ABCMdb

PMID: 11063720

Unterrainer G, Molzer B, Forss-Petter S, Berger J
Co-expression of mutated and normal adrenoleukodystrophy protein reduces protein function: implications for gene therapy of X-linked adrenoleukodystrophy.
Hum Mol Genet. 2000 Nov 1;9(18):2609-16., [PubMed]

Sentences

No. Mutations Sentence Comment

43 ABCD1 p.Ala626Thr In accordance with these data, transient transfection of ALDP-deficient fibroblasts (A626T) with normal ALD cDNA resulted in restoration of β- oxidation. Login to comment 45 ABCD1 p.Asp194His We also transfected one of these cell lines, D194H, with an haemagglutinin (HA)-tagged normal construct to investigate the influence of mutated ALDP on correct peroxisomal targeting of normal ALDP when both are present in the same cell, a situation expected after gene transfer into autologous bone marrow cells of some X-ALD patients. Login to comment 49 ABCD1 p.Asn148Ser ABCD1 p.Ser213Cys ABCD1 p.Asp194His Average transfection efficiency was determined in three independent experiments and was 14% for the ALDP-deficient cell line (range 10-17%), 20% for the D194H cell line (range 15-23%), 26% for the S213C cells and 24% for the N148S cell line (range 22-30%). Login to comment 51 ABCD1 p.Asn148Ser ABCD1 p.Ala626Thr ABCD1 p.Asp194His Transient transfection of normal ALD cDNA into X-ALD fibroblast cell lines: A626T lack detectable ALDP, whereas N148S, D194H and S312C cell lines produce stable, mutated ALDP. Login to comment 56 ABCD1 p.Asp194His To be able to further distinguish between endogenously expressed normal ALDP and mutated ALDP, a 9 amino acid motif from the influenza haemagglutinin protein was added to the C-terminus of D194H ALDP. Login to comment 59 ABCD1 p.Asp194His HeLa Tet-on cells were transfected with HA-tagged D194H-ALD cDNA and stable transformants were selected. Login to comment 60 ABCD1 p.Asp194His PCR analysis confirmed the presence of both endogenous ALD gene and integrated D194H-ALD cDNA. Login to comment 61 ABCD1 p.Asp194His Based on indirect immunofluorescence studies one clone, HeLa-D194H, which showed low background expression and high inducibility, was finally selected. Login to comment 63 ABCD1 p.Asp194His No staining outside the peroxisomes above typical cellular background staining could be observed, indicating that overexpression of D194H-ALDP did not alter its localization. Login to comment 66 ABCD1 p.Asp194His Induction of D194H-ALD expression was verified by analysing mRNA and protein levels of both normal and mutated ALDP. Login to comment 67 ABCD1 p.Asp194His Northern blot analysis of the HeLa-D194H clone at various concentrations of doxycycline showed robust induction even at the lowest concentration (Fig. 3a). Login to comment 70 ABCD1 p.Asp194His Concerning the ratio of normal to mutated ALD mRNA, in HeLa-D194H without doxycycline the level of mutated ALD mRNA was ~30% of the level of endogenous ALD mRNA. Login to comment 75 ABCD1 p.Asp194His Western blot analysis was performed with HeLa-D194H cells at varying concentrations of doxycycline (Fig. 4). Login to comment 77 ABCD1 p.Asp194His ABCD1 p.Asp194His Indirect immunofluorescence analysis of the HA-tagged D194H-ALDP in doxycycline-induced HeLa-D194H cells. Login to comment 80 ABCD1 p.Asp194His Northern blot analysis of HeLa cells stably expressing mutated D194H-ALDP. Login to comment 99 ABCD1 p.Asp194His Mutated D194H-ALDP was induced by different doxycycline concentrations (Fig. 5a). Login to comment 100 ABCD1 p.Asp194His ABCD1 p.Asp194His A slight decrease in β-oxidation was observed for uninduced HeLa-D194H cells and after induction with the lowest doxycycline concentration. Login to comment 101 ABCD1 p.Asp194His Compared with normal HeLa Tet-on cells, the decrease for both uninduced and slightly induced HeLa-D194H cells was not statistically significant. Login to comment 102 ABCD1 p.Asp194His When expression of D194H-ALDP was further upregulated by higher doxycycline concentrations the rate of β-oxidation was markedly reduced and this reduction was statistically significant compared with normal HeLa Tet-on cells. Login to comment 103 ABCD1 p.Asp194His Thus, overexpression of mutated D194H-ALDP is sufficient to reduce β-oxidation in HeLa Tet-on cells. Login to comment 104 ABCD1 p.Asp194His In the induced HeLa-D194H cells the overall reduction in the rate of β-oxidation was ~45%, which is a less severe effect than the reduction of ~80% for X-ALD fibroblasts compared with healthy fibroblasts (Fig. 5a). Login to comment 106 ABCD1 p.Asp194His Western blot analysis of HeLa-D194H cells induced with increasing concentrations of doxycycline. Login to comment 118 ABCD1 p.Asp194His (b) Gas chromatographic analysis of VLCFA levels displayed as ratio of C26:0/C22:0 accumulation in HeLa-D194H and HeLa Tet-on control cells cultured in the presence of the indicated doxycycline concentrations for 1 week. Login to comment 120 ABCD1 p.Asp194His ABCD1 p.Asp194His ABCD1 p.Asp194His ABCD1 p.Asp194His To establish whether the decreased rate of β-oxidation also leads to intracellular accumulation of very long chain fatty acids, expression of D194H-ALDP was induced in HeLa-D194H cells for 1 week and the content of VLCFA was determined by gas chromatography. Login to comment 123 ABCD1 p.Asp194His In contrast, more pronounced expression of mutated D194H-ALDP at 2 or 5 µg/ml doxycycline resulted in a 7-fold increase in the accumulation of VLCFA compared with untreated cells. Login to comment 137 ABCD1 p.Asp194His To further elucidate the mechanism of interaction between normal and mutated ALDP, we constructed a HeLa Tet-on cell line with D194H mutated ALDP under a strong inducible promoter. Login to comment 138 ABCD1 p.Asp194His Northern blot analysis of HeLa-D194H cells at different levels of induction revealed that the amount of endogenous ALD mRNA remains constant, independent of the level of overexpression of mutated ALD mRNA. Login to comment 148 ABCD1 p.Asp194His However, direct interaction between D194H mutated ALDP and normal ALDP is still hypothetical and there is no direct evidence that such heterodimers are completely non-functional. Login to comment 149 ABCD1 p.Ser213Cys ABCD1 p.Asp194His ABCD3 p.Asn148Ser Of the three mutations analysed in transient transfection assays, N148S and S213C are located in transmembrane domains, whereas D194H is in the first cytosolic loop of the ALDP (Fig. 6). Login to comment 150 ABCD1 p.Ser213Cys ABCD1 p.Asp194His ABCD3 p.Asn148Ser In yeast two-hybrid experiments performed with ALDP lacking the N-terminal 361 amino acids, homodimers still formed (13), arguing against the possibility that the three mutations N148S, D194H and S213C interfere with dimerization of ALDP. Login to comment 155 ABCD1 p.Ser213Cys ABCD1 p.Asp194His ABCD3 p.Asn148Ser Missense mutations N148S and S213C are both located within putative transmembrane domains and D194H is located in the first cytosolic loop. Login to comment 160 ABCD1 p.Asn148Ser ABCD1 p.Asp194His Three different fibroblast cell lines described as having normal levels of non-functional ALDP were obtained: fibroblasts N148S (26-29, patient 55 in ref. 26) were kindly provided by Dr Aubourg (Paris), D194H (kindred no. Login to comment 161 ABCD1 p.Ser213Cys 12 in ref. 30) were provided by Dr Wanders (Amsterdam) and cell line S213C (31) was obtained from Drs Holzinger and Roscher (Munich). Login to comment 162 ABCD1 p.Ala626Thr SV40-transformed X-ALD fibroblasts lacking ALDP (mutation A626T) were kindly provided by Drs Smith and Braiterman (Baltimore, MD) (32). Login to comment 169 ABCD1 p.Asp194His To introduce the point mutation D194H into the pTRE-ALD expression plasmid, in vitro mutagenesis was performed using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Login to comment 176 ABCD1 p.Asp194His ABCD1 p.Asp194His For stable transfection of 1 × 106 HeLa Tet-on cells, 40 µg of D194H-ALD expression plasmid plus 2 µg of hygromycin resistance-conferring plasmid (pTK-Hyg; Clontech) were co-transfected at 220 V with a capacitance of 950 µF (Gene pulser and capacitance extender plus; BioRad, Hercules, CA) in 0.4 cm cuvettes. Login to comment 180 ABCD1 p.Asp194His Identification of transfected clones by PCR DNA from 24 HeLa Tet-on clones was subjected to PCR analysis, resulting in two fragments of different sizes: the endogenously expressed genomic ALD DNA was amplified as a 334 bp fragment (including 145 bp from intron 6) whereas clones with the D194H-ALD cDNA gave an additional fragment of 189 bp. Login to comment 182 ABCD1 p.Asp194His Of 24 clones analysed, 10 carried the mutated D194H-ALD cDNA. Login to comment 185 ABCD1 p.Asp194His Finally, one clone, HeLa-D194H, was selected that showed high inducibility and no background expression. Login to comment 186 ABCD1 p.Asp194His Northern blot D194H-ALDP expression was induced by different doxycycline concentrations. Login to comment