Proc. Nati. Acad. Sci. USA Vol. 82, pp. 7081-7085, October 1985 Medical Sciences Molecular heterogeneity of variant isovaleryl-CoA dehydrogenase from -cultured isovaleric acidemia fibroblasts (inborn metabolic disorder/leucine metabolism/posttranslational processing/precursor enzyme/immunoprecipitation) YASUYUKI IKEDA*, SUSAN M. KEESE, AND KAY TANAKA Yale University School of Medicine, Department of Human Genetics, New Haven, CT 06510 Communicated by Edward E. Adelberg, June 24, 1985 ABSTRACT Variants of isovaleryl-CoA dehydrogenase (IVDHase, EC 1.3.99.10) in 15 isovaleric acidemia fibroblast lines were analyzed using [3sS]methionine labeling, immuno- precipitation with anti-rat IVDHase antiserum, and NaDod- S04/polyacrylamide gel electrophoresis. Five distinct variants of IVDHase were detected. The molecular size of variant 1 (43 kDa) was indistinguishable from that of normal IVDHase (43 kDa), although the activity of this enzyme was as deficient (0-2.2% of normal control) as that of any other variant. It was synthesized as a precursor (45 kDa), which is the case for normal IVDHase. Variant 2 was synthesized as a 42-kDa precursor, but only a small portion of it was processed to the mature variant form (40 kDa). Variant 3 (41 kDa) was synthesized as a 43-kDa precursor. Variant 4 (40 kDa) was synthesized as a 42-kDa precursor that was readily processed to the mature form. In cells with variant 5, no material that crossreacted with the anti-rat IVDHase antibody was detected. These results suggest that variant 1 may be due to a point mutation, while variants 2-4 may be encoded by a different mutant IVDHase allele that causes the premature termination of translation, although other complex mechanisms are possi- ble. A deletion, a nonsense mutation close to the NH2 terminus or an extremely labile mRNA may give rise to variant 5. Isovaleric acidemia (IVA), an inborn error of leucine catab- olism, is clinically characterized by the accumulation of isovaleric acid and its derivatives in the serum and urine of affected patients, which leads to episodic vomiting, lethargy, coma, and ketoacidosis (1, 2). In patients with this disease, no other short-chain fatty acids accumulate in the body fluids. Results from the initial biochemical studies on patients with isovaleric acidemia (1, 2) suggested the deficiency of isovaleryl-CoA dehydrogenase (IVDHase, EC 1.3.99.10). Cultured skin fibroblasts from patients with IVA were used to demonstrate that IVA is, in fact, due to a deficiency of this enzyme (3). Subsequently, we have purified IVDHase from rat liver mitochondria, to homogeneity along with four other distinct acyl-CoA dehydrogenases and extensively charac- terized each (4-9). IVDHase is a FAD-containing enzyme (175 kDa) consist- ing of four subunits of equal size (5). The other four enzymes include short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases (7) plus another newly identified enzyme, 2-methyl-branched chain acyl-CoA dehydrogenase (6). We have shown that although all these enzymes are similar in molecular size, structure, and reaction mechanism (8), their substrate specificities are distinctly different. Isovaleryl-CoA (derived from leucine) is dehydrogenated only by IVDHase, whereas a-methyl-substituted acyl-CoAs such as isobutyryl- (derived from valine) and S-2-methylbutyryl-CoAs (derived from isoleucine) are dehydrogenated solely by 2-methyl- branched chain acyl-CoA dehydrogenase. The other three acyl-CoA dehydrogenases catalyze the first step of the ,8-oxidation cycle in the catabolism of fatty acids. We have also shown that these five acyl-CoA dehydrogenases are immunologically distinct. The antibodies raised against each enzyme did not crossreact with other acyl-CoA dehydrogen- ases from the same source (5-7). Two different phenotypes in the clinical expression of IVA have been noted. In some patients, this disorder manifests itself within a few days after birth, and its subsequent course is acute and often fatal in the neonatal period. In others, clinical manifestations are first recognized much later and are only episodic (10). When patients with the severe type of IVA survive the first 2 weeks of life the subsequent course of the disease is similar to that of patients with the mild type. To characterize the defect in IVDHase we have first tested the residual IVDHase activity in nine cell lines derived from IVA patients, by the improved tritium release assay. IVDHase activity was found to be uniformly low, regardless of the clinical phenotype (11). We then did genetic complementa- tion studies using polyethylene glycol-induced heterokary- ons but failed to demonstrate the existence of genetic heterogeneity (12). However, these results did not exclude the possibility that more than one mutant allele exists, each encoding for a varient IVDHase that is equally deficient in activity. In this study we have examined the molecular basis of IVA by investigating the biosynthesis of IVDHase in cultured IVA fibroblasts using immunoprecipitation with monospecific anti-rat IVDHase antibody (5) and NaDodSO4/PAGE. With this approach, we have identified at least five heterogeneous variants of IVDHase, which differ from each other in their subunit molecular size. MATERIALS AND METHODS Materials. L-[35S]Methionine (>600 Ci/mmol; 1 Ci = 37 GBq) was purchased from Amersham. Inactivated Staphy- lococcus aureus cells were obtained from Bethesda Research Laboratories. Rhodamine 6G was procured from Eastman. Cell culture materials were from GIBCO. Preparation of Antibodies Against Four Acyl-CoA Dehy- drogenases. Isovaleryl-CoA-, short-chain acyl-CoA-, medi- um-chain acyl-CoA-, and long-chain acyl-CoA dehydrogen- ases were purified to homogeneity from rat liver mitochon- dria as described (5, 7). Antisera against each of the four rat acyl-CoA dehydrogenases were raised in rabbits and partially purified by twice fractionating with 50% saturated ammoni- um sulfate. These antibodies are monospecific to the corre- sponding enzymes, and each is capable of precipitating at Abbreviations: IVA, isovaleric acidemia; IVDHase, isovaleryl-CoA dehydrogenase (EC 1.3.99.10). *Present address: National Cardiovascular Center Research Insti- tute, Suita 565, Osaka, Japan. 7081 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 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Molecular heterogeneity of variant isovaleryl-CoA dehydrogenase from cultured isovaleric acidemia fibroblasts.Proc. Nati. Acad. Sci. USA Vol. 82, pp. 7081-7085, October 1985 Medical Sciences
Molecular heterogeneity of variant isovaleryl-CoA dehydrogenase from-cultured isovaleric acidemia fibroblasts
(inborn metabolic disorder/leucine metabolism/posttranslational processing/precursor enzyme/immunoprecipitation)
YASUYUKI IKEDA*, SUSAN M. KEESE, AND KAY TANAKA Yale University School of Medicine, Department of Human Genetics, New Haven, CT 06510
Communicated by Edward E. Adelberg, June 24, 1985
ABSTRACT Variants of isovaleryl-CoA dehydrogenase (IVDHase, EC 1.3.99.10) in 15 isovaleric acidemia fibroblast lines were analyzed using [3sS]methionine labeling, immuno- precipitation with anti-rat IVDHase antiserum, and NaDod- S04/polyacrylamide gel electrophoresis. Five distinct variants of IVDHase were detected. The molecular size of variant 1 (43 kDa) was indistinguishable from that of normal IVDHase (43 kDa), although the activity of this enzyme was as deficient (0-2.2% of normal control) as that of any other variant. It was synthesized as a precursor (45 kDa), which is the case for normal IVDHase. Variant 2 was synthesized as a 42-kDa precursor, but only a small portion of it was processed to the mature variant form (40 kDa). Variant 3 (41 kDa) was synthesized as a 43-kDa precursor. Variant 4 (40 kDa) was synthesized as a 42-kDa precursor that was readily processed to the mature form. In cells with variant 5, no material that crossreacted with the anti-rat IVDHase antibody was detected. These results suggest that variant 1 may be due to a point mutation, while variants 2-4 may be encoded by a different mutant IVDHase allele that causes the premature termination of translation, although other complex mechanisms are possi- ble. A deletion, a nonsense mutation close to the NH2 terminus or an extremely labile mRNA may give rise to variant 5.
Isovaleric acidemia (IVA), an inborn error of leucine catab- olism, is clinically characterized by the accumulation of isovaleric acid and its derivatives in the serum and urine of affected patients, which leads to episodic vomiting, lethargy, coma, and ketoacidosis (1, 2). In patients with this disease, no other short-chain fatty acids accumulate in the body fluids. Results from the initial biochemical studies on patients with isovaleric acidemia (1, 2) suggested the deficiency of isovaleryl-CoA dehydrogenase (IVDHase, EC 1.3.99.10). Cultured skin fibroblasts from patients with IVA were used to demonstrate that IVA is, in fact, due to a deficiency of this enzyme (3). Subsequently, we have purified IVDHase from rat liver mitochondria, to homogeneity along with four other distinct acyl-CoA dehydrogenases and extensively charac- terized each (4-9). IVDHase is a FAD-containing enzyme (175 kDa) consist-
ing of four subunits of equal size (5). The other four enzymes include short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases (7) plus another newly identified enzyme, 2-methyl-branched chain acyl-CoA dehydrogenase (6). We have shown that although all these enzymes are similar in molecular size, structure, and reaction mechanism (8), their substrate specificities are distinctly different. Isovaleryl-CoA (derived from leucine) is dehydrogenated only by IVDHase, whereas a-methyl-substituted acyl-CoAs such as isobutyryl- (derived from valine) and S-2-methylbutyryl-CoAs (derived from isoleucine) are dehydrogenated solely by 2-methyl-
branched chain acyl-CoA dehydrogenase. The other three acyl-CoA dehydrogenases catalyze the first step of the ,8-oxidation cycle in the catabolism of fatty acids. We have also shown that these five acyl-CoA dehydrogenases are immunologically distinct. The antibodies raised against each enzyme did not crossreact with other acyl-CoA dehydrogen- ases from the same source (5-7). Two different phenotypes in the clinical expression ofIVA
have been noted. In some patients, this disorder manifests itself within a few days after birth, and its subsequent course is acute and often fatal in the neonatal period. In others, clinical manifestations are first recognized much later and are only episodic (10). When patients with the severe type ofIVA survive the first 2 weeks of life the subsequent course of the disease is similar to that of patients with the mild type. To characterize the defect in IVDHase we have first tested the residual IVDHase activity in nine cell lines derived from IVA patients, by the improved tritium release assay. IVDHase activity was found to be uniformly low, regardless of the clinical phenotype (11). We then did genetic complementa- tion studies using polyethylene glycol-induced heterokary- ons but failed to demonstrate the existence of genetic heterogeneity (12). However, these results did not exclude the possibility that more than one mutant allele exists, each encoding for a varient IVDHase that is equally deficient in activity.
In this study we have examined the molecular basis ofIVA by investigating the biosynthesis ofIVDHase in cultured IVA fibroblasts using immunoprecipitation with monospecific anti-rat IVDHase antibody (5) and NaDodSO4/PAGE. With this approach, we have identified at least five heterogeneous variants of IVDHase, which differ from each other in their subunit molecular size.
MATERIALS AND METHODS
Materials. L-[35S]Methionine (>600 Ci/mmol; 1 Ci = 37 GBq) was purchased from Amersham. Inactivated Staphy- lococcus aureus cells were obtained from Bethesda Research Laboratories. Rhodamine 6G was procured from Eastman. Cell culture materials were from GIBCO.
Preparation of Antibodies Against Four Acyl-CoA Dehy- drogenases. Isovaleryl-CoA-, short-chain acyl-CoA-, medi- um-chain acyl-CoA-, and long-chain acyl-CoA dehydrogen- ases were purified to homogeneity from rat liver mitochon- dria as described (5, 7). Antisera against each of the four rat acyl-CoA dehydrogenases were raised in rabbits and partially purified by twice fractionating with 50% saturated ammoni- um sulfate. These antibodies are monospecific to the corre- sponding enzymes, and each is capable of precipitating at
Abbreviations: IVA, isovaleric acidemia; IVDHase, isovaleryl-CoA dehydrogenase (EC 1.3.99.10). *Present address: National Cardiovascular Center Research Insti- tute, Suita 565, Osaka, Japan.
7081
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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7082 Medical Sciences: Ikeda et al.
least 15 ± 5 pug of the corresponding enzyme per 100 u1 of antiserum (5, 7).
Cell Culture. Fibroblasts from skin biopsy specimens of 15 IVA patients, six parents of these patients, and four normal individuals were cultured in Eagle's minimum essential medium supplemented with 10% fetal calf serum, nonessen- tial amino acids, and kanamycin. They were routinely main- tained at 370C in a 5% C02/95% air atmosphere.
Labeling and Extraction of Fibroblast Proteins. The growth media were removed from confluent monolayers of fibro- blasts (5 x 106 cells per 6-cm plastic dish). Each dish was washed twice with 2 ml of phosphate-buffered saline, pH 7.4 (Pi/NaCI), then 5 ml of labeling medium [60% Puck's saline F (vol/vol)/15% fetal calfserum (vol/vol)/10o 0.5M glucose (vol/vol)/15% H20 (vol/vol)] was added to each, and the dishes were incubated at 370C for 60 min. The medium was aspirated and replaced with 2.5 ml of fresh labeling medium containing L-[35S]methionine (50-100 ,uCi). Again, the dishes were incubated at 370C for 60 min. The medium was re- moved, the cell layers were rinsed twice with Pi/NaCl, and then solubilized by the addition of 1.0 ml of 150 mM NaCl/10 mM EDTA/0.5% Triton X-100/and 0.25% NaDodSO4, pH 7.2, containing 2% unlabeled methionine (wt/vol). The re- sulting disrupted cells were then immunoprecipitated. When rhodamine 6G was used, it was added (final concentration, 2.1 ,uM) 30 min before the medium was changed to labeling medium containing the same concentration ofrhodamine 6G. Cells were then labeled and extracted as described above.
Immunoprecipitation of Radiolabeled IVDHase and Other Acyl-CoA Dehydrogenases. One millimeter of cell extract from one dish was centrifuged for 30 min at 105,000 x g. The supernatant was mixed with 10 ,ul of anti-IVDHase antibody and the mixture was incubated at 4°C for 12 hr. The antigen-antibody complexes were recovered by adding 10 vol of S. aureus cell suspension (10%, wt/vol) per vol of antibody, followed by centrifugation at 1700 x g for 10 min. The pellets were first washed four times with 2 ml of 150 mM NaCl/10 mM Tris.HCl/1% Triton X-100 (vol/vol)/1% de- oxycholate (vol/vol)/0.1% NaDodSO4, pH 7.2, then the pellets were further washed two times with 10 ml of H20 to remove radiolabeled actin bound to S. aureus cells. The washed immunoprecipitates were solubilized in 20 ,ul of 0.1 M Tris HCl, pH 6.8/3% NaDodSO4 (wt/vol)/37% glycerol (vol/vol)/15% 2-mercaptoethanol (vol/vol)/0.019% bromophenol blue (wt/vol) and boiled for 5 min. The sample mixtures were centrifuged at 4000 x g for 10 min to collect S. aureus cells, and the supernatant was subjected to slab NaDodSO4/PAGE. The immunoprecipitations of the other acyl-CoA dehydrogenases were performed using the respec- tive monospecific antibodies under the same experimental conditions. PAGE and Fluorography. Slab NaDodSO4/PAGE was
performed using 9% gels (0.8 mm thick) according to the method ofLaemmli (13). The gels were stained with Coomas-
W_-9
68
1 2- 3
FIG. 1. Slab gel electrophoresis ofIVDHase immunoprecipitated from extracts of cultured human fibroblasts using anti-rat IVDHase antiserum. The confluent monolayers in 6-cm dishes were labeled with [35S]methionine for 60 min and then solubilized. The extracts (1 ml each) were immunoprecipitated by the monospecific anti-rat IVDHase antibodies (10 ,ul each). The immunoprecipitates were analyzed by NaDodSO4/PAGE. Lane 1, IVDHase immunoprecipit- ated with 10 ,ul anti-IVDHase antibody; lane 2, same as in lane 1, except that 7,g of unlabeled rat liver IVDHase was added before the addition of antiserum; lane 3, control immunoprecipitates with 10 j.1 of nonimmune rabbit serum. The tick mark on the left indicates the position of the IVDHase polypeptides. Numbers on the right are molecular size markers expressed in kDa.
sie brilliant blue, treated for fluorography with Auto-fluor (National Diagnostics, Somerville, NJ), dried, and fluoro- graphed according to the supplier's directions. X-ray film (XAR-5, Kodak) was used to detect the radiolabeled protein bands from the fluorographed gels. The following 14C-labeled proteins were used as molecular size markers: phosphorylase b (94 kDa), bovine serum albumin (68 kDa), ovalbumin (46 kDa), and a-chymotrypsinogen (26 kDa).
RESULTS
Crossreactivity of the Antisera Raised Against Rat Liver Acyl-CoA Dehydrogenases to the Corresponding Human Liver and Fibroblast Enzymes. When supernatants of sonicated rat and human liver homogenates were incubated with mono- specific antisera raised against the individual rat liver acyl-
Normal Fibroblasts Isovaleric Acidemia Fibroblosts Nonimmune |Ir--~~~ ~~~- ~ ~ -- ~- IgG
1- 1 ~~~22 3 4 5
2 3 4 5 6 7 8 9 10 12 13 14 15 16
FIG. 2. Electrophoretic demonstration of five variant IVDHase polypeptides from IVA fibroblasts. The experimental conditions were as described in Fig. 1. Lanes 1 and 2, normal fibroblasts; lanes 3-16, IVA fibroblasts from different patients. Our cell line numbers, which appear in lanes 1-16 are 1074, 1159, 501, 502, 743, 763, 262, 1302, 1310, 834, 747, 765, 766, 778, 1339, and 1311, respectively. The right-hand lane is immunoprecipitates with nonimmune serum.
Proc. Natl. Acad. Sci. USA 82 (1985)
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Proc. Natl. Acad. Sci. USA 82 (1985) 7083
Table 1. Classification and molecular features of normal and variant human IVDHase Bands synthesized, kDa
No rhodamine 6G Rhodamine 6G Class (mature enzyme) (precursor) Cell line(s)
Normal IVDHase 43 45 1074, 1159 Variant IVDHase Homozygotes
Variant 1 43 45 262, 501, 502, 743, 763, 1302, 1310 Variant 2 42 (major) 42 747
40 (minor) Variant 3 41 43 765, 766 Variant 4 40 42 778 Variant5 * * 1311, 1339
Compound heterozygote Variant 1-variant 2 43, 42, and 40 45 and 42 834, 1374
Heterozygotes Normal-variant 1 43 45 1389 (father of 834), 1376 (mother of 1374) Normal-variant 2 43, 42, and 40 45 and 42 1375 (father of 1374), 1390 (mother of 834)
*No crossreactive material was precipitated with the antiserum.
CoA dehydrogenases only homologous enzymes were im- munoprecipitated (data not shown). The molecular size ofthe human enzymes was identical to those of the corresponding rat enzymes with the exception of medium-chain acyl-CoA dehydrogenase. Human-medium-chain acyl-CoA dehydro- genase was detected as doublet bands, which were -0.5 and 1 kDa, respectively, larger than the rat-medium-chain acyl- CoA dehydrogenase (45 kDa). The doublet presumably resulted from partial proteolysis during the postmortem period. The molecular size of human IVDHase was equiva- lent to that of the rat enzyme (43 kDa). These results demonstrate that the antibodies raised against the individual rat enzymes specifically precipitate the corresponding human enzymes.
Immunoprecipitation of human fibroblast extracts with the anti-rat IVDHase antibody identified a 43-kDa protein, which appeared to be identical to that immunoprecipitated from human and rat livers (Fig. 1, lane 1). This band disappeared when excess unlabeled rat IVDHase was added to the immunoprecipitation reaction mixture (lane 2) or when la- beled cell extract was incubated with nonimmunized rabbit serum (lane 3). These results confirm the identity of the immunoprecipitated IVDHase, and indicate that cultured fibroblasts are suitable for the molecular studies of acyl-CoA dehydrogenases.
Heterogeneity of the Subunit Molecular Weight of Variant IVDHase from IVA Fibroblasts. The extracts from fibroblasts derived from two normal persons and fifteen individuals with IVA were immunoprecipitated using anti-rat IVDHase anti- body and were analyzed by NaDodSO4/PAGE (Fig. 2). Normal IVDHase (43 kDa) from two normal cell lines is shown in lanes 1 and 2. The molecular size of IVDHase immunoprecipitated from seven IVA cell lines (lanes 3-9) is 43 kDa and indistinguishable from normal IVDHase, al- though these cell lines are as deficient in IVDHase activity as any of the other IVA cell lines (11). We designated this variant 1. In lane 11, one major band (42 kDa) and one faint smaller band (40 kDa) were detected. We designated this variant 2. Lane 10 shows a compound heterozygote of variants 1 and 2. In lanes 12 and 13, there is only one major IVDHase band (41 kDa). We named this variant 3. In addition, there is a faint band that is approximately equal to normal IVDHase. These two cell lines were derived from two Senegalese siblings (14). Variant 4 (lane 14) has a major band at 40 kDa. There is, in addition, a faint band (42 kDa) that is 1 kDa less than normal IVDHase and approximately equal to variant 2. In lanes 15 and 16, no crossreactive material was detected. We named these mutants variant 5. Thus, five variants of IVDHase were identified by NaDodSO4/PAGE.
A NORMAL ISOVALERIC ACIDEMIA FIBROBLASTS FIBROBLAST 1 1-2 3 4 5
r --7
I1 + - A + - + - + - + - + -
2 3 4 5 6 7 8 9 10 !1 1I? 13 4
NF 2 [ + - - -
45- 43,
2 3 4
FIG. 3. Comparison of the molecular weight of precursor IVDHase in normal and IVA fibroblasts to their mature counterparts. Extracts of fibroblasts labeled with [35S]methionine in the presence (+) and absence (-) of rhodamine 6G were prepared. Equal volumes of the extracts were treated with anti-IVDHase antibody or nonimmune serum. (A) The cell line numbers analyzed were as follows: lanes 1 and 2, 1074; lanes 3 and 4, 502; lanes 5 and 6, 834; lanes 7 and 8, 765; lanes 9 and 10, 778; lanes 11 and 12, 1311. In lanes 13 and 14 cell extracts were immunoprecipitated with nonimmune serum. (B) The cell line numbers analyzed were as follows: lanes 1 and 2, 1074, lanes 3 and 4, 747. Precursor and mature IVDHases are indicated by arrows. Numbers at left are molecular size markers, kDa.
Medical Sciences: Ikeda et al.
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ISOVALERIC ACIDEMIA FIBROBLASTS NORMAL r
FIBROBLAST 1 1-2 I ~ ~ i r- ---
pIVD IVD pIVO IVD pIVD IVD _ + _ + _ + - + - + - +
45 - _ 4 43 - 4nmitm
3 1 ---~~~~~~~~~~~~~~~~~~~~~I pIVD IVD
2 3 4 '5 6 7 8 9 10 I I 12 13 14 15 16
FIG. 4. Competitive inhibition of the immunoprecipitation of the labeled precursor (pIVD) and mature (IVD) forms of normal and mutant human IVDHase by pure unlabeled rat IVDHase. Cell lines used are: lanes 1-4, 1074; lanes 5-8, 743; lanes 9-12, 834; and lanes 13-16, 765. Pure rat IVDHase (7 ,ug) was added as indicated (+). Precursor and mature IVDHases are indicated by arrows. Molecular size markers on left are in kDa's.
The molecular features of the variant IVDs are summarized in Table 1. When short-chain acyl-CoA dehydrogenase was analyzed
in all IVA cell lines it was indistinguishable from that in normal cell lines (data not shown). The Precursor of Variant IVDHase. We have recently
shown that acyl-CoA dehydrogenases are synthesized in the cytosol as precursors which are 2-5 kDa larger than the corresponding mature enzyme (15, 16). The extended se- quence in the precursor enzymes is located at the amino terminus (17-19). The extended sequence of the precursor enzymes is then clipped as the enzyme is imported into the mitochondria. This posttranslational processing is energy dependent (20) and is inhibited with rhodamine 6G (21), which inhibits mitochondrial energy production. We studied the precursors of the variant IVDHases and
their processing using rhodamine 6G, as shown in Fig. 3A. The precursor of variant 1 is 2 kDa larger than the mature variant 1 (lane 3), as in the case of the normal counterpart. Likewise, the precursor of variant 3 (lanes 7 and 8) and that of variant 4 (lanes 9 and 10) were also 2 kDa larger than the respective mature enzymes. In contrast, when variant 2 homozygote cells were labeled in the presence of rhodamine 6G (Fig. 3B), only a single 42-kDa band, which was identical to the major variant 2 band synthesized in the absence of rhodamine 6G, was detected. The minor 40-kDa band was not detected. Likewise, when the variant 1-variant 2 compound heterozygote cells were labeled in the presence of rhodamine 6G (Fig. 3A), precursor of variant 1 (45 kDa) was detected. However, the size of the variant 2 major band (42 kDa) remained unchanged while the variant 2 minor band (40 kDa) disappeared (lanes 5 and 6; see Fig. 5, lanes 1, 2, 7, and 8). When variant 5 homozygote cells were labeled in the pres- ence of rhodamine 6G, no crossreactive material was syn- thesized (lanes 11 and 12). The pattern of each cell line was highly reproducible. The molecular features ofthe precursors of the variant IVDHases are summarized in Table 1. The identity of the variant IVDHases and their precursors was confirmed by the competition experiments using the pure rat IVDHase (Fig. 4). These labeled bands were not im-
1-2 (834)
39-
munoprecipitated when an excess of pure rat IVDHase was added to the homogenates prior to addition of the antibody. Family Studies. Among the IVA cell lines which we
studied, two cell lines (nos. 834 and 1374) were found to be variant 1-2 compound heterozygotes (Figs. 3 and 5). When the cells from the parents of these patients were studied, cells from the mother of no. 834 and the father of no. 1374 exhibited the same pattern as those ofthe respective proband cells, indicating that they are normal-variant 2 heterozy- gotes. The cells from the father of no. 834 and those from the mother of no.…
Molecular heterogeneity of variant isovaleryl-CoA dehydrogenase from-cultured isovaleric acidemia fibroblasts
(inborn metabolic disorder/leucine metabolism/posttranslational processing/precursor enzyme/immunoprecipitation)
YASUYUKI IKEDA*, SUSAN M. KEESE, AND KAY TANAKA Yale University School of Medicine, Department of Human Genetics, New Haven, CT 06510
Communicated by Edward E. Adelberg, June 24, 1985
ABSTRACT Variants of isovaleryl-CoA dehydrogenase (IVDHase, EC 1.3.99.10) in 15 isovaleric acidemia fibroblast lines were analyzed using [3sS]methionine labeling, immuno- precipitation with anti-rat IVDHase antiserum, and NaDod- S04/polyacrylamide gel electrophoresis. Five distinct variants of IVDHase were detected. The molecular size of variant 1 (43 kDa) was indistinguishable from that of normal IVDHase (43 kDa), although the activity of this enzyme was as deficient (0-2.2% of normal control) as that of any other variant. It was synthesized as a precursor (45 kDa), which is the case for normal IVDHase. Variant 2 was synthesized as a 42-kDa precursor, but only a small portion of it was processed to the mature variant form (40 kDa). Variant 3 (41 kDa) was synthesized as a 43-kDa precursor. Variant 4 (40 kDa) was synthesized as a 42-kDa precursor that was readily processed to the mature form. In cells with variant 5, no material that crossreacted with the anti-rat IVDHase antibody was detected. These results suggest that variant 1 may be due to a point mutation, while variants 2-4 may be encoded by a different mutant IVDHase allele that causes the premature termination of translation, although other complex mechanisms are possi- ble. A deletion, a nonsense mutation close to the NH2 terminus or an extremely labile mRNA may give rise to variant 5.
Isovaleric acidemia (IVA), an inborn error of leucine catab- olism, is clinically characterized by the accumulation of isovaleric acid and its derivatives in the serum and urine of affected patients, which leads to episodic vomiting, lethargy, coma, and ketoacidosis (1, 2). In patients with this disease, no other short-chain fatty acids accumulate in the body fluids. Results from the initial biochemical studies on patients with isovaleric acidemia (1, 2) suggested the deficiency of isovaleryl-CoA dehydrogenase (IVDHase, EC 1.3.99.10). Cultured skin fibroblasts from patients with IVA were used to demonstrate that IVA is, in fact, due to a deficiency of this enzyme (3). Subsequently, we have purified IVDHase from rat liver mitochondria, to homogeneity along with four other distinct acyl-CoA dehydrogenases and extensively charac- terized each (4-9). IVDHase is a FAD-containing enzyme (175 kDa) consist-
ing of four subunits of equal size (5). The other four enzymes include short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases (7) plus another newly identified enzyme, 2-methyl-branched chain acyl-CoA dehydrogenase (6). We have shown that although all these enzymes are similar in molecular size, structure, and reaction mechanism (8), their substrate specificities are distinctly different. Isovaleryl-CoA (derived from leucine) is dehydrogenated only by IVDHase, whereas a-methyl-substituted acyl-CoAs such as isobutyryl- (derived from valine) and S-2-methylbutyryl-CoAs (derived from isoleucine) are dehydrogenated solely by 2-methyl-
branched chain acyl-CoA dehydrogenase. The other three acyl-CoA dehydrogenases catalyze the first step of the ,8-oxidation cycle in the catabolism of fatty acids. We have also shown that these five acyl-CoA dehydrogenases are immunologically distinct. The antibodies raised against each enzyme did not crossreact with other acyl-CoA dehydrogen- ases from the same source (5-7). Two different phenotypes in the clinical expression ofIVA
have been noted. In some patients, this disorder manifests itself within a few days after birth, and its subsequent course is acute and often fatal in the neonatal period. In others, clinical manifestations are first recognized much later and are only episodic (10). When patients with the severe type ofIVA survive the first 2 weeks of life the subsequent course of the disease is similar to that of patients with the mild type. To characterize the defect in IVDHase we have first tested the residual IVDHase activity in nine cell lines derived from IVA patients, by the improved tritium release assay. IVDHase activity was found to be uniformly low, regardless of the clinical phenotype (11). We then did genetic complementa- tion studies using polyethylene glycol-induced heterokary- ons but failed to demonstrate the existence of genetic heterogeneity (12). However, these results did not exclude the possibility that more than one mutant allele exists, each encoding for a varient IVDHase that is equally deficient in activity.
In this study we have examined the molecular basis ofIVA by investigating the biosynthesis ofIVDHase in cultured IVA fibroblasts using immunoprecipitation with monospecific anti-rat IVDHase antibody (5) and NaDodSO4/PAGE. With this approach, we have identified at least five heterogeneous variants of IVDHase, which differ from each other in their subunit molecular size.
MATERIALS AND METHODS
Materials. L-[35S]Methionine (>600 Ci/mmol; 1 Ci = 37 GBq) was purchased from Amersham. Inactivated Staphy- lococcus aureus cells were obtained from Bethesda Research Laboratories. Rhodamine 6G was procured from Eastman. Cell culture materials were from GIBCO.
Preparation of Antibodies Against Four Acyl-CoA Dehy- drogenases. Isovaleryl-CoA-, short-chain acyl-CoA-, medi- um-chain acyl-CoA-, and long-chain acyl-CoA dehydrogen- ases were purified to homogeneity from rat liver mitochon- dria as described (5, 7). Antisera against each of the four rat acyl-CoA dehydrogenases were raised in rabbits and partially purified by twice fractionating with 50% saturated ammoni- um sulfate. These antibodies are monospecific to the corre- sponding enzymes, and each is capable of precipitating at
Abbreviations: IVA, isovaleric acidemia; IVDHase, isovaleryl-CoA dehydrogenase (EC 1.3.99.10). *Present address: National Cardiovascular Center Research Insti- tute, Suita 565, Osaka, Japan.
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The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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7082 Medical Sciences: Ikeda et al.
least 15 ± 5 pug of the corresponding enzyme per 100 u1 of antiserum (5, 7).
Cell Culture. Fibroblasts from skin biopsy specimens of 15 IVA patients, six parents of these patients, and four normal individuals were cultured in Eagle's minimum essential medium supplemented with 10% fetal calf serum, nonessen- tial amino acids, and kanamycin. They were routinely main- tained at 370C in a 5% C02/95% air atmosphere.
Labeling and Extraction of Fibroblast Proteins. The growth media were removed from confluent monolayers of fibro- blasts (5 x 106 cells per 6-cm plastic dish). Each dish was washed twice with 2 ml of phosphate-buffered saline, pH 7.4 (Pi/NaCI), then 5 ml of labeling medium [60% Puck's saline F (vol/vol)/15% fetal calfserum (vol/vol)/10o 0.5M glucose (vol/vol)/15% H20 (vol/vol)] was added to each, and the dishes were incubated at 370C for 60 min. The medium was aspirated and replaced with 2.5 ml of fresh labeling medium containing L-[35S]methionine (50-100 ,uCi). Again, the dishes were incubated at 370C for 60 min. The medium was re- moved, the cell layers were rinsed twice with Pi/NaCl, and then solubilized by the addition of 1.0 ml of 150 mM NaCl/10 mM EDTA/0.5% Triton X-100/and 0.25% NaDodSO4, pH 7.2, containing 2% unlabeled methionine (wt/vol). The re- sulting disrupted cells were then immunoprecipitated. When rhodamine 6G was used, it was added (final concentration, 2.1 ,uM) 30 min before the medium was changed to labeling medium containing the same concentration ofrhodamine 6G. Cells were then labeled and extracted as described above.
Immunoprecipitation of Radiolabeled IVDHase and Other Acyl-CoA Dehydrogenases. One millimeter of cell extract from one dish was centrifuged for 30 min at 105,000 x g. The supernatant was mixed with 10 ,ul of anti-IVDHase antibody and the mixture was incubated at 4°C for 12 hr. The antigen-antibody complexes were recovered by adding 10 vol of S. aureus cell suspension (10%, wt/vol) per vol of antibody, followed by centrifugation at 1700 x g for 10 min. The pellets were first washed four times with 2 ml of 150 mM NaCl/10 mM Tris.HCl/1% Triton X-100 (vol/vol)/1% de- oxycholate (vol/vol)/0.1% NaDodSO4, pH 7.2, then the pellets were further washed two times with 10 ml of H20 to remove radiolabeled actin bound to S. aureus cells. The washed immunoprecipitates were solubilized in 20 ,ul of 0.1 M Tris HCl, pH 6.8/3% NaDodSO4 (wt/vol)/37% glycerol (vol/vol)/15% 2-mercaptoethanol (vol/vol)/0.019% bromophenol blue (wt/vol) and boiled for 5 min. The sample mixtures were centrifuged at 4000 x g for 10 min to collect S. aureus cells, and the supernatant was subjected to slab NaDodSO4/PAGE. The immunoprecipitations of the other acyl-CoA dehydrogenases were performed using the respec- tive monospecific antibodies under the same experimental conditions. PAGE and Fluorography. Slab NaDodSO4/PAGE was
performed using 9% gels (0.8 mm thick) according to the method ofLaemmli (13). The gels were stained with Coomas-
W_-9
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1 2- 3
FIG. 1. Slab gel electrophoresis ofIVDHase immunoprecipitated from extracts of cultured human fibroblasts using anti-rat IVDHase antiserum. The confluent monolayers in 6-cm dishes were labeled with [35S]methionine for 60 min and then solubilized. The extracts (1 ml each) were immunoprecipitated by the monospecific anti-rat IVDHase antibodies (10 ,ul each). The immunoprecipitates were analyzed by NaDodSO4/PAGE. Lane 1, IVDHase immunoprecipit- ated with 10 ,ul anti-IVDHase antibody; lane 2, same as in lane 1, except that 7,g of unlabeled rat liver IVDHase was added before the addition of antiserum; lane 3, control immunoprecipitates with 10 j.1 of nonimmune rabbit serum. The tick mark on the left indicates the position of the IVDHase polypeptides. Numbers on the right are molecular size markers expressed in kDa.
sie brilliant blue, treated for fluorography with Auto-fluor (National Diagnostics, Somerville, NJ), dried, and fluoro- graphed according to the supplier's directions. X-ray film (XAR-5, Kodak) was used to detect the radiolabeled protein bands from the fluorographed gels. The following 14C-labeled proteins were used as molecular size markers: phosphorylase b (94 kDa), bovine serum albumin (68 kDa), ovalbumin (46 kDa), and a-chymotrypsinogen (26 kDa).
RESULTS
Crossreactivity of the Antisera Raised Against Rat Liver Acyl-CoA Dehydrogenases to the Corresponding Human Liver and Fibroblast Enzymes. When supernatants of sonicated rat and human liver homogenates were incubated with mono- specific antisera raised against the individual rat liver acyl-
Normal Fibroblasts Isovaleric Acidemia Fibroblosts Nonimmune |Ir--~~~ ~~~- ~ ~ -- ~- IgG
1- 1 ~~~22 3 4 5
2 3 4 5 6 7 8 9 10 12 13 14 15 16
FIG. 2. Electrophoretic demonstration of five variant IVDHase polypeptides from IVA fibroblasts. The experimental conditions were as described in Fig. 1. Lanes 1 and 2, normal fibroblasts; lanes 3-16, IVA fibroblasts from different patients. Our cell line numbers, which appear in lanes 1-16 are 1074, 1159, 501, 502, 743, 763, 262, 1302, 1310, 834, 747, 765, 766, 778, 1339, and 1311, respectively. The right-hand lane is immunoprecipitates with nonimmune serum.
Proc. Natl. Acad. Sci. USA 82 (1985)
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Proc. Natl. Acad. Sci. USA 82 (1985) 7083
Table 1. Classification and molecular features of normal and variant human IVDHase Bands synthesized, kDa
No rhodamine 6G Rhodamine 6G Class (mature enzyme) (precursor) Cell line(s)
Normal IVDHase 43 45 1074, 1159 Variant IVDHase Homozygotes
Variant 1 43 45 262, 501, 502, 743, 763, 1302, 1310 Variant 2 42 (major) 42 747
40 (minor) Variant 3 41 43 765, 766 Variant 4 40 42 778 Variant5 * * 1311, 1339
Compound heterozygote Variant 1-variant 2 43, 42, and 40 45 and 42 834, 1374
Heterozygotes Normal-variant 1 43 45 1389 (father of 834), 1376 (mother of 1374) Normal-variant 2 43, 42, and 40 45 and 42 1375 (father of 1374), 1390 (mother of 834)
*No crossreactive material was precipitated with the antiserum.
CoA dehydrogenases only homologous enzymes were im- munoprecipitated (data not shown). The molecular size ofthe human enzymes was identical to those of the corresponding rat enzymes with the exception of medium-chain acyl-CoA dehydrogenase. Human-medium-chain acyl-CoA dehydro- genase was detected as doublet bands, which were -0.5 and 1 kDa, respectively, larger than the rat-medium-chain acyl- CoA dehydrogenase (45 kDa). The doublet presumably resulted from partial proteolysis during the postmortem period. The molecular size of human IVDHase was equiva- lent to that of the rat enzyme (43 kDa). These results demonstrate that the antibodies raised against the individual rat enzymes specifically precipitate the corresponding human enzymes.
Immunoprecipitation of human fibroblast extracts with the anti-rat IVDHase antibody identified a 43-kDa protein, which appeared to be identical to that immunoprecipitated from human and rat livers (Fig. 1, lane 1). This band disappeared when excess unlabeled rat IVDHase was added to the immunoprecipitation reaction mixture (lane 2) or when la- beled cell extract was incubated with nonimmunized rabbit serum (lane 3). These results confirm the identity of the immunoprecipitated IVDHase, and indicate that cultured fibroblasts are suitable for the molecular studies of acyl-CoA dehydrogenases.
Heterogeneity of the Subunit Molecular Weight of Variant IVDHase from IVA Fibroblasts. The extracts from fibroblasts derived from two normal persons and fifteen individuals with IVA were immunoprecipitated using anti-rat IVDHase anti- body and were analyzed by NaDodSO4/PAGE (Fig. 2). Normal IVDHase (43 kDa) from two normal cell lines is shown in lanes 1 and 2. The molecular size of IVDHase immunoprecipitated from seven IVA cell lines (lanes 3-9) is 43 kDa and indistinguishable from normal IVDHase, al- though these cell lines are as deficient in IVDHase activity as any of the other IVA cell lines (11). We designated this variant 1. In lane 11, one major band (42 kDa) and one faint smaller band (40 kDa) were detected. We designated this variant 2. Lane 10 shows a compound heterozygote of variants 1 and 2. In lanes 12 and 13, there is only one major IVDHase band (41 kDa). We named this variant 3. In addition, there is a faint band that is approximately equal to normal IVDHase. These two cell lines were derived from two Senegalese siblings (14). Variant 4 (lane 14) has a major band at 40 kDa. There is, in addition, a faint band (42 kDa) that is 1 kDa less than normal IVDHase and approximately equal to variant 2. In lanes 15 and 16, no crossreactive material was detected. We named these mutants variant 5. Thus, five variants of IVDHase were identified by NaDodSO4/PAGE.
A NORMAL ISOVALERIC ACIDEMIA FIBROBLASTS FIBROBLAST 1 1-2 3 4 5
r --7
I1 + - A + - + - + - + - + -
2 3 4 5 6 7 8 9 10 !1 1I? 13 4
NF 2 [ + - - -
45- 43,
2 3 4
FIG. 3. Comparison of the molecular weight of precursor IVDHase in normal and IVA fibroblasts to their mature counterparts. Extracts of fibroblasts labeled with [35S]methionine in the presence (+) and absence (-) of rhodamine 6G were prepared. Equal volumes of the extracts were treated with anti-IVDHase antibody or nonimmune serum. (A) The cell line numbers analyzed were as follows: lanes 1 and 2, 1074; lanes 3 and 4, 502; lanes 5 and 6, 834; lanes 7 and 8, 765; lanes 9 and 10, 778; lanes 11 and 12, 1311. In lanes 13 and 14 cell extracts were immunoprecipitated with nonimmune serum. (B) The cell line numbers analyzed were as follows: lanes 1 and 2, 1074, lanes 3 and 4, 747. Precursor and mature IVDHases are indicated by arrows. Numbers at left are molecular size markers, kDa.
Medical Sciences: Ikeda et al.
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ISOVALERIC ACIDEMIA FIBROBLASTS NORMAL r
FIBROBLAST 1 1-2 I ~ ~ i r- ---
pIVD IVD pIVO IVD pIVD IVD _ + _ + _ + - + - + - +
45 - _ 4 43 - 4nmitm
3 1 ---~~~~~~~~~~~~~~~~~~~~~I pIVD IVD
2 3 4 '5 6 7 8 9 10 I I 12 13 14 15 16
FIG. 4. Competitive inhibition of the immunoprecipitation of the labeled precursor (pIVD) and mature (IVD) forms of normal and mutant human IVDHase by pure unlabeled rat IVDHase. Cell lines used are: lanes 1-4, 1074; lanes 5-8, 743; lanes 9-12, 834; and lanes 13-16, 765. Pure rat IVDHase (7 ,ug) was added as indicated (+). Precursor and mature IVDHases are indicated by arrows. Molecular size markers on left are in kDa's.
The molecular features of the variant IVDs are summarized in Table 1. When short-chain acyl-CoA dehydrogenase was analyzed
in all IVA cell lines it was indistinguishable from that in normal cell lines (data not shown). The Precursor of Variant IVDHase. We have recently
shown that acyl-CoA dehydrogenases are synthesized in the cytosol as precursors which are 2-5 kDa larger than the corresponding mature enzyme (15, 16). The extended se- quence in the precursor enzymes is located at the amino terminus (17-19). The extended sequence of the precursor enzymes is then clipped as the enzyme is imported into the mitochondria. This posttranslational processing is energy dependent (20) and is inhibited with rhodamine 6G (21), which inhibits mitochondrial energy production. We studied the precursors of the variant IVDHases and
their processing using rhodamine 6G, as shown in Fig. 3A. The precursor of variant 1 is 2 kDa larger than the mature variant 1 (lane 3), as in the case of the normal counterpart. Likewise, the precursor of variant 3 (lanes 7 and 8) and that of variant 4 (lanes 9 and 10) were also 2 kDa larger than the respective mature enzymes. In contrast, when variant 2 homozygote cells were labeled in the presence of rhodamine 6G (Fig. 3B), only a single 42-kDa band, which was identical to the major variant 2 band synthesized in the absence of rhodamine 6G, was detected. The minor 40-kDa band was not detected. Likewise, when the variant 1-variant 2 compound heterozygote cells were labeled in the presence of rhodamine 6G (Fig. 3A), precursor of variant 1 (45 kDa) was detected. However, the size of the variant 2 major band (42 kDa) remained unchanged while the variant 2 minor band (40 kDa) disappeared (lanes 5 and 6; see Fig. 5, lanes 1, 2, 7, and 8). When variant 5 homozygote cells were labeled in the pres- ence of rhodamine 6G, no crossreactive material was syn- thesized (lanes 11 and 12). The pattern of each cell line was highly reproducible. The molecular features ofthe precursors of the variant IVDHases are summarized in Table 1. The identity of the variant IVDHases and their precursors was confirmed by the competition experiments using the pure rat IVDHase (Fig. 4). These labeled bands were not im-
1-2 (834)
39-
munoprecipitated when an excess of pure rat IVDHase was added to the homogenates prior to addition of the antibody. Family Studies. Among the IVA cell lines which we
studied, two cell lines (nos. 834 and 1374) were found to be variant 1-2 compound heterozygotes (Figs. 3 and 5). When the cells from the parents of these patients were studied, cells from the mother of no. 834 and the father of no. 1374 exhibited the same pattern as those ofthe respective proband cells, indicating that they are normal-variant 2 heterozy- gotes. The cells from the father of no. 834 and those from the mother of no.…
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