Proc. Nati. Acad. Sci. USA Vol. 83, pp. 9193-9196, December 1986 Medical Sciences Presence of the peroxisomal 22-kDa integral membrane protein in the liver of a person lacking recognizable peroxisomes (Zellweger syndrome) (peroxisome disease/organelie biogenesis/sodium carbonate membrane isolation/immunoblots/kidney) PAUL B. LAZAROW*t, YUKIO FuJIKI*t, GILLIAN M. SMALL*, PAUL WATKINS§, AND HUGO MOSER§ *The Rockefeller University, New York, NY 10021; and §The John F. Kennedy Institute and Department of Neurology, The University, , MD 21205 Communicated by DeWitt Stetten, Jr., August 11, 1986

ABSTRACT Peroxisomes have not been detected in liver consequences. Two possible defects are in the assembly of and kidney of patients with Zellweger syndrome. Some peroxi- the peroxisome membrane or in the import of matrix pro- some proteins are missing; others are present in normal teins. This paper communicates our first step in investigating amounts but are located in the cytosol. We have prepared an peroxisome membrane proteins in Zellweger syndrome. We antiserum against the 22-kDa integral membrane protein show that normal human liver contains an integral membrane characteristic of rat liver peroxisomes. The antiserum cross- protein that is the same size as, and cross-reacts immuno- reacts with the human liver counterpart, which likewise has a logically with, the 22-kDa integral membrane protein (22IMP) mass of 22 kDa. By immunoblot analysis, we demonstrate that that we previously demonstrated to be located exclusively in the 22-kDa protein is present in normal amount in Zellweger peroxisomes in rat liver (17). Rat 22IMP is synthesized on liver and is integral to a membrane. The result suggests that free polysomes at its final size (18). We report immunoblot peroxisome membranes are assembled in Zellweger syndrome analyses of this and other peroxisomal proteins in Zellweger but may be defective for the import of matrix proteins. As a liver and kidney. result, newly synthesized proteins are left in the cytosol, where some persist and others are degraded. Lacking their usual CASE HISTORY content, such aberrant peroxisomal membranes would be Patient RA presented as a classic example of Zellweger unrecognizable morphologically. Immunoblot analyses also syndrome. This girl was the first child of unrelated healthy showed that the peroxisomal hydratase-dehydrogenase is de- parents. Pregnancy was uneventful and birth weight was 3.0 ficient in Zellweger kidney as well as liver, but catalase is kg. The diagnosis of Zellweger syndrome was suggested by present in both organs. severe hypotonia and characteristic dysmorphic features including high forehead, wide-open fontanels, posteriorly Zellweger syndrome (1-4) is a fatal disease in which peroxi- rotated ears, high arched palate, simian creases, and equinus some assembly appears to be defective. Peroxisomes are deformity of feet. Chondrodysplasia calcificans was present abundant in normal human liver and kidney but have not been in the patellae and hips. Seizures were first noted at 1.5 weeks detected in these organs in Zellweger patients despite careful and were treated with phenobarbital. She required feeding by searches by electron microscopy and by electron microscop- nasogastric tube and intermittent oxygen therapy. She re- ic cytochemistry that reveals catalase (a characteristic mained unresponsive and severely hypotonic. She had sev- peroxisomal enzyme) (5-7). Peroxisomal proteins are syn- eral episodes of massive gastrointestinal bleeding. Pneumo- thesized at normal rates in patients with Zellweger syndrome nia caused her death at 4 months. Postmortem examination (8), but some fail to accumulate. These include the - showed patchy periportal and sinusoidal hepatic fibrosis. The oxidation enzymes (7, 9) and the first enzyme in the synthesis kidneys were studded with multiple cortical cysts. Adrenal of plasmalogens, dihydroxyacetone phosphate acyltransfer- cortical cells showed cytoplasmic inclusions characteristic of ase (10, 11). Very long chain fatty acids (C24, C26) accumulate Zellweger syndrome or . (12, 13) and plasmalogens are deficient (14); death usually Control patients were a 2.5-year-old boy who died with no occurs within the first year after birth, often within weeks or history of liver disease (C in ref. 7), a 5.5-year-old female months. Farber disease patient (acid ceramidase deficiency), a 6-year- Other peroxisomal enzymes-e.g., catalase-accumulate old male X-linked adrenoleukodystrophy patient, and a to normal levels. However, the catalase is neither latent (15) woman who died of cancer at age 75. Postmortem liver and nor sedimentable (7), indicating that the enzyme is located in kidney biopsy samples were kept frozen at -80°C. the cytosol ofthe cell. These observations are consistent with our current understanding of peroxisome biogenesis (16): METHODS peroxisomal proteins are synthesized on free polyribosomes Biochemical Methods. Pieces of frozen tissue were sepa- and are imported posttranslationally into preexisting rated with a serrated knife, thawed, and homogenized in 4 vol peroxisomes, generally without proteolytic processing. Old of 0.25 M sucrose/5 mM imidazole buffer, pH 7.0/0.1% peroxisomes divide to form new ones. In the absence of ethanol with a Potter-Elvehjem homogenizer. Protein was peroxisomes, the newly synthesized proteins would be left in determined according to Lowry et al. (19) with bovine serum the cytosol, where some would be degraded, perhaps by the albumin as standard. Very long chain fatty acids (12), bile ubiquitin-mediated system, while others might survive. acids (20), and pipecolic acid (21) were measured as de- Zellweger syndrome is an autosomal recessive disease, scribed. which implies that a single genetic defect causes all the above Abbreviation: 22IMP, 22-kDa integral membrane protein of perox- isomes. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" *Present address: Meiji Institute of Health Science, 540 Naruda, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Odawara, 250 Japan.

9193 Downloaded by guest on September 23, 2021 Proc. Natl. Acad. Sci. USA 83 (1986)

Liver Kidney rated by NaDodSO4/PAGE in 7-15% polyacrylamide slab R C C Z C C Z CC gels (22) and immunoblotted by a modification (18) of Burnette's procedure (25). The antibody raised against peroxisomal 22IMP (rabbit no. 10-270) detected one major band (with a mass of22 kDa) in blots ofrat liver homogenates, and this antigen cosedimented with peroxisomes in cell fractionation experiments (not shown). The antiserum cross- reacted with a protein ofthe same size in control human liver

68 (Fig. 1). This human protein was an integral membrane protein by the criterion of not being solubilized by the carbonate procedure (22). Fibroblast Culture and Analyses. Skin fibroblasts were cultured as described (26). Fibroblasts from two 10-cm dishes were harvested by trypsinization, washed in 0.25 M sucrose/20 mM 4-morpholinepropanesulfonic acid (Mops), pH 7, and lysed with digitonin at 100 gg/ml in 800 /.l of the above medium. Half of the cell lysate was centrifuged for 10 min in a Fisher microcentrifuge. Catalase was assayed (27) (in 26-k the presence of0.1% Triton X-100) in the pellet, supernatant, and starting material. One unit ofcatalase decreases the H202 concentration to 1/10th in 1 min in a volume of 50 ml (28). Materials. 1251-labeled staphylococcal protein A was from New England Nuclear. Molecular mass standards were from

.e-'i8^3'% 22LMIP Bethesda Research Laboratories: rabbit muscle phosphoryl- ase b (97.4 kDa), bovine serum albumin (68 kDa), ovalbumin 18-. U~ (45 kDa), a-chymotrypsinogen (25.7 kDa), bovine milk f- lactoglobulin (18.4 kDa), and egg white lysozyme (14.3 kDa). All other reagents were of analytical grade.

RESULTS Patient RA had greatly increased concentrations of very long 1 2 3 4 5 6 7 8 9 chain fatty acids (C26 and C24) in plasma and in cultured skin FIG. 1. Immunoblot analysis of the peroxisomal 22IMP. Mem- fibroblasts (Table 1). An intermediate in the conversion of branes were isolated (22) from 200 ,g of homogenate (human liver cholesterol to bile acids, trihydroxycholestanoic acid, had and kidney) or 100 ug of rat liver. Z, Zellweger patient; C, human accumulated to very high levels in serum (Table 1). The blood controls; R, rat. The molecular mass markers are indicated in kDa to pipecolic acid concentration was slightly elevated. These the left. biochemical abnormalities are characteristic of Zellweger syndrome (12, 13, 21, 29). Total membranes were purified from the homogenates by Cultured skin fibroblasts from RA contained normal the carbonate procedure described previously (22). Treat- amounts of catalase activity, but the enzyme was not ment with 0.1 M Na2CO3 at 0°C for 30 min causes organelle sedimentable (Table 2), indicating that it was located in the membranes to rupture, releases matrix proteins, and strips cell cytosol, not in peroxisomes. off peripheral membrane proteins. The membranes, recov- By immunoblotting analysis, RA's liver lacked the ered by high-speed centrifugation, retain their phospholipids, peroxisomal bifunctional hydratase-dehydrogenase (Fig. 2 integral proteins, and characteristic trilamellar appearance. Left, lane 4) but contained normal amounts of catalase (Fig. Antiserum Preparation and Immunoblots. Rabbit antiserum 2 Right, lane 3). All four control livers contained both the was raised against 22IMP of highly purified rat liver hydratase-dehydrogenase and catalase. The patient's defi- peroxisomes in exactly the same fashion as described previ- ciency of hydratase-dehydrogenase and sufficiency of ously (18). Rabbit anti-rat liver catalase (23) and rabbit catalase agree with previous observations on two percuta- anti-rat liver bifunctional hydratase-dehydrogenase (24) neous Zellweger liver biopsies (7) and three postmortem were as described. Cross-reaction of the latter with human Zellweger liver samples (9). Taken together with the other hydratase-dehydrogenase has been demonstrated (7). clinical observations, these biochemical results indicate un- Homogenate proteins and membrane proteins were sepa- ambiguously that RA had Zellweger syndrome. Table 1. Very long chain fatty acids, bile acid intermediate, and pipecolic acid in patients and controls Fatty acids* Plasma Fibroblasts trihydroxy-Serum Serum 26:0, 26:0/22:00 24:0/22:0 26:0, 26:0/22:0 cholestanoic acid,t pipecolic acid,* Subjects Ag/ml (wt/wt) (wt/wt) yg/ml (wt/wt) AM AM Patient RA 4.06 0.68 1.85 0.54 0.46 16.4 8 Other Zellweger patients (n = 36) 2.5 ± 0.85 0.49 ± 0.03 2.0 ± 0.24 0.68 ± 0.21 2.0 ± 1.2 12 (n = 1) 7-188 (n = 5) Controls (n = 66) 0.33 ± 0.15 0.014 ± 0.076 0.83 ± 0.15 0.079 ± 0.066 0.080 ± 0.029 Not detectable <4 (n = 16) *Results are means ± SD. 26:0, hexacosanoic acid; 24:0, tetracosanoic acid; 22:0, docosanoic acid. Control values are from ref. 12. t3a,7a,12a-Trihydroxy-53-cholestan-26-oic acid. Other patient and control values are from ref. 29. *Range is presented for other Zellweger patients. Other patient and control values are from ref. 21. Downloaded by guest on September 23, 2021 Medical Sciences: Lazarow et al. Proc. Natl. Acad. Sci. USA 83 (1986) 9195

Table 2. Fibroblast catalase sedimentability Catalase activity Soluble, Sedimentable, Homogenate, % of % of Recovery, Subjects mU/mg recovered recovered % Patient RA 6.3 87 13 117 Other Zeliweger patients (n = 9) 9.8 ± 4.7 93 ± 3 7 ± 3 108 ± 7 Controls (n = 6) 5.1 ± 1.6 9 ± 3 91 ± 3 122 ± 19 Results are means ± SD.

The patient's liver contained a normal amount of peroxi- (7, 9), human hydratase-dehydrogenase is slightly bigger somal 22IMP (Fig. 1, lane 4). There was an appreciable than the rat protein (Fig. 2 Left, lane 1 vs lanes 2, 3, 5, 6, 8, variability in the intensity of the immunoblot signal for this and 9). Two closely spaced bands were observed in the protein among the four control livers; the Zellweger sample catalase immunoblots oftwo ofthe control human livers (Fig. fell in the middle of this range. The immunoblot analysis for 2 Right, lanes 1 and 2). The amounts of the lower band 22IMP was carried out on membranes isolated from the correlate with the length of time the homogenates were stored homogenates by the carbonate procedure (22). Thus this prior to analysis. Rat liver catalase is susceptible to proteo- result implies that 22IMP not only is present in Zellweger lytic nicking, with the loss ofan =4-kDa peptide (30, 31). We liver but also is integrally embedded in a membrane. suspect that a similar phenomenon accounts for the smaller Similar immunoblot analyses were carried out on kidney catalase band in the two human samples. samples (Figs. 1 and 2). Control human kidney contains hydratase-dehydrogenase and catalase, but in smaller con- centrations than in liver. Zellweger kidney contains catalase DISCUSSION but lacks hydratase-dehydrogenase, as does liver. 22IMP We have taken particular care with the diagnosis of this was not detected in control or Zellweger kidney (Fig. 1, lanes patient because there are several peroxisomal diseases with 7-9). However, because of the generally lower amounts of similarities to Zellweger syndrome (2-4). We (7) and peroxisomal proteins in kidney and the presence of a blotch Goldfischer et al. (32) have described "pseudoZellweger" in this region of the fluorogram, we cannot be certain that patients, who present with some clinical similarities to 22IMP is entirely absent from control kidney. Zellweger syndrome but who have a normal abundance of Human 22IMP comigrates in NaDodSO4/PAGE with rat hepatic peroxisomes (7, 32), normal amounts of hepatic 22IMP (Fig. 1, lane 1 vs lanes 2-6). As observed previously hydratase-dehydrogenase (7), but two deficient oxidases Liver Kidney Liver Kidney R CC Z CC Z C C c c Z c cI Z C C

97-+ .. 97--

i8-:":* 68-* 68-- +-Cat kw" - _ _

45 - 4.5-

26 - 26 -

...... De po siti on! 18- 1 4 14-

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 FIG. 2. Immunoblot analyses of peroxisomal hydratase-dehydrogenase (HD; Left) and catalase (Cat; Right) in liver and kidney (100 ,ug of homogenate protein). Symbols as in Fig. 1. Downloaded by guest on September 23, 2021 9196 Medical Sciences: Lazarow et al. Proc. Natl. Acad. Sci. USA 83 (1986) (32). The clinical and biochemical data on our current 9. Tager, J. M., van der Beek, W. A. T. H., Wanders, R. J. A., subject, in particular the nonsedimentability of the fibroblast Hashimoto, T., Heymans, H. S. A., van den Bosch, H., catalase, the absence of hepatic hydratase-dehydrogenase, Schutgens, R. B. H. & Schram, A. W. (1985) Biochem. and the presence of renal cysts, clearly establish her as Biophys. Res. Commun. 126, 1269-1275. 10. Datta, N. S., Wilson, G. N. & Hajra, A. K. (1984) N. Engl. J. having a classical Zellweger syndrome. Med. 311, 1080-1083. The finding of normal amounts of hepatic 22IMP in a 11. Schutgens, R. B. H., Romeyn, G. J., Wanders, R. J. A., van disease in which liver peroxisomes are apparently missing is den Bosch, H., Schrakamp, G. & Heymans, H. S. A. (1984) clearly a surprise. Moreover, the 22IMP in this patient is Biochem. Biophys. Res. Commun. 120, 179-184. integral to a membrane. The result suggests that peroxisome 12. Moser, A. E., Singh, I., Brown, F. R., III, Solish, G. I., membranes are present in Zellweger liver but there is a defect Kelley, R. I., Benke, P. J. & Moser, H. W. (1984) N. Engl. J. in the machinery for the import ofmatrix proteins. As a result Med. 310, 1141-1146. the membranes lack (or are greatly deficient in) content 13. Bakkeren, J. A. J. M., Monnens, L. A. H., Trijbels, J. M. F. proteins and therefore they are unrecognizable morphologi- & Maas, J. M. (1984) Clin. Chim. Acta 138, 325-331. 14. Heymans, H. S. A., Schutgens,. R. B. H., Tan, R., van den cally and cytochemically. Bosch, H. & Borst, P. (1983) Nature (London) 306, 69-70. Some support for this interpretation comes from morpho- 15. Wanders, R. J. A., Kos, M., Roest, B., Meijer, A. J., logical observations suggesting that the deficiency of perox- Schrakamp, G., Heymans, H. S. A., Tegelaers, W. H. H., isomes in Zellweger syndrome may not be absolute. In van den Bosch, H., Schutgens, R. B. H. & Tager, J. M. (1984) intestine of two Zellweger patients, occasional small (0.04- to Biochem. Biophys. Res. Commun. 123, 1054-1061. 0.13-pum) bodies with a slight positive cytochemical reaction 16. Lazarow, P. B. & Fujiki, Y. (1985) Annu. Rev. Cell Biol. 1, for catalase have been observed (7). A few peroxisomes 489-530. (7-9% of normal) were reported in Zellweger fibroblasts by 17. Fujiki, Y., Fowler, S., Shio, H., Hubbard, A. L. & Lazarow, Arias et al. (33); however, these were not found by Santos et P. B. (1982) J. Cell Biol. 93, 103-110. 18. Fujiki, Y., Rachubinski, R. A. & Lazarow, P. B. (1984) Proc. al. (34). On the other hand, one cannot categorically exclude Natl. Acad. Sci. USA 81, 7127-7131. the possibility that 22IMP inserts erroneously into some other 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, intracellular membrane due to the absence of peroxisome R. J. (1951) J. Biol. Chem. 193, 265-275. membranes. 20. Ali, S. S. & Javitt, N. B. (1970) Can. J. Biochem. 48, Recent measurements of hepatic 22IMP in other patients 1054-1057. with various peroxisomal diseases support the present results 21. Kelley, R. I. & Moser, H. W. (1984) Am. J. Med. Genet. 19, and will be communicated subsequently. 791-795. 22. Fujiki, Y., Hubbard, A. L., Fowler, S. & Lazarow, P. B. We thank Ms. Judiann McGhee for expert technical assistance. (1982) J. Cell Biol. 93, 97-102. We thank Dr. Norman B. Javitt for performing the bile acid assays. 23. Lazarow, P. B. & de Duve, C. (1973) J. Cell Biol. 59, 491-506. This research was supported by National Institutes of Health Grants 24. Mortensen, R. M. (1983) Thesis (The Rockefeller Univ., New AM19394 and HD10981. P.B.L. was supported by an Established York). Fellowship from the New York Heart Association. 25. Burnette, W. N. (1981) Anal. Biochem. 112, 195-203. 26. Singh, I., Moser, A. E., Moser, H. W. & Kishimoto, Y. (1984) 1. Kelley, R. I. (1983) Am. J. Med. Genet. 16, 503-517. Pediatr. Res. 18, 286-290. 2. Goldfischer, S. & Reddy, J. K. (1984) Int. Rev. Exp. Pathol. 27. Aebi, H. (1974) in Methods of Enzymatic Analysis, ed. 26, 45-84. Bergmeyer, H. U. (Academic, New York), 2nd Ed., Vol. 2, 3. Moser, H. W. & Goldfischer, S. L. (1985) Hosp. Pract. 20, pp. 673-678. 61-70. 28. Baudhuin, P., Beaufay, H., Rahman-Li, Y., Sellinger, 0. Z., 4. Schutgens, R. B. H., Heymans, H. S. A., Wanders, R. J. A., Wattiaux, R., Jacques, P. & de Duve, C. (1964) Biochem. J. van den Bosch, H. & Tager, J. M. (1986) Eur. J. Pediatr. 144, 92, 179-184. 430-440. 29. Kase, B. F., Bjorkhem, I., Hga, P. & Pedersen, J. I. (1985) J. 5. Goldfischer, S., Moore, C. L., Johnson, A. B., Spiro, A. J., Clin. Invest. 75, 427-435. Valsamis, M. P., Wisniewski, H. K., Ritch, R. H., Norton, 30. Robbi, M. & Lazarow, P. B. (1978) Proc. Natl. Acad. Sci. W. T., Rapin, I. & Gartner, L. M. (1973) Science 182, 62-64. USA 75, 4344-4348. 6. Mooi, W. J., Dingemans, K. P., van den Bergh Weerman, 31. Mainferme, F. & Wattiaux, R. (1982) Ann. N.Y. Acad. Sci. M. A., Jobsis, A. C., Heymans, H. S. A. & Barth, P. G. 386, 507-509. (1983) Ultrastruct. Pathol. 5, 135-144. 32. Goldfischer, S., Collins, J., Rapin, I., Neumann, P., Neglia, 7. Lazarow, P. B., Black, V., Shio, H., Fujiki, Y., Hajra, A. K., W., Spiro, A. J., Ishii, T., Roels, F., Vamecq, J. & Van Hoof, Datta, N. S., Bangaru, B. S. & Dancis, J. (1985) Pediatr. Res. F. (1986) J. Pediatr. 108, 25-32. 19, 1356-1364. 33. Arias, J. A., Moser, A. B. & Goldfischer, S. L. (1985) J. Cell 8. Schram, A. W., Strijland, A., Hashimoto, T., Wanders, Biol. 100, 1789-1792. R. J. A., Schutgens, R. B. H., van den Bosch, H. & Tager, 34. Santos, M. J., Ojeda, J. M., Garrido, J. & Leighton, F. (1985) J. M. (1986) Proc. Natl. Acad. Sci. USA 83, 6156-6158. Proc. Natl. Acad. Sci. USA 82, 6556-6560. Downloaded by guest on September 23, 2021