Biochemical characterization of X-linked adrenoleukodystrophy using a fibroblast cell model and a yeast model
Date Issued
2007
Date
2007
Author(s)
Chen, Yu-Jen
DOI
en-US
Abstract
X-linked adrenolukodystrophy (X-ALD) is impaired β-oxidation of very long chain fatty acids resulted from mutations of the ABCD1 gene which encodes a peroxisomal membrane protein (ALDP) with an ATP binding cassette. The principal biochemical characterization is abnormality of X-ALD which is the accumulation of saturated very long chain fatty acids (VLCFAs, ≧C22:0, mainly C26:0) in patient’s tissues, plasma and skin fibroblasts. ALDP is a putative transporter of very long chain fatty acids, but the detailed molecular mechanism of the disease and physiological effects of the accumulated VLCFAs are still not clear. To investigate possible physiological effects of the accumulated VLCFAs, fibroblast cell models were used to examine gene expression profiles before and after hexacosanoic acid (C26:0, 1µg/ml) challenge. Before hexacosanoic acid challenge, the intracellular level of C26:0 in X-ALD fibroblasts (0.02687% of total fatty acids) was about 3 folds to that of two fibroblast cell lines derived from normal controls (0.0095% & 0.01053% of total fatty acids). After hexacosanoic acid challenge, about 2-fold increase of C26:0 level was determined in X-ALD (0.052% of total fatty acids) compared to that of normal fibroblasts (0.021% of total fatty acids). Gene expression profiles were also compared between normal and X-ALD fibroblasts before and after VLCFA challenge using a microarray strategy. There was no significant difference in gene expression profiles before and after VLCFA challenge as analyzed by cDNA microarray of approximately 8,000 unique genes. Nevertheless, there were 6 differentially expressed genes of >2-fold differences between cultured X-ALD and normal skin fibroblasts by both cDNA microarray and real-time PCR. It was estimated an 8-15-fold increase of TFPI2 protein and a 1.5-3-fold decrease of DDEF2 protein expression in X-ALD compared to that in normal fibroblasts using Western blotting. After treatment of 1,2-dioleoylglycerol (1,2-DOG), the accumulation of VLCFAs was reduced by 14~20% (the 20% reduction of the intracellular C26:0 level in X-ALD fibroblasts after 1,2-DOG treatment was approximately one-third of the difference between normal and X-ALD skin fibroblasts before treatment). The mechanism of this reduction may possibly be due to the ability of phosphatidic acid (PA) and diacylglycerol (DAG) to initiate peroxisome division and proliferation resulting in the increased activity of VLCFAs β-oxidation or to activate the activity of ARF1 GAP involved in peroxisomal biogenesis. The above results suggest that mutations in the ABCD1 gene resulted in differential gene expression, and 1,2-DOG is useful for the reduction of VLCFAs in X-ALD skin fibroblasts model in vitro.
In the yeast model, we confirmed that deletion of the FAT1 gene resulted in the accumulation of VLCFAs in vivo. The addition of His tag at the carboxyl terminus of Fat1p, though not significantly affecting cellular VLCFA metabolism, does show different influences on cellular VLCFA accumulation of different carbon chain lengths. We had successfully expressed and purified recombinant Fat1p and used it to produce anti-Fat1p antibody with high purity for use in the detection of Fat1p in yeast protein extract. In order to verify whether purified Fat1p has VLCS activity in vitro, MBP-FAT1-6His fusion protein expressed in prokaryotic over-expression system was subjected to activity evaluation. There was no apparent fatty acyl-CoA synthetase activity for purified MBP-FAT1-6His using both C18:0 and C26:0 as substrates by both HPLC and radioactive isotope based analyses. The cause for the lack of VLCS activity for MBP-FAT1-6His will be further investigated.
Subjects
腎上腺大腦白質退化症
極長鏈乙醯基輔酶
A合成酶
極長鏈飽和脂肪酸
基因表現
微陣列晶片
即時定量聚合酶
鏈反應
融合蛋白質
X-linked adrenolukodystrophy
Very long chain acyl-CoA synthetase
Very long chain fatty acid
Gene expression profil
, Microarray
Real-time PCR
MBP-FAT1- 6His fusion protein
Type
thesis