Oct 30, 1986 - analysis of the peroxisomal 13-oxidation enzymes revealed an almost complete lack of the bifunctional enzyme in neonatal. ALD liver, similar ...
Proc. Natl. Acad. Sci. USA Vol. 84, pp. 1425-1428, March 1987 Medical Sciences
Peroxisomal 13-oxidation enzyme proteins in adrenoleukodystrophy: Distinction between X-linked adrenoleukodystrophy and neonatal adrenoleukodystrophy WINSTON W. CHEN*, PAUL A. WATKINS*, TAKASHI OSUMIt, TAKASHI HASHIMOTOt,
AND
HUGO W. MOSER*:
*Kennedy Institute and Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and tDepartment of Biochemistry, Shinshu University School of Medicine, Matsumoto, Nagano 390, Japan
Communicated by John W. Littlefield, October 30, 1986
Very long chain fatty acids, which accumuABSTRACT late in plasma and tissues in X-linked adrenoleukodystrophy (ALD), neonatal ALD, and the Zellweger cerebrohepatorenal syndrome, are degraded by the peroxisomal ,8-oxidation pathway, consisting of acyl-CoA oxidase, the bifunctional enoylCoA hydratase/3-hydroxyacyl-CoA dehydrogenase, and 13ketothiolase. A marked deficiency of all three enzyme proteins was reported in livers from patients with the Zellweger syndrome, a disorder in which peroxisomes are decreased or absent. Peroxisomes are not as markedly decreased in neonatal ALD and appear normal in X-linked ALD. Immunoblot analysis of the peroxisomal 13-oxidation enzymes revealed an almost complete lack of the bifunctional enzyme in neonatal ALD liver, similar to the finding in Zellweger tissue. In contrast, acyl-CoA oxidase and f3-ketothiolase were present in neonatal ALD liver, although the thiolase appeared to be in precursor form (2-3 kDa larger than the mature enzyme) in neonatal ALD. Unlike either neonatal ALD or Zellweger syndrome, all three peroxisomal 13-oxidation enzymes were present in X-linked ALD liver. Despite the absence in neonatal ALD liver of bifunctional enzyme protein, its mRNA was detected by RNA blot analysis in fibroblasts from these patients. These observations suggest that lack of bifunctional enzyme protein in neonatal ALD results from either abnormal translation of the mRNA or degradation of the enzyme prior to its entry into peroxisomes.
The term adrenoleukodystrophy (ALD) refers to a group of degenerative neurological disorders characterized by demyelination and by cytoplasmic lipid inclusions in the central nervous system, adrenal cortical cells, and Schwann cells (1-3). Several forms of the disorder have been described. In neonatal ALD, clinical symptoms are present at birth and an autosomal recessive mode of inheritance is followed (4). This differs from the better known childhood form of ALD, an X-linked recessive disorder, in which symptoms appear after 3-5 years of normal development (2). The cytoplasmic lipid inclusions observed in these disorders were shown to be cholesterol esterified to very long chain fatty acids (VLCFA) (5); both disorders are characterized biochemically by increased levels of VLCFA in both plasma and cultured skin fibroblasts (6). It is currently believed that both neonatal ALD and X-linked ALD are disorders of the peroxisome. Peroxisomes are spherical or ovoid organelles (diameter, 0.1-1.5 ,um) limited by a single membrane that are ubiquitous in mammalian cells (7-10). These organelles participate in a number of metabolic cellular processes-e.g., 13-oxidation of fatty acids (11, 12), plasmalogen biosynthesis (13, 14), and bile acid synthesis (15, 16). Peroxisomes are decreased or absent in
liver and kidney from patients with the Zellweger cerebrohepatorenal syndrome (17, 18), which is characterized biochemically by accumulation of VLCFA in plasma, tissues, and fibroblasts (19-21); decreased plasmalogen content of tissues (22, 23); and accumulation of pipecolic acid (24, 25) and bile acid synthesis intermediates (26-28) in tissues. The Zellweger syndrome is usually fatal within the first year of life (21), suggesting that peroxisomes play a vital role in normal cellular metabolism. Neonatal ALD bears many similarities to the Zellweger syndrome, both clinically and biochemically (4). Peroxisomes were decreased in both size and number in neonatal ALD liver biopsies, although not as markedly as in the Zellweger syndrome (29), whereas normal numbers ofnormal appearing peroxisomes were present in liver biopsies from X-linked ALD patients (29). Increased VLCFA (4, 19), pipecolic acid (4, 30), and bile acid intermediates (29), as well as decreased plasmalogens (4), are features of both neonatal ALD and Zellweger syndrome. However, the longer life-span and milder clinical course observed in neonatal ALD (4), along with the fact that peroxisomes are not totally absent (29), suggest that neonatal ALD may be a less severe variant of the Zellweger syndrome. In contrast to the multiple defects seen in neonatal ALD and Zellweger, only increased VLCFA have been observed in X-linked ALD (6, 29, 31). Previous results support the hypothesis that there is decreased capacity to degrade VLCFA by 1-oxidation in all three disorders (32, 33). The recent finding that a VLCFA, lignoceric acid, is oxidized primarily in peroxisomes in rat liver homogenates suggests that X-linked ALD may also be a peroxisomal disorder (34). The peroxisomal p-oxidation pathway, while functionally similar to the better known mitochondrial system, consists primarily of three enzymes that are immunologically distinct from their mitochondrial counterparts. They include acylCoA oxidase (35, 36), a bifunctional enzyme containing enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities (37, 38), and 13-ketothiolase (39). Recently, Tager et al. (40) reported that there was a marked deficiency of all three peroxisomal f3-oxidation enzymes in liver samples from three Zellweger patients, whereas the amount of catalase present was normal; these studies were done by immunoblot techniques with use of antibodies prepared against the purified rat liver proteins. In this report, we have used these antibodies to detect peroxisomal p-oxidation enzymes in liver samples from both neonatal ALD and X-linked ALD patients. MATERIALS AND METHODS Materials and General Methods. Protein A was obtained from Sigma; carrier-free Nal25I was from Amersham; nitro-
The publication costs of this article were defrayed in part by page charge
Abbreviations: ALD, adrenoleukodystrophy; VLCFA, very long chain fatty acid. tTo whom reprint requests should be addressed.
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1425
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cellulose filters were from Schleicher & Schuell; reagents for electrophoresis were from Bio-Rad; sodium sarcosine was from Eastman Kodak. Samples of postmortem liver were obtained from patients diagnosed as having either X-linked ALD, neonatal ALD, or the Zellweger syndrome within 12 hr of death and were stored at -80TC. Protein A was labeled with 125I according to the procedure of Greenwood et al. (41). Antibodies to purified rat liver acyl-CoA oxidase, bifunctional protein, and g-ketothiolase were prepared as described (36, 38, 39). A recombinant cDNA clone of rat peroxisomal bifunctional enzyme (pMJ26) was prepared as described (42). Protein was determined by the method of Lowry et al. (43). Immunoblot Analysis. Liver samples, stored at -80'C, were allowed to thaw in ice-cold 1 mM potassium phosphate (pH 7.4) (25 mg of tissue per ml), and then homogenized in a Ten-Broeck tissue grinder. Phenazinemethylsulfonyl fluoride was added to a final concentration of 1 mM, and 1-ml portions of homogenate were sonicated by five pulses of 20 sec each in a Heat System/Ultrasonics (Plainview, NY) sonicator operated at 60% full power. After centrifugation for 5 min in a Fisher Microcentrifuge in the cold, the supernatant fraction was removed and trichloroacetic acid was added to a final concentration of 10%. Precipitated proteins were collected by centrifugation as described above, solubilized in 1% sodium dodecyl sulfate containing 50 mM dithiothreitol, and subjected to electrophoresis on 10% acrylamide gels according to the method of Laemmli (44). Each gel lane contained protein corresponding to 17.5 mg (wet weight) of liver. Proteins were then transferred from gels to nitrocellulose filters in 25 mM Tris-HCl (pH 8.3), containing 192 mM glycine, 0.01% sodium dodecyl sulfate, and 20% (vol/vol) methanol, at 4 mA/cm2 for 8 hr. Nitrocellulose filters were incubated with buffer (10 mM sodium phosphate, pH 7.5/0.15 M NaCl/0.2% Triton X-100/1 mM EGTA/1 mM NaN3) containing 4% bovine serum albumin for 30 min at room temperature, after which antibody was added and the incubation was continued overnight. Filters were washed with several changes of buffer to remove antibody, and then incubated for 2 hr with 125I-labeled protein A (-106 cpm/ml). Filters were washed exhaustively to reduce background radiation, dried overnight, and exposed to Kodak X-Omat AR film for 1-4 days. RNA Blot Analysis of Peroxisomal Bifunctional Enzyme mRNA. The cDNA coding for the peroxisomal bifunctional enzyme (42) was used to determine mRNA levels in cultured fibroblasts from normal controls and from patients with X-linked ALD or neonatal ALD. Cultured cells (2 x grown to 85% confluence in roller bottles in minimal essential medium containing 10%6 fetal bovine serum, were harvested by scraping in 5 ml of 4 M guanidine isothiocyanate containing 14% 2-mercaptoethanol and 0.1 M Tris HCl (pH 7.4) and homogenized with 10 strokes of a tight-fitting pestle in a glass vessel. Cellular RNA was isolated from the homogenate by precipitation with 38% ethanol containing 0.1 M sodium acetate and 0.25% sodium sarcosine at -20°C for 20 hr and purified by centrifugation in a 5.7 M CsCl solution for 20 hr at 200C in a swinging bucket rotor operated at 100,000 x g according to the procedure of Chirgwin et al. (45). Cellular RNA was modified by glyoxalation by the procedure of Thomas (46), and the modified RNA (5 ,g per gel lane) was analyzed on a 1.0% agarose gel in 10 mM sodium phosphate (pH 7.0) at 2 V/cm for 4 hr. RNA was then transferred to a nitrocellulose filter for RNA blot analysis. 32P-labeled cDNA (1.2-4.7 x 108 cpm/,ug) coding for the bifunctional enzyme was prepared by using the large fragment of DNA polymerase I with a 2.3-kilobase (kb) single-stranded fragment as a template and random oligonucleotides (hexamers) as primers (47). Hybridization with the nitrocellulose filter was as described (42) using 0.05 ug of 32P-labeled cDNA. Filters were exposed to Kodak X-Omat AR film for 1-2 days.
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RESULTS Immunoblot Analysis of Peroxisomal ,3-Oxidation Enzymes. Peroxisomal fatty acid 8-oxidation enzymes in liver samples from both neonatal ALD and X-linked ALD patients were detected by immunoblot analysis with antibodies raised against the purified rat liver proteins. These antibodies had previously been shown to cross-react with the human liver enzymes (40). Studies of liver samples from two X-linked ALD patients showed that all three proteins, the 78-kDa bifunctional enzyme (Fig. 1), the 41-kDa ,-ketothiolase (Fig. 2), and acyl-CoA oxidase (consisting of 52-kDa and 72-kDa peptides, Fig. 3), were present. In contrast to X-linked ALD, the bifunctional enzyme was markedly deficient in neonatal ALD liver samples (Fig. 1); the level of this enzyme was below the detection limit of the immunoblot analysis. In agreement with the findings of Tager et al. (40), bifunctional enzyme was also deficient in the liver of patients with Zellweger syndrome (Fig. 1). 3-KetoacylCoA thiolase was detected in neonatal ALD liver samples (Fig. 2); however, the molecular mass of this enzyme was apparently 2-3 kDa larger than that of the thiolase in control and X-linked ALD liver samples. A small amount of protein that comigrated with the normal enzyme was also present in neonatal ALD liver. Fatty acyl-CoA oxidase was also detected in both neonatal ALD liver samples (Fig. 3). In control and X-linked ALD samples, the 52-kDa subunit appeared to be present at the same or a higher level than the 72-kDa subunit (Fig. 3); in contrast, more 72-kDa than 52-kDa subunit was observed in neonatal ALD livers. RNA Blot Analysis of Bifunctional Enzyme mRNA. The absence of bifunctional enzyme in neonatal ALD liver correlates with the accumulation of VLCFA and impaired VLCFA oxidation observed in this disorder. However, a primary defect in the bifunctional enzyme gene is not likely since multiple peroxisomal processes are affected in this disease. Bifunctional enzyme has also been detected by immunoblot analysis in normal human skin fibroblasts; as observed in liver, the enzyme was not present in neonatal ALD fibroblasts (unpublished data). Using a 2.3-kb rat bifunctional enzyme cDNA as a probe, bifunctional enzyme mRNA was detected in both normal, X-linked ALD, and neonatal ALD fibroblasts by RNA blot analysis (Fig. 4). The kDa 94 67 -
No
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FIG. 1. Immunoblot analysis of peroxisomal bifunctional enzyme. Autopsy liver samples were prepared, subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and blotted onto nitrocellulose membranes as described. After incubation with antibody specific for the peroxisomal bifunctional enzyme, bound immunoglobulins were detected by I251-labeled protein A binding and autoradiography. Lanes: C, liver sample from control patient; Z, Zellweger syndrome; Ni and N2, neonatal ALD; Al and A2, X-linked ALD. Amounts of sample protein applied to the gel are as follows (mg per gel lane): lane C, 0.78; lane Ni, 1.39; lane N2, 1.29; lane Al, 1.22; lane A2, 1.10; lane Z, 1.45. Positions of molecular size markers are indicated on the left.
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Proc. Natl. Acad. Sci. USA 84 (1987)
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size of the mRNA from neonatal ALD fibroblasts was the same as from controls and X-linked ALD and was 3.0 kb. DISCUSSION The absence of peroxisomes in liver from patients with the Zellweger syndrome is accompanied by an accumulation of VLCFA in various lipids and a number of other biochemical abnormalities (19-28). An impairment in oxidation of VLCFA in Zellweger syndrome fibroblasts (21), and the lack of immunologically detectable peroxisomal /-oxidation enzymes in Zellweger syndrome liver (40), is in agreement with the hypothesis that the peroxisome is the primary site of VLCFA oxidation (34). VLCFA accumulation and impaired VLCFA oxidation are features of both X-linked ALD and neonatal ALD (4, 6, 19, 29, 31-33), suggesting that these diseases are also peroxisomal disorders. As indicated in the Introduction, neonatal ALD and the Zellweger syndrome exhibit many other clinical and biochemical similarities (4, 6, 17-30). Thus, it was of importance to determine whether any of the peroxisomal /oxidation enzymes were abnormal in neonatal ALD or X-linked ALD. In X-linked ALD liver samples, acyl-CoA oxidase, bifunctional enzyme, and thiolase were all clearly present. The presence of immunologically cross-reacting proteins, however, does not rule out the possibility that one or more of these enzymes may be catalytically defective in this disorder. Immunoblot analyses of neonatal ALD liver enzymes clearly differed from those of both X-linked ALD and the Zellweger syndrome. Acyl-CoA oxidase and ,B-ketothiolase were immunologically detected in neonatal ALD but not in Zellweger
I 67-
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_
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FIG. 4. RNA blot analysis of fibroblast mRNA using cDNA coding for bifunctional enzyme as probe. Cellular mRNA was isolated from cultured skin fibroblasts of controls and patients with either neonatal ALD or X-linked ALD. RNA blot analysis was performed as described using a labeled 2.3-kb cDNA insert of the plasmid pMJ26 that codes for the bifunctional enzyme. Lanes: C, control fibroblast mRNA; N, neonatal ALD mRNA; A, X-linked ALD mRNA. Molecular size markers (kb) are indicated on the left; sedimentation coefficients are shown on the right.
syndrome liver, whereas bifunctional enzyme was absent in both disorders. It has been shown that in rat liver, all three peroxisomal /3-oxidation enzymes are synthesized in the cytosol on free polyribosomes; these enzymes are then transported into preexisting peroxisomes (48-50). Acyl-CoA oxidase and bifunctional enzyme were synthesized in a cell-free protein translation system as mature enzymes identical to those of liver peroxisomes (48, 49). On the other hand, f3-ketothiolase was synthesized as a precursor that was both more basic and =3 kDa larger than the mature protein (48, 50). The larger thiolase observed in neonatal ALD liver samples appeared to have the same mobility as the thiolase precursor synthesized in vitro (48, 50). The presence of very low concentrations of high molecular mass /3-ketothiolase in some Zellweger syndrome liver samples was also reported (40). These findings support the possibility that the thiolase present in neonatal ALD and some Zellweger syndrome liver samples is the precursor rather than the mature enzyme. The presence of mRNA of the bifunctional enzyme in neonatal ALD fibroblasts in conjunction with the absence of the protein measured by immunoblot analysis could be caused by either a primary defect in the enzyme or a secondary phenomenon. Decreased bifunctional enzyme protein could result from a genetic mutation that causes either a lack of translation or increased susceptibility to proteolysis prior to transport into peroxisomes. A secondary phenomenon seems much more likely because of the existence in neonatal ALD of multiple biochemical abnormalities, all of which appear to be related to defective peroxisomal structure.
1
C
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FIG. 2. Immunoblot analysis of peroxisomal /-ketothiolase. Nitrocellulose membranes as described in Fig. 1 were incubated with antibody specific for peroxisomal /-ketothiolase; detection of immunoreactive proteins was as in Fig. 1.
kDa 94
6 0
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FIG. 3. Immunoblot analysis of peroxisomal acyl-CoA oxidase. Nitrocellulose membranes as described in Fig. 1 were incubated with antibody specific for peroxisomal acyl-CoA oxidase; detection of immunoreactive proteins was as in Fig. 1.
Liver peroxisomes in neonatal ALD, while diminished, are not as severely decreased as in the Zellweger syndrome (29). This, as well as the significantly milder course and longer life-span associated with neonatal ALD (26), suggests that neonatal ALD may be a milder variant of the Zellweger syndrome. The absence of all three peroxisomal /3-oxidation enzymes in the Zellweger syndrome has been attributed to the susceptibility of these proteins to proteolytic degradation in the absence of peroxisomal structure (40). The absence of bifunctional enzyme protein despite the presence of its mRNA in neonatal ALD fibroblasts suggests that the protein
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may be rapidly degraded in the cytoplasm prior to its entry into peroxisomes. The presence of smaller fragments in the immunoblot shown in Fig. 1 is consistent with this hypothesis. The finding that in neonatal ALD only the bifunctional protein is markedly deficient suggests that this protein is more affected by a reduced number of peroxisomes than are acyl-CoA oxidase and 8-ketothiolase. Since all three enzymes are synthesized on free polyribosomes and then transported into peroxisomes, a systematic analysis of the defect in neonatal ALD may shed light on how these enzymes are targeted to and enter peroxisomes. We thank Steven M. Willi and Edward V. Ferrell, Jr., for expert technical assistance. This work was supported by Grant HD 10981 from the National Institutes of Health. 1. Schaumburg, H. H., Richardson, E. P., Suzuki, K. & Raine, C. S. (1974) Arch. Neurol. 31, 210-213. 2. Schaumburg, H. H., Powers, J. M., Raine, C. S., Suzuki, K. & Richardson, E. P., Jr. (1975) Arch. Neurol. 32, 577-591. 3. Powers, J. M. & Schaumburg, H. H. (1974) Arch. Neurol. 30, 406-408. 4. Kelley, R. I., Datta, N. S., Dobyns, W. B., Hajra, A. K., Moser, A. B., Noetzel, M. J., Zackai, E. H. & Moser, H. W. (1986) Am. J. Med. Genet. 23, 869-901. 5. Johnson, A. B., Schaumburg, H. H. & Powers, J. M. (1976) J. Histochem. Cytochem. 24, 725-730. 6. Moser, H. W., Moser, A. B., Singh, I. & O'Neill, B. P. (1984) Ann. Neurol. 16, 628-641. 7. de Duve, C. & Baudhuin, P. (1966) Physiol. Rev. 46, 327-357. 8. de Duve, C. (1969) Proc. Roy. Soc. London Ser. B 173, 71-83. 9. Hogg, J. F., ed. (1969) Ann. N. Y. Acad. Sci. 168, 209-381. 10. Tolbert, N. E. (1981) Annu. Rev. Biochem. 50, 133-157. 11. Lazarow, P. B. & de Duve, C. (1976) Proc. Nati. Acad. Sci. USA 73, 2043-2046. 12. Lazarow, P. B. (1978) J. Biol. Chem. 253, 1522-1528. 13. Hajra, A. K. & Bishop, J. E. (1982) Ann. N. Y. Acad. Sci. 386, 170-182. 14. Ballas, L. M., Lazarow, P. B. & Bell, R. M. (1984) Biochim. Biophys. Acta 795, 297-300. 15. Pedersen, J. I. & Gustafsson, J. (1983) FEBS Lett. 121, 345-348. 16. Kase, B. F., Bjorkhem, I. & Pedersen, J. I. (1983) J. Lipid Res. 24, 1560-1567. 17. Goldfischer, S., Moore, C. L., Johnson, A. B., Spiro, A. L., Valsamis, M. P., Wisniewski, H. M., Ritch, R. H., Norton, W. T., Rapin, I. & Gartner, L. M. (1973) Science 182, 62-64. 18. Mooi, W. J., Dingemans, K. P., van den Bergh Weerman, M. A., Jobsis, A. C., Heymans, H. S. & Barth, P. G. (1983) Ultrastruct. Pathol. 5, 135-144. 19. Brown, F. R., III, McAdams, A. J., Cummins, J. W., Konkol, R., Singh, I., Moser, A. B. & Moser, H. W. (1982) Johns Hopkins Med. J. 151, 344-351. 20. Goldfischer, S., Powers, J. M., Johnson, A. B., Axe, S., Brown, F. R. & Moser, H. W. (1983) Virchows Arch. A 401, 355-361. 21. Moser, A. E., Singh, I., Brown, F. R., Solish, G. I., Kelley, R. I., Benke, P. J., Burton, B. K. & Moser, H. W. (1984) N. Engl. J. Med. 310, 1141-1146. 22. Heymans, H. S. A., Schutgens, R. B. H., Tan, R., van den Bosch, H. & Borst, P. (1983) Nature (London) 306, 69-70.
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