Vol. 172, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
October 15, 1990
Pages 364-369
PEROXISOMAL DISORDERS: COMPLEMENTATION ANALYSIS USING BETA-OXIDATION OF VERY LONG CHAIN FATTY ACIDS M.C. McGuinness, A . B . Moser, H.W. Moser a n d P . A . Watkins K e n n e d y I n s t i t u t e a n d the Department of N e u r o l o g y , J o h n s Hopkins U n i v e r s i t y School of Medicine, Baltimore, MD Received August 31, 1990
Complementation s t u d i e s , u s i n g f u s e d cell lines from p a t i e n t s with peroxisomal d i s o r d e r s , have shown c o r r e c t i o n of defective plasmalogen s y n t h e s i s a n d p h y t a n i c acid oxidation as well as an i n c r e a s e in the n u m b e r of peroxisomes. At least six complementation g r o u p s have been r e p o r t e d . We d e m o n s t r a t e h e r e t h a t complementing cell lines also a c q u i r e the ability to oxidize v e r y long chain f a t t y acids (VLCFA), a n d t h a t complementation g r o u p s defined with this t e c h n i q u e are identical to t h o s e r e p o r t e d p r e v i o u s l y when plasmalogen s y n t h e s i s was u s e d as the criterion f o r complementation. This VLCFA complementation t e c h n i q u e is of p a r t i c u l a r value in the s t u d y of p a t i e n t s in whom defective VLCFA is the only or major enzymatic d e f e c t , and we show complementation between cell lines from two patients each with an isolated defect in one of the peroxisomal f a t t y acid beta-oxidation enzymes. ~, 199oAoad~m~oPress, ~no.
Disorders of peroxisomal b i o g e n e s i s , i n c l u d i n g the Zellweger s y n d r o m e , neonatal a d r e n o l e u k o d y s t r o p h y , infantile Refsum s y n d r o m e a n d h y p e r p i p e c o l i c acidemia a r e c h a r a c t e r i z e d b y an a b s e n c e or diminution in the n u m b e r of morphologically d e t e c t a b l e peroxisomes in t i s s u e s from a f f e c t e d p a t i e n t s .
The
a b s e n c e of peroxisomes in t h e s e p a t i e n t s is a s s o c i a t e d with a n u m b e r of biochemical abnormalities ( r e v i e w e d in r e f e r e n c e 1) i n c l u d i n g impaired b e t a oxidation of v e r y long chain f a t t y acids (VLCFA), impaired s y n t h e s i s of plasmalogens, and accumulation of bile acid i n t e r m e d i a t e s , pipecolic acid and p h y t a n i c acid.
The biochemical and clinical distinctions between t h e s e
p h e n o t y p e s , h o w e v e r , a r e u n c l e a r a n d it is not known w h e t h e r t h e s e d i v e r s e d i s o r d e r s r e p r e s e n t allelic or non-allelic mutations of peroxisomai b i o g e n e s i s . Complementation analysis has been u s e d to examine the relationship between p h e n o t y p e a n d g e n o t y p e in the peroxisomal d i s o r d e r s .
F i b r o b l a s t s from two
d i f f e r e n t p a t i e n t s , b o t h deficient in a peroxisomal metabolic p r o c e s s , a r e i n d u c e d to f u s e ; the r e s u l t i n g multinuclear cells are examined f o r t h e i r ability to c a r r y out this metabolic f u n c t i o n (2).
R e s t o r a t i o n of a c t i v i t y can o n l y o c c u r if each
p a r e n t a l cell line p r o v i d e s the gene p r o d u c t defective in the o t h e r .
Other
i n v e s t i g a t o r s h a v e u s e d d__eenov____0_oplasmalogen b i o s y n t h e s i s (3), d i h y d r o x y a c e t o n e p h o s p h a t e a c y t t r a n s f e r a s e a c t i v i t y (4), particle b o u n d catalase ( 4 , 5 ) o r p h y t a n i c acid oxidase a c t i v i t y (6) as c r i t e r i a f o r complementation. 0006-291x/90 $1.50 Copyright © 1990 by Academic Press, lnc. All rights of reproduction in any form reserved.
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3ingh et al. (7) showed t h a t b e t a - o x i d a t i o n of VLCFA, e . g . llgnoceric acid ~.C24=o) t a k e s place in peroxisomes r a t h e r t h a n mitochondria. the f i r s t to u s e b e t a - o x i d a t i o n of VLCFA
The p r e s e n t s t u d y is
as the c r i t e r i o n for complementation
when g r o u p i n g cell lines from patients with peroxisomal d i s o r d e r s .
Many of the
cell lines u s e d in this s t u d y were also u s e d b y R o s c h e r and coworkers (3) in complementation s t u d i e s where plasmalogen b i o s y n t h e s i s was the criterion f o r complementation.
It was of i n t e r e s t , t h e r e f o r e , to compare complementation
g r o u p s o b t a i n e d u s i n g b o t h b e t a - o x i d a t i o n of VLCFA a n d plasmalogen b i o s y n t h e s i s as indices of complementation to see if the same genetic d e f e c t a f f e c t s b o t h metabolic p a t h w a y s . Complementation a n a l y s i s , u s i n g b e t a - o x i d a t i o n as the c r i t e r i o n f o r complementation, may be u s e f u l in the s t u d y of p a t i e n t s who are s u s p e c t e d of h a v i n g a single enzyme defect in the peroxisomal b e t a - o x i d a t i o n p a t h w a y .
In
many of t h e s e p a t i e n t s , all t h r e e b e t a - o x i d a t i o n enzyme p r o t e i n s ( a c y l - C o A oxidase, bifunctional enzyme a n d b e t a - k e t o t h i o l a s e ) are detectable b y immunoblot analysis (McGuinness a n d Watkins, u n p u b l i s h e d o b s e r v a t i o n s ) .
These p a t i e n t s may
have an inactive form of one of the enzymes which could be d e t e c t e d b y complementation s t u d i e s u s i n g cell lines known to be deficient in each of the b e t a - o x i d a t i o n enzyme activities.
MATERIALS AND METHODS Materials: Cell c u l t u r e r e a g e n t s were o b t a i n e d from GIBCO L a b o r a t o r i e s ( G r a n d I s l a n d , NY). [1-~4C]IAgnoceric acid (54.0 mCi/mmol) was o b t a i n e d from R e s e a r c h P r o d u c t s I n t e r n a t i o n a l C o r p o r a t i o n a n d [ 1- ~4C ]palmitic acid (58.0 mCi/mmol) from New England Nuclear. Ficoll 400 was obtained from Pharmacia Fine Chemicals ( P i s c a t a w a y , NJ) a n d p o l y e t h y l e n e glycol 4000 from Merck (Rahway, NJ). All o t h e r r e a g e n t s were of analytical g r a d e and were obtained from commercial s o u r c e s . Cell lines: C u l t u r e d skin f i b r o b l a s t s from twelve p a t i e n t s with d i s o r d e r s of peroxisomal biogenesis were originally obtained f o r diagnostic p u r p o s e s . They were maintained in Eagle's Minimum Essential Medium with 10% fetal bovine serum, 1% gtutamine and p e n i c i l l i n / s t r e p t o m y c i n . Clinical f i n d i n g s f o r p a t i e n t s SED1 and SED2, with single enzyme d e f e c t s , h a v e been r e p o r t e d p r e v i o u s l y b y Watkins et al. (8) a n d Poll-The et al. (9) r e s p e c t i v e l y . The major clinical f i n d i n g s of patients Z2, Z4, Z5, Z6, ZT, N7 a n d N8 i n c l u d e d in this s t u d y , w h e r e Z a n d N r e p r e s e n t Zellweger a n d neonatal a d r e n o l e u k o d y s t r o p h y p a t i e n t s , r e s p e c t i v e l y , were r e p o r t e d b y R o s c h e r et al. (3). Skin f i b r o b l a s t s from p a t i e n t s Z9, Z10 a n d Z l l were also s t u d i e d . Cell f u s i o n : Two p a r e n t a l cell lines (1.5 X 10 e cells each) were s e e d e d in 10cm dishes in a 1:1 ratio a n d f u s e d with p o l y e t h y l e n e glycol 4000 u s i n g the method of R o s c h e r et al. (3). Multi- and m o n o - n u c l e a t e d cells were s e p a r a t e d on a d i s c o n t i n u o u s Ficoll g r a d i e n t . A f t e r s e p a r a t i o n of the cells, "fused" a n d " u n f u s e d " cell f r a c t i o n s were examined microscopically. In the p r e s e n t s t u d y at least 80% of the cells in the f u s e d cell f r a c t i o n were multinucleate. The u n f u s e d cell f r a c t i o n c o n t a i n e d g r e a t e r t h a n 95% mononuclear cells. This f r a c t i o n was e q u i v a l e n t to c o c u l t u r e d cells. F u s e d cells a n d t h o s e t h a t failed to f u s e were r e c u l t i v a t e d a n d b e t a - o x i d a t i o n m e a s u r e d a f t e r a 48-h r e c o v e r y period. 365
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F a t t y acid oxidation: Oxidation of [1-14Clpalmitic acid a n d [1-14C1 lignoceric acid to w a t e r soluble p r o d u c t s was m e a s u r e d as p r e v i o u s l y d e s c r i b e d (10).
RESULTS AND DISCUSSION F a t t y acid b e t a - o x i d a t i o n was measured in f i b r o b l a s t s from controls and from two p a t i e n t s k n o w n to be deficient in one of the peroxisomal b e t a oxidation e n z y m e s (Table 1).
The ratio of oxidation of lignoceric acid (C24:o) to
palmitic acid (C16:o) was t a k e n as an indication of t h e ability of the cells to oxidize VLCFA ( 8 , 1 0 , 1 1 ) .
VLCFA oxidation in SED1 (immunologically deficient in
peroxisomal bifunctional enzyme) a n d SED2 (immunologicaUy deficient in peroxisomal a c y l - C o A oxidase) was 11 and 16% of control, r e s p e c t i v e l y (Table 1), in a g r e e m e n t with p r e v i o u s l y p u b l i s h e d r e s u l t s ( 8 , 9 ) . To determine w h e t h e r complementation analysis could be used in the s t u d y of p a t i e n t s with peroxisomal b e t a - o x i d a t i o n d i s o r d e r s , we i n v e s t i g a t e d the ability of celt lines SED1 a n d SED2 to complement each o t h e r .
F i b r o b l a s t s were b r i e f l y
TABLE 1 B e t a - o x i d a t i o n of V L C F A in f i b r o b l a s t s b e f o r e a n d a f t e r f u s i o n Beta-Oxidation of Fatty Acids
C++
C2,
Ls24~Cls
% of Control
(pmol/mg/min) Ct
62.9
14.3
0.228
(100)
C2
61.9
15.2
0.245
93
C1XC2 cc
49.8
11.1
0,222
91
C1XC2 fused
57.1
14.5
0.253
103
SED1
55.7
1.4
0,026
11
SED2
82.9
3.1
0.038
16
SED1XSED2 cc
72.6
4.1
0.056
23
SED1XSED2 fused
64.3
14.3
0,223
91
Oxidation of palmltlc acid (Cle) and Ilgnocerlc acid (C24) w a s measured In parental fibroblast cell lines, fused and cocultured (cc) cells as described in Methods. In the last column, the C241 C+e ratio is expressed as percentage of control C1. C1 and C2, Controls; SED1, bifunctional protein single enzyme defect; SED2, acyI-CoA oxidase single enzyme defect.
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TABLE 2 Complementation analysis of fibroblasts from patients with peroxisomal disorders
using beta-oxidation of VLCFA as the criterion for complementation 1
2
3
~
z2 zs N71zol ....
6
4
-
Zll
N8
-
[/ ff
Y
J'lf __z7
_
%Y Y Y
81
The ratio of oxidation of lignoceric acid (C24) to palmitic acid (C16) is expressed as percentage of a control measured in the same experiment. Numbers above the diagonal represent cocultured cells and those below this line represent fused cells. Z, Zellweger syndrome; N, neonatal ALD. The numbers above the brackets refer to the original group numbers of Roscher et aL (3).
incubated with polyethylene VLCFA in the cocultured, cell f r a c t i o n w a s m e a s u r e d .
g l y c o l to i n d u c e c e l l f u s i o n .
Beta-oxidation of
mononucleate cell fraction and the
fused,
multinucleate
C o c u l t u r e d SED1 a n d SED2 f i b r o b l a s t s h a d a low
o x i d a t i o n r a t i o b u t t h e f u s e d c e i l s o x i d i z e d V L C F A a t 91% o f t h e c o n t r o l r a t i o (Table 1), indicating that complementation had occurred. Having determined that restoration of VLCFA oxidation was a valid i n d i c a t o r o f c o m p l e m e n t a t i o n , we i n v e s t i g a t e d patients with disorders Results are expressed
o f p e r o x i s o m a l b i o g e n e s i s to c o m p l e m e n t ( T a b l e 2 ) . as a percentage
of the oxidation ratio in control cells.
The numbers above the diagonal represent line represent
the ability of fibroblasts from
fused cells.
cocultured
Oxidation ratios in parent
cells and those below this cell l i n e s ( d a t a n o t s h o w n ) ,
c o c u l t u r e d c e l l s o r n o n - c o m p l e m e n t i n g c e l l l i n e s w e r e g e n e r a l l y l e s s t h a n 30% o f the ratio of normal control cells.
An increase of at least 2.5-fold in the
oxidation ratio of fused cells over coeultured
367
cells was considered to indicate
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complementation.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fusion of c o n t r o l cells (Table 1) o r p a t i e n t s ' cells (Table 2)
with themselves did not a f f e c t the oxidation ratio. Cell lines r e p r e s e n t i n g five of the six complementation g r o u p s r e p o r t e d b y R o s c h e r et al. (3) were s t u d i e d .
As can be seen from Table 2, complementing
cell lines u s i n g VLCFA oxidation as the c r i t e r i o n s e g r e g a t e d into g r o u p s t h a t were identical to the g r o u p s obtained p r e v i o u s l y ( i n d i c a t e d b y b r a c k e t s at the top of Table 2) when plasmalogen s y n t h e s i s was u s e d as the criterion f o r complementation (3).
U n f o r t u n a t e l y , f i b r o b l a s t s from p a t i e n t Z8
(complementation g r o u p 5 of R o s c h e r a n d c o w o r k e r s ) were no l o n g e r available. Complementation analysis was also p e r f o r m e d on cell lines from t h r e e Zellweger p a t i e n t s which were not included in the s t u d y of R o s c h e r et al. (3) ( d e s i g n a t e d Z9, Z10 a n d Z l l ) .
Both VLCFA oxidation a n d plasmalogen s y n t h e s i s
were m e a s u r e d in f u s e d a n d c o c u l t u r e d cells.
Patient Z9 and patients Z10 a n d
Z l l were f o u n d to b e l o n g to complementation g r o u p s 1 a n d 4, r e s p e c t i v e l y , of R o s c h e r a n d colleagues when b o t h b e t a - o x i d a t i o n of VLCFA (Table 2) and r e s t o r a t i o n of plasmatogen s y n t h e s i s ( d a t a not shown) were u s e d as c r i t e r i a f o r complementation. To d a t e , no correlation between g e n o t y p e a n d p h e n o t y p e has been f o u n d in studies of f i b r o b l a s t s from p a t i e n t s with peroxisomal d i s o r d e r s .
R o s c h e r e t al. (3)
showed that all known p h e n o t y p e s were r e p r e s e n t e d within t h e i r complementation g r o u p 1 and the most common p h e n o t y p e , classical Zellweger s y n d r o m e , was f o u n d in five of t h e i r six complementation g r o u p s .
T h e r e is genetic h e t e r o g e n e i t y ,
t h e r e f o r e , n o t o n l y within the g r o u p of peroxisomal d i s o r d e r s b u t also between d i f f e r e n t Zellweger cell lines. Complementation
g r o u p s obtained in the p r e s e n t
s t u d y , when b e t a - o x i d a t i o n of VLCFA was u s e d as the c r i t e r i o n of complementation, were f o u n d to be identical to the complementation g r o u p s obtained b y R o s c h e r a n d c o w o r k e r s , s u p p o r t i n g the h y p o t h e s i s t h a t defects in peroxisome a s s e m b l y a f f e c t b o t h VLCFA oxidation a n d plasmalogen b i o s y n t h e s i s . Imanaka et al. (12) h a v e shown t h a t import of peroxisomal matrix p r o t e i n s r e q u i r e s the p r e s e n c e of c e r t a i n membrane p r o t e i n s , p o s s i b l y a c t i n g as r e c e p t o r s . It is p r o b a b l e , t h e r e f o r e , t h a t a single import mutation may a f f e c t b o t h metabolic pathways. Complementation a n a l y s i s , u s i n g b e t a - o x i d a t i o n of VLCFA as the c r i t e r i o n for complementation, will p r o v e u s e f u l in the s t u d y of p a t i e n t s who, b a s e d on clinical d i a g n o s i s , a r e s u s p e c t e d of h a v i n g a single enzyme defect in the peroxisomal b e t a - o x i d a t i o n p a t h w a y .
The l a b o r a t o r y f i n d i n g s common to t h e s e
patients include e l e v a t e d plasma VLCFA, impaired VLCFA oxidation in f i b r o b l a s t s and normal plasmalogen s y n t h e s i s ( 8 , 9 , 1 3 ) .
Some p a t i e n t s , with s u s p e c t e d single
enzyme d e f e c t s , were f o u n d to be immunologically positive f o r all t h r e e b e t a oxidation e n z y m e s (11, a n d McGuinness a n d Watkins, u n p u b l i s h e d o b s e r v a t i o n s ) .
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These patients p r e s u m a b l y have an inactive form
of one of the oxidation
enzymes. Fusing f i b r o b l a s t s from p a t i e n t s , with s u s p e c t e d single enzyme defects, with fibroblasts from patients SED1 and SED2 will allow identification of patients who are likely to be deficient in the third peroxisomal beta-oxidation enzyme, b e t a ketothiolase.
A beta-ketothiolase deficient cell line, along with the two lines
known to be deficient in acyl-CoA oxidase and bifunctional enzyme activities, will enable us to s t u d y and classify f i b r o b l a s t s from patients who a r e s u s p e c t e d of having a single enzyme defect in the peroxisomal beta-oxidation pathway. ACKNOWLEDGMENTS The a u t h o r s wish to thank Ms. Angela Liu for e x p e r t technical a s s i s t a n c e , and Dr. Bwee-Tien Poli-The for s u p p l y i n g the cell line SED2. This work was s u p p o r t e d in p a r t b y g r a n t s HD 10981 and HD 24061 from the National I n s t i t u t e s of Health. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13.
Wanders, R . J . A . , Heymans, H . S . A . , S c h u t g e n s , R . B . H . , Barth, P . G . , van den Bosch, H . , and T a g e r , J.M. (1988) J. Neurological Sci. 88, 1-39. Gravel, R . A . , Mahoney, M . J . , Ruddle, F . H . , and R o s e n b e r g , L.E. (1975) Proc. Natl. Acad. Sci. (USA) 72, 3181-3185. Roscher, A . A . , Hoefler, S . , Hoefler, G . , Paschke, E., Paltauf, F . , Moser, A . , and Moser, H. (1989) Pediatric. Res. 26, 67-72. Brul, S . , Westerveld, A . , Strijland, A . , Wanders, R . J . A . , Schram, A.W., Heymans, H . S . A . . , S c h u t g e n s , R . B . H . , van den Bosch, H . , and T a g e r , J.M. (1988) J . Clin. I n v e s t . 81, 1710-1715. Singh, A . K . , Kutvatunyou, N . , Singh, I . , and Stanley, W.S. (1989) Hum. Hered. 39, 298-301. Poll-The, B . T . , Skjeldal, O . H . , Stokke, O . , Poulos, A . , Demaugre, F . , and S a u d u b r a y , J.M. (1989) Hum. Genet. 81, 175-181. Singh, I . , Moser, A . E . , Goldfischer, S. and Moser, H.W. (1984) Proc. Natl. Acad. Sci. (USA) 81, 4203-4207. Watkins, P . A . , Chen, W.W., Harris, C . J . , Hoefler, G., Hoefler, S . , Blake, D . C . , J r . , Balfe, A . , Kelley, R . I . , Moser, A . B . , Beard, M.E., and Moser, H.W. (1989) J. Clin. I n v e s t . 83, 771-777. Poll-The, B . T . , Roels, F . , Ogier, H . , Scotto, J . , Vamecq, J . , S c h u t g e n s , R . B . H . , Wanders, R . J . A . , v a n Roermund, C . W . T . , van Wijland, M . J . A . , Schram A.W., T a g e r , J . M . , and S a u d u b r a y , J.M. (1988) Am. J. Hum. Genet. 42, 422434. Hoefler, G . , Hoefler, S . , Watkins, P . A . , Chen, W.W., Moser, A . , Baldwin, V . , McGillivary, B . , Charrow, J . , Friedman, J . M . , Rutledge, L . , Hashimoto, T . , ant Moser, H.W. (1988) J. Pediatr. 112, 726-733. Naldu, S . , Hoefler, G . , Watkins, P . A . , Chen, W.W., Moser, A . B . , Hoefler, S . , Rance, N . E . , Powers, J . M . , Beard, M., Green, W.R., Hashimoto, T . , and Moser, H.W. (1988) Neurology 38, 1100-1107. Imanaka, T . , Small, G.M. and Lazarow, P . B . (1987) J. Cell Biol., 105, 2915-2922. Schram, A.W., Goldfischer, S . , van Roermund, C . W . T . , B r o u w e r - K e l d e r , E.M., Collins, J . , Hashimoto, T . , Heymans, H . S . A . , van den Bosch, H . , S c h u t g e n s , R . B . H . , T a g e r , J . M . , and Wanders, R . J . A . (1987) Proc. Natl. Acad. Sci. (USA) 84, 2494-2496.
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Pages 364-369
PEROXISOMAL DISORDERS: COMPLEMENTATION ANALYSIS USING BETA-OXIDATION OF VERY LONG CHAIN FATTY ACIDS M.C. McGuinness, A . B . Moser, H.W. Moser a n d P . A . Watkins K e n n e d y I n s t i t u t e a n d the Department of N e u r o l o g y , J o h n s Hopkins U n i v e r s i t y School of Medicine, Baltimore, MD Received August 31, 1990
Complementation s t u d i e s , u s i n g f u s e d cell lines from p a t i e n t s with peroxisomal d i s o r d e r s , have shown c o r r e c t i o n of defective plasmalogen s y n t h e s i s a n d p h y t a n i c acid oxidation as well as an i n c r e a s e in the n u m b e r of peroxisomes. At least six complementation g r o u p s have been r e p o r t e d . We d e m o n s t r a t e h e r e t h a t complementing cell lines also a c q u i r e the ability to oxidize v e r y long chain f a t t y acids (VLCFA), a n d t h a t complementation g r o u p s defined with this t e c h n i q u e are identical to t h o s e r e p o r t e d p r e v i o u s l y when plasmalogen s y n t h e s i s was u s e d as the criterion f o r complementation. This VLCFA complementation t e c h n i q u e is of p a r t i c u l a r value in the s t u d y of p a t i e n t s in whom defective VLCFA is the only or major enzymatic d e f e c t , and we show complementation between cell lines from two patients each with an isolated defect in one of the peroxisomal f a t t y acid beta-oxidation enzymes. ~, 199oAoad~m~oPress, ~no.
Disorders of peroxisomal b i o g e n e s i s , i n c l u d i n g the Zellweger s y n d r o m e , neonatal a d r e n o l e u k o d y s t r o p h y , infantile Refsum s y n d r o m e a n d h y p e r p i p e c o l i c acidemia a r e c h a r a c t e r i z e d b y an a b s e n c e or diminution in the n u m b e r of morphologically d e t e c t a b l e peroxisomes in t i s s u e s from a f f e c t e d p a t i e n t s .
The
a b s e n c e of peroxisomes in t h e s e p a t i e n t s is a s s o c i a t e d with a n u m b e r of biochemical abnormalities ( r e v i e w e d in r e f e r e n c e 1) i n c l u d i n g impaired b e t a oxidation of v e r y long chain f a t t y acids (VLCFA), impaired s y n t h e s i s of plasmalogens, and accumulation of bile acid i n t e r m e d i a t e s , pipecolic acid and p h y t a n i c acid.
The biochemical and clinical distinctions between t h e s e
p h e n o t y p e s , h o w e v e r , a r e u n c l e a r a n d it is not known w h e t h e r t h e s e d i v e r s e d i s o r d e r s r e p r e s e n t allelic or non-allelic mutations of peroxisomai b i o g e n e s i s . Complementation analysis has been u s e d to examine the relationship between p h e n o t y p e a n d g e n o t y p e in the peroxisomal d i s o r d e r s .
F i b r o b l a s t s from two
d i f f e r e n t p a t i e n t s , b o t h deficient in a peroxisomal metabolic p r o c e s s , a r e i n d u c e d to f u s e ; the r e s u l t i n g multinuclear cells are examined f o r t h e i r ability to c a r r y out this metabolic f u n c t i o n (2).
R e s t o r a t i o n of a c t i v i t y can o n l y o c c u r if each
p a r e n t a l cell line p r o v i d e s the gene p r o d u c t defective in the o t h e r .
Other
i n v e s t i g a t o r s h a v e u s e d d__eenov____0_oplasmalogen b i o s y n t h e s i s (3), d i h y d r o x y a c e t o n e p h o s p h a t e a c y t t r a n s f e r a s e a c t i v i t y (4), particle b o u n d catalase ( 4 , 5 ) o r p h y t a n i c acid oxidase a c t i v i t y (6) as c r i t e r i a f o r complementation. 0006-291x/90 $1.50 Copyright © 1990 by Academic Press, lnc. All rights of reproduction in any form reserved.
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3ingh et al. (7) showed t h a t b e t a - o x i d a t i o n of VLCFA, e . g . llgnoceric acid ~.C24=o) t a k e s place in peroxisomes r a t h e r t h a n mitochondria. the f i r s t to u s e b e t a - o x i d a t i o n of VLCFA
The p r e s e n t s t u d y is
as the c r i t e r i o n for complementation
when g r o u p i n g cell lines from patients with peroxisomal d i s o r d e r s .
Many of the
cell lines u s e d in this s t u d y were also u s e d b y R o s c h e r and coworkers (3) in complementation s t u d i e s where plasmalogen b i o s y n t h e s i s was the criterion f o r complementation.
It was of i n t e r e s t , t h e r e f o r e , to compare complementation
g r o u p s o b t a i n e d u s i n g b o t h b e t a - o x i d a t i o n of VLCFA a n d plasmalogen b i o s y n t h e s i s as indices of complementation to see if the same genetic d e f e c t a f f e c t s b o t h metabolic p a t h w a y s . Complementation a n a l y s i s , u s i n g b e t a - o x i d a t i o n as the c r i t e r i o n f o r complementation, may be u s e f u l in the s t u d y of p a t i e n t s who are s u s p e c t e d of h a v i n g a single enzyme defect in the peroxisomal b e t a - o x i d a t i o n p a t h w a y .
In
many of t h e s e p a t i e n t s , all t h r e e b e t a - o x i d a t i o n enzyme p r o t e i n s ( a c y l - C o A oxidase, bifunctional enzyme a n d b e t a - k e t o t h i o l a s e ) are detectable b y immunoblot analysis (McGuinness a n d Watkins, u n p u b l i s h e d o b s e r v a t i o n s ) .
These p a t i e n t s may
have an inactive form of one of the enzymes which could be d e t e c t e d b y complementation s t u d i e s u s i n g cell lines known to be deficient in each of the b e t a - o x i d a t i o n enzyme activities.
MATERIALS AND METHODS Materials: Cell c u l t u r e r e a g e n t s were o b t a i n e d from GIBCO L a b o r a t o r i e s ( G r a n d I s l a n d , NY). [1-~4C]IAgnoceric acid (54.0 mCi/mmol) was o b t a i n e d from R e s e a r c h P r o d u c t s I n t e r n a t i o n a l C o r p o r a t i o n a n d [ 1- ~4C ]palmitic acid (58.0 mCi/mmol) from New England Nuclear. Ficoll 400 was obtained from Pharmacia Fine Chemicals ( P i s c a t a w a y , NJ) a n d p o l y e t h y l e n e glycol 4000 from Merck (Rahway, NJ). All o t h e r r e a g e n t s were of analytical g r a d e and were obtained from commercial s o u r c e s . Cell lines: C u l t u r e d skin f i b r o b l a s t s from twelve p a t i e n t s with d i s o r d e r s of peroxisomal biogenesis were originally obtained f o r diagnostic p u r p o s e s . They were maintained in Eagle's Minimum Essential Medium with 10% fetal bovine serum, 1% gtutamine and p e n i c i l l i n / s t r e p t o m y c i n . Clinical f i n d i n g s f o r p a t i e n t s SED1 and SED2, with single enzyme d e f e c t s , h a v e been r e p o r t e d p r e v i o u s l y b y Watkins et al. (8) a n d Poll-The et al. (9) r e s p e c t i v e l y . The major clinical f i n d i n g s of patients Z2, Z4, Z5, Z6, ZT, N7 a n d N8 i n c l u d e d in this s t u d y , w h e r e Z a n d N r e p r e s e n t Zellweger a n d neonatal a d r e n o l e u k o d y s t r o p h y p a t i e n t s , r e s p e c t i v e l y , were r e p o r t e d b y R o s c h e r et al. (3). Skin f i b r o b l a s t s from p a t i e n t s Z9, Z10 a n d Z l l were also s t u d i e d . Cell f u s i o n : Two p a r e n t a l cell lines (1.5 X 10 e cells each) were s e e d e d in 10cm dishes in a 1:1 ratio a n d f u s e d with p o l y e t h y l e n e glycol 4000 u s i n g the method of R o s c h e r et al. (3). Multi- and m o n o - n u c l e a t e d cells were s e p a r a t e d on a d i s c o n t i n u o u s Ficoll g r a d i e n t . A f t e r s e p a r a t i o n of the cells, "fused" a n d " u n f u s e d " cell f r a c t i o n s were examined microscopically. In the p r e s e n t s t u d y at least 80% of the cells in the f u s e d cell f r a c t i o n were multinucleate. The u n f u s e d cell f r a c t i o n c o n t a i n e d g r e a t e r t h a n 95% mononuclear cells. This f r a c t i o n was e q u i v a l e n t to c o c u l t u r e d cells. F u s e d cells a n d t h o s e t h a t failed to f u s e were r e c u l t i v a t e d a n d b e t a - o x i d a t i o n m e a s u r e d a f t e r a 48-h r e c o v e r y period. 365
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F a t t y acid oxidation: Oxidation of [1-14Clpalmitic acid a n d [1-14C1 lignoceric acid to w a t e r soluble p r o d u c t s was m e a s u r e d as p r e v i o u s l y d e s c r i b e d (10).
RESULTS AND DISCUSSION F a t t y acid b e t a - o x i d a t i o n was measured in f i b r o b l a s t s from controls and from two p a t i e n t s k n o w n to be deficient in one of the peroxisomal b e t a oxidation e n z y m e s (Table 1).
The ratio of oxidation of lignoceric acid (C24:o) to
palmitic acid (C16:o) was t a k e n as an indication of t h e ability of the cells to oxidize VLCFA ( 8 , 1 0 , 1 1 ) .
VLCFA oxidation in SED1 (immunologically deficient in
peroxisomal bifunctional enzyme) a n d SED2 (immunologicaUy deficient in peroxisomal a c y l - C o A oxidase) was 11 and 16% of control, r e s p e c t i v e l y (Table 1), in a g r e e m e n t with p r e v i o u s l y p u b l i s h e d r e s u l t s ( 8 , 9 ) . To determine w h e t h e r complementation analysis could be used in the s t u d y of p a t i e n t s with peroxisomal b e t a - o x i d a t i o n d i s o r d e r s , we i n v e s t i g a t e d the ability of celt lines SED1 a n d SED2 to complement each o t h e r .
F i b r o b l a s t s were b r i e f l y
TABLE 1 B e t a - o x i d a t i o n of V L C F A in f i b r o b l a s t s b e f o r e a n d a f t e r f u s i o n Beta-Oxidation of Fatty Acids
C++
C2,
Ls24~Cls
% of Control
(pmol/mg/min) Ct
62.9
14.3
0.228
(100)
C2
61.9
15.2
0.245
93
C1XC2 cc
49.8
11.1
0,222
91
C1XC2 fused
57.1
14.5
0.253
103
SED1
55.7
1.4
0,026
11
SED2
82.9
3.1
0.038
16
SED1XSED2 cc
72.6
4.1
0.056
23
SED1XSED2 fused
64.3
14.3
0,223
91
Oxidation of palmltlc acid (Cle) and Ilgnocerlc acid (C24) w a s measured In parental fibroblast cell lines, fused and cocultured (cc) cells as described in Methods. In the last column, the C241 C+e ratio is expressed as percentage of control C1. C1 and C2, Controls; SED1, bifunctional protein single enzyme defect; SED2, acyI-CoA oxidase single enzyme defect.
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TABLE 2 Complementation analysis of fibroblasts from patients with peroxisomal disorders
using beta-oxidation of VLCFA as the criterion for complementation 1
2
3
~
z2 zs N71zol ....
6
4
-
Zll
N8
-
[/ ff
Y
J'lf __z7
_
%Y Y Y
81
The ratio of oxidation of lignoceric acid (C24) to palmitic acid (C16) is expressed as percentage of a control measured in the same experiment. Numbers above the diagonal represent cocultured cells and those below this line represent fused cells. Z, Zellweger syndrome; N, neonatal ALD. The numbers above the brackets refer to the original group numbers of Roscher et aL (3).
incubated with polyethylene VLCFA in the cocultured, cell f r a c t i o n w a s m e a s u r e d .
g l y c o l to i n d u c e c e l l f u s i o n .
Beta-oxidation of
mononucleate cell fraction and the
fused,
multinucleate
C o c u l t u r e d SED1 a n d SED2 f i b r o b l a s t s h a d a low
o x i d a t i o n r a t i o b u t t h e f u s e d c e i l s o x i d i z e d V L C F A a t 91% o f t h e c o n t r o l r a t i o (Table 1), indicating that complementation had occurred. Having determined that restoration of VLCFA oxidation was a valid i n d i c a t o r o f c o m p l e m e n t a t i o n , we i n v e s t i g a t e d patients with disorders Results are expressed
o f p e r o x i s o m a l b i o g e n e s i s to c o m p l e m e n t ( T a b l e 2 ) . as a percentage
of the oxidation ratio in control cells.
The numbers above the diagonal represent line represent
the ability of fibroblasts from
fused cells.
cocultured
Oxidation ratios in parent
cells and those below this cell l i n e s ( d a t a n o t s h o w n ) ,
c o c u l t u r e d c e l l s o r n o n - c o m p l e m e n t i n g c e l l l i n e s w e r e g e n e r a l l y l e s s t h a n 30% o f the ratio of normal control cells.
An increase of at least 2.5-fold in the
oxidation ratio of fused cells over coeultured
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cells was considered to indicate
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fusion of c o n t r o l cells (Table 1) o r p a t i e n t s ' cells (Table 2)
with themselves did not a f f e c t the oxidation ratio. Cell lines r e p r e s e n t i n g five of the six complementation g r o u p s r e p o r t e d b y R o s c h e r et al. (3) were s t u d i e d .
As can be seen from Table 2, complementing
cell lines u s i n g VLCFA oxidation as the c r i t e r i o n s e g r e g a t e d into g r o u p s t h a t were identical to the g r o u p s obtained p r e v i o u s l y ( i n d i c a t e d b y b r a c k e t s at the top of Table 2) when plasmalogen s y n t h e s i s was u s e d as the criterion f o r complementation (3).
U n f o r t u n a t e l y , f i b r o b l a s t s from p a t i e n t Z8
(complementation g r o u p 5 of R o s c h e r a n d c o w o r k e r s ) were no l o n g e r available. Complementation analysis was also p e r f o r m e d on cell lines from t h r e e Zellweger p a t i e n t s which were not included in the s t u d y of R o s c h e r et al. (3) ( d e s i g n a t e d Z9, Z10 a n d Z l l ) .
Both VLCFA oxidation a n d plasmalogen s y n t h e s i s
were m e a s u r e d in f u s e d a n d c o c u l t u r e d cells.
Patient Z9 and patients Z10 a n d
Z l l were f o u n d to b e l o n g to complementation g r o u p s 1 a n d 4, r e s p e c t i v e l y , of R o s c h e r a n d colleagues when b o t h b e t a - o x i d a t i o n of VLCFA (Table 2) and r e s t o r a t i o n of plasmatogen s y n t h e s i s ( d a t a not shown) were u s e d as c r i t e r i a f o r complementation. To d a t e , no correlation between g e n o t y p e a n d p h e n o t y p e has been f o u n d in studies of f i b r o b l a s t s from p a t i e n t s with peroxisomal d i s o r d e r s .
R o s c h e r e t al. (3)
showed that all known p h e n o t y p e s were r e p r e s e n t e d within t h e i r complementation g r o u p 1 and the most common p h e n o t y p e , classical Zellweger s y n d r o m e , was f o u n d in five of t h e i r six complementation g r o u p s .
T h e r e is genetic h e t e r o g e n e i t y ,
t h e r e f o r e , n o t o n l y within the g r o u p of peroxisomal d i s o r d e r s b u t also between d i f f e r e n t Zellweger cell lines. Complementation
g r o u p s obtained in the p r e s e n t
s t u d y , when b e t a - o x i d a t i o n of VLCFA was u s e d as the c r i t e r i o n of complementation, were f o u n d to be identical to the complementation g r o u p s obtained b y R o s c h e r a n d c o w o r k e r s , s u p p o r t i n g the h y p o t h e s i s t h a t defects in peroxisome a s s e m b l y a f f e c t b o t h VLCFA oxidation a n d plasmalogen b i o s y n t h e s i s . Imanaka et al. (12) h a v e shown t h a t import of peroxisomal matrix p r o t e i n s r e q u i r e s the p r e s e n c e of c e r t a i n membrane p r o t e i n s , p o s s i b l y a c t i n g as r e c e p t o r s . It is p r o b a b l e , t h e r e f o r e , t h a t a single import mutation may a f f e c t b o t h metabolic pathways. Complementation a n a l y s i s , u s i n g b e t a - o x i d a t i o n of VLCFA as the c r i t e r i o n for complementation, will p r o v e u s e f u l in the s t u d y of p a t i e n t s who, b a s e d on clinical d i a g n o s i s , a r e s u s p e c t e d of h a v i n g a single enzyme defect in the peroxisomal b e t a - o x i d a t i o n p a t h w a y .
The l a b o r a t o r y f i n d i n g s common to t h e s e
patients include e l e v a t e d plasma VLCFA, impaired VLCFA oxidation in f i b r o b l a s t s and normal plasmalogen s y n t h e s i s ( 8 , 9 , 1 3 ) .
Some p a t i e n t s , with s u s p e c t e d single
enzyme d e f e c t s , were f o u n d to be immunologically positive f o r all t h r e e b e t a oxidation e n z y m e s (11, a n d McGuinness a n d Watkins, u n p u b l i s h e d o b s e r v a t i o n s ) .
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These patients p r e s u m a b l y have an inactive form
of one of the oxidation
enzymes. Fusing f i b r o b l a s t s from p a t i e n t s , with s u s p e c t e d single enzyme defects, with fibroblasts from patients SED1 and SED2 will allow identification of patients who are likely to be deficient in the third peroxisomal beta-oxidation enzyme, b e t a ketothiolase.
A beta-ketothiolase deficient cell line, along with the two lines
known to be deficient in acyl-CoA oxidase and bifunctional enzyme activities, will enable us to s t u d y and classify f i b r o b l a s t s from patients who a r e s u s p e c t e d of having a single enzyme defect in the peroxisomal beta-oxidation pathway. ACKNOWLEDGMENTS The a u t h o r s wish to thank Ms. Angela Liu for e x p e r t technical a s s i s t a n c e , and Dr. Bwee-Tien Poli-The for s u p p l y i n g the cell line SED2. This work was s u p p o r t e d in p a r t b y g r a n t s HD 10981 and HD 24061 from the National I n s t i t u t e s of Health. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
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