b (open circles), measured as described in “Material & Methods”, were plotted against a number of atoms of carbon of acyl-CoA from C4 to C18. Values represent the mean of three assays (± SD). Assay contained acyl-CoA at 50 μM and 1 μg of ACOX1a or ACOX1b.

Figure 4

ACOX1 isoforms expression in COS-7 cells. Western blot analysis and corrected specific activity measurements in COS-7 crude extracts (100 μg proteins). ACOX activities were standardized by beta-galactosidase activity and corrected by the amount of ACOX1 expressed in COS-7 cells evaluated by densitometry. The ratio was calculated by dividing the value of corrected specific activity of lane 3 by the value of lane 2 or lane 4. Lane 1, control COS-7 cells transfected with pTriex4 empty vector; lanes 2, 3 and 4 COS-7 cells transfected with pTriEx-4-ACOX1a, pTriEx-4-ACOX1b or both vectors respectively; lane 5, control recombinant ACOX1b produced in bacteria with the pTriEx-4-ACOX1b vector.