Quantitative Analysis of Mitochondrial Protein Expression in

Quantitative Analysis of Mitochondrial Protein Expression in Methylmalonic Acidemia by Two-Dimensional Difference Gel Electrophoresis Eva Richard,*,† Lucia Monteoliva,‡ Silvia Juarez,§ Bele´ n Pe´ rez,† Lourdes R. Desviat,† Magdalena Ugarte,† and Juan Pablo Albar§ Centro de Biologıá Molecular “Severo Ochoa”, CSIC-UAM, Universidad Autońoma de Madrid, Departamento de Microbiologıá, Facultad de Farmacia, Universidad Complutense de Madrid, and Centro Nacional de Biotecnologıá, CSIC, Madrid, Spain Received December 23, 2005

Isolated methylmalonic acidemia (MMA) is a rare metabolic disease due to the deficient activity of L-methylmalonyl-CoA mutase (MCM). This mitochondrial enzyme converts L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (Adocbl) as cofactor. Isolated MMA is subdivided into five forms: mut MMA associated with MCM deficiency, three different defects related to mitochondrial Adocbl formation (cblA, cblB, and cblH), and cblD variant 2. We performed proteomic analysis on mitochondria from an individual with cblH/cblD disorder using 2-D DIGE to identify differentially expressed proteins in this disease. Comparative analysis of control/patient mitochondrial proteome allowed us to identify differential expression of 10 proteins. The most notable groups included proteins involved in apoptosis (cytochrome c), oxidative stress (manganese superoxide dismutase) and cell metabolism (succinyl-CoA ligase (GDP forming) and mitochondrial glycerophosphate dehydrogenase). Immunoblot analysis further validated 2-D DIGE results of two of these proteins in multiple MMA patients, suggesting that the differences in expression are a general effect in this disorder. It is feasible that the differential proteins identified in this study have a biological significance and might be related to the pathophysiology of MMA. Keywords: methylmalonic acidemia • cblA • cblB • cblH • cblD • mitochondria • proteome • two-dimensional difference gel electrophoresis

Introduction Isolated methylmalonic acidemia (MMA) is an inborn error of metabolism caused by the impaired isomerization of Lmethylmalonyl-CoA to succinyl-CoA. This reaction is part of the catabolic pathway of odd-chain fatty acids, certain branched chain amino acids, and cholesterol through propionyl-CoA to the Krebs cycle, and it is catalyzed by the mitochondrial protein L-methylmalonyl-CoA mutase (MCM, EC 5.4.99.2), using adenosylcobalamin (Adocbl) as cofactor1 (Figure 1). Isolated MMA is due to defects in the MCM apoenzyme (mut complementation group) or defects in enzymes required for the mitochondrial formation of the active form of Adocbl (cbl complementation group). Biochemical and complementation studies with cultured patient fibroblasts have defined different mutants belong to cbl complementation group: cblA (OMIM 607 481), cblB (OMIM 607 568) and cblH (OMIM 606 169) groups, and * To whom correspondence should be addressed: Centro de Biologıá Molecular “Severo Ochoa”. Mo´dulo CX, laboratorio 210. Facultad de Ciencias. Universidad Autońoma de Madrid, 28049 Madrid, Spain. Tel: +34914974868. Fax: +34917347797. E-mail: [email protected]. † Universidad Autońoma de Madrid. ‡ Universidad Complutense de Madrid. § CSIC.

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Journal of Proteome Research 2006, 5, 1602-1610

Published on Web 06/17/2006

one case reported into cblD variant 2,2 in all of them the synthesis of Adocbl is blocked. Functional deficiencies of these enzymes lead to isolated MMA deficiency, and the clinical presentation of this disorder may vary from a fatal neonatal presentation to an infantile form. Although the etiology of methylmalonic aciduria is heterogeneous, the clinical symptoms of affected patients are quite similar, and commonly include lethargy, coma, failure to thrive, recurrent vomiting, muscular hypotonia, and developmental retardation.1 It has been suggested that the neuropathological changes are caused by the accumulation of the toxic organic acids.3 The mature MCM behaves as a homodimer of 718-residue.4 The MMAA gene (cblA disorder) encodes a predicted polypeptide of 418-residue and its function is unknown.5,6 The MMAB gene (cblB complementation group) encodes cob(I)alamin ATP transferase of 27 kDa.7 At this time, the gene involved in cblH and the gene and its localization of cblD variant 2 complementation groups have not been identified8,9 (Figure 1). Genetic analysis of patients with isolated MMA has been extensively performed and a large number of allelic variants have been identified in MCM, MMAA, and MMAB genes.5,7,10-15 MCM, MMAA, MMAB proteins, the unknown cblH protein and maybe the unknown cblD variant 2 gene product are 10.1021/pr050481r CCC: $33.50

 2006 American Chemical Society

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QA of Mitochondrial Protein Expression

Figure 1. Pathway of the conversion of propionyl-CoA to succinyl-CoA and intracellular metabolism of cobalamin (OH-Cbl). PCC, propionyl-CoA carboxylase; OH-Cbl, hydroxycobalamin; Adocbl, adenosylcobalamin; MSR, methionine synthase reductase. Genes and complementation groups associated with isolated methylmalonic acidemia are: MCM (mut), MMAA (cblA), MMAB (cblB), and unknown genes (cblH and cblD variant 2). Question marks indicate unknown gene product functions (MMAA), unknown genes (cblH and cblD variant 2) or poorly defined cellular reactions (process catalyzed by mitochondrial isoform of methionine synthase reductase).

nuclear-encoded enzymes and transported into mitochondria. Mitochondria exert important functions in biological pathways, and their dysfunction is associated with a large number of pathologies that range from inborn errors of metabolism to etiologically complex diseases with a mitochondrial association.16 Over the past few years, a variety of approaches such as genetics, genomics, bioinformatics and proteomics have been applied to identify human mitochondrial molecular constituents from different source tissues.16-21 The proteomic studies of mammalian mitochondria have yielded more than 1400 proteins. In the next years, mitochondrial proteomes of heart, liver, and brain should be near completion. However, it is likely that the function of the majority of these proteins will be unknown. Despite the information already emerged from genetic studies of MMA disease, additional investigation, especially at the protein level, is essential to characterize this disorder. Clearly, the identification of (i) new mitochondrial gene products (i.e., proteins responsible for cblH and cblD variant 2 groups), and (ii) differentially expressed proteins to elucidate the secondary metabolic effects in MMA mitochondria, is important for understanding the molecular bases and pathophysiology of MMA disease. In this study, our goal was to identify the proteins indicated above in a patient with cblH/ cblD disorder using a subproteomic approach. Recent developments in quantitative proteomic techniques have allowed samples to be compared simultaneously, such as 2-D DIGE, one of the major advances in this field. This technique was originally developed by Unlu et al.,22 and was validated and optimized by Tonge et al.23 This technology is based on covalent labeling of three protein mixtures with fluorescent cyanine dyes, and the labeled proteins can be mixed and electrophoresed in the same 2-D gel. In this work, we have applied the proteomic approach 2-D DIGE to detect variations in mitochondrial protein expression in a MMA patient of cblH/cblD complementation group. We have identified significant proteomic differences between control and patient individuals. The most notable differentially expressed proteins are related to apoptosis, oxidative stress and cell metabolism, proteins that might participate in the patho-

physiology of methylmalonic acidemia. This study represents the first analysis of patients with isolated MMA at the protein level.

Materials and Methods MMA Patients. 2-D DIGE study included the one available methylmalonic acidemia patient with cblH or cblD disorder at this time (10 657). This MMA patient was referred to our lab and was diagnosed by measuring metabolites in physiological fluids, and determination of methylmalonyl-CoA mutase activity and propionate uptake in cultured fibroblasts. Complementation studies in fibroblasts measuring [14C] propionate incorporation revealed that this patient did not belong to mut, cblA, or cblB complementation groups. In addition, MMAA and MMAB genes were sequenced and no mutation was identified in this patient. On the basis of these results, 10 657 patient could belong to cblH or cblD variant 2 groups, and was designated as cblH/cblD in this study. 2-D DIGE study could not be performed using more MMA patients because a limited quantity of these cell lines was available. However, the limited quantity of these fibroblasts was sufficient to complement the protein expression analysis by Western Blot. GM9503 (control), GM1674 (cblB), GM595 (cblA), and GM306 (cblA) cell lines were provided by Coriell Institute for Medical Research, NIGMS Human Genetic Cell Repository. 17547 (cblB) and 17017 (cblC) cell lines were classified by complementation analysis by our group. Two-Dimensional Gel Electrophoresis. 2-DE method and preparation of mitochondrial extracts for 2-DE are available in the Supporting Information experimental procedures. Purification of Mitochondria and Preparation of Protein Samples for 2-D DIGE. Skin fibroblasts from control individual and cblH/cblD patient were cultured using T150 flasks according to standard procedures in MEM supplemented with 1% glutamine, 10% fetal calf serum, and antibiotics, in a humidified atmosphere containing 5% CO2. Cells from confluent T150 flasks were harvested by trypsinization. Fibroblast pellets were washed twice in PBS buffer, and then resuspended in a buffer containing 0.25 M sucrose, 10 mM Tris-HCl pH 7.4 and 2 mM EDTA. Cells were disrupted with a Teflon Potter homogenizer, Journal of Proteome Research • Vol. 5, No. 7, 2006 1603

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Table 1. Gel Setup for 2D-DIGE Experiments and Fluorophore Labeling Schemea

Gel 1 Gel 2 Gel 3 Gel 4

Cy2

Cy3

Cy5

pooled P1, C1 pooled P1, C1 pooled P2, C2 pooled P2, C2

control C1 control C1 cblH/cblD patient P2 cblH/cblD patient P2

cblH/cblD patient P1 cblH/cblD patient P1 controlC2 controlC2

a 50 µg of each mitochondrial protein extract from control (C1 and C2) and cblH/cblD patient (P1 and P2) fibroblasts were labeled with Cy3 or Cy5 dyes as indicated. 50 µg of the internal standard composed with equal amounts of different samples were labeled with Cy2.

and mitochondria were isolated according to Rabilloud T.,19 and stored at -70 °C. Mitochondrial proteins were solubilized in a lysis buffer containing 30 mM Tris-HCl pH 8.5, 7 M urea, 2 M thiourea, 4% CHAPS and 50 mM DTT. Samples were agitated for 1 h at room temperature, cleaned with PlusOne 2-DE Cleanup Kit (GE Healthcare), and resuspended in the same buffer but not containing DTT. After 2 h agitation at room temperature, samples were dialyzed against lysis buffer and protein content was determined using the RC DC Protein Assay (Bio-Rad). Two-Dimensional Difference Gel Electrophoresis (2-D DIGE). Two different extractions of purified mitochondrial proteins of human fibroblasts from control (C1 and C2) and cblH/cblD patient (P1 and P2) were labeled with Cy2, Cy3, or Cy5 following the instructions described in the Ettan DIGE User Mannual (GE Healthcare). 2-D DIGE experimental design is shown in Table 1. Briefly, 50 µg of lysate of either sample (control and cblH/cblD patient) was labeled with 400 pmol of either Cy3 or Cy5, whereas the same amount of the pooled standard containing equal amounts of the two samples was labeled with Cy2. Labeling reactions were carried out on ice and in the dark for 30 min before being quenched with 1 µL of 10 mM lysine for 10 min on ice. The three labeled samples were then combined (150 µg) and diluted with rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% IPG buffer, 10 mM DTT). The IPG strips (18 cm, pH 3-11 NL) were rehydrated overnight with rehydration buffer described above, but with 0.5% IPG buffer and DTT was replaced with 12 µL/mL of DeStreak (GE Healthcare). The labeled sample was loaded using the cup loading method on universal strip holders, and the strips were subjected to electrophoresis using an Ettan IPGphor Isoelectric Focusing system (GE Healthcare). Focusing was then carried out using the following conditions: (i) 300 V, 3 h, step; (ii) 1000 V, 3 h, gradient; (iii) 8000 V, 3 h, gradient; and (iv) 8000 V, 3 h, step. Prior to the second dimension, the IPG strips were equilibrated twice for 15 min with solutions containing 50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and traces of bromophenol blue. Dithiothreitol (1%) and iodoacetamide (4%) were added in the solutions of the first and second equilibration steps, respectively. Equilibrated IPG strips were transferred onto 12.5% homogeneous polyacrylamide gels casted in low fluorescence glass plates using an Ettan-DALT Six system (GE Healthcare). Electrophoresis was conducted for 30 min at 2 W/gel and for 3 h 30 min at 20 W/gel constant current at 15 °C. Image and Data Analysis. Gels were scanned directly between the glass plates using a Typhoon 9400 (GE Healthcare) laser scanner according to the manufacturer’s recommendations. Cy2-, Cy3-, and Cy5-labeled images of each gel were acquired using excitation/emission values of 488/520, 523/580, and 633/670 nm, respectively. DIGE images were previewed 1604

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and checked with ImageQuant software. After image acquisition, the gels were subsequently subjected to silver staining. Determination of protein spot abundance and statistics were performed automatically using DeCyder version 5.0 (GE Healthcare). Spot detection was carried out automatically using Differential In-gel Analysis (DIA) module. The estimated number of spots was set at 4000 and the excluded filter was set as follows: slope > 1.8, area < 100, and peak height < 100. For inter-gel matching and calculation of average abundance and statistics, four replicate gels (two labeled with Cy3 and two with Cy5) containing identical sample were grouped and analyzed with the biological variation analysis (BVA) module. Selection criteria for the detection of significant changed protein spots were: protein spots presented in three of the four analyzed gels (in 9 of 12 analyzed images) and the standardized average spot volume ratios exceed 1.7 with statistical significance (student’s t-test e 0.03). Differentially expressed selected proteins were excised directly from the post-stained 2-D DIGE gels. To obtain higher amount of some protein spots for MS identification, the same spot was excised from several gels and then mixed before trypsin digestion. Mass Spectrometry. Three-mm3 silver-stained spots were excised from gels manually, deposited in 96-well plates, and then processed automatically using an Investigator ProGest protein digestion station (Genomic Solutions, Cambridgeshire, UK), where samples were in-gel-reduced, alkylated with iodoacetamide, and digested with trypsin as previously described.24 For MALDI-TOF analysis of peptides, 0.3 µL of matrix solution (5 mg/mL 2,5-dihydrobenzoic acid in 33% [vol/vol] aqueous acetonitrile and 0.1% [vol/vol] trifluoroacetic acid) was added onto an AnchorChip MALDI target (Bruker Daltonics GmbH, Bremen, Germany) and allowed to dry at room temperature. A 0.3 µL-aliquot of each peptide mixture was then deposited onto these matrix surfaces and dried out at room temperature. Peptide spectra were obtained on a Reflex IV MALDI-TOF mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) equipped with a SCOUT source in positiveion reflector mode. Ion acceleration voltage was set at 23 kV. Spectra were processed using Xtof 5.1.1 software that analyzes raw XMASS data generated by FLEXControl 1.1 (Bruker Daltonics GmbH, Bremen, Germany). For peak list generation, each spectrum was processed by subtracting the baseline and then internally calibrating using trypsin autoproteolysis signals, specifically 842.510 and 2211.105 Da peptides, in the 800-2600 m/z range. Also, all known contaminant ions were excluded during the process. The parameters used to analyze the data were: a signal-to-noise threshold of 20, and the resolution higher than 4000 with a mass accuracy of 30 ppm. For protein identification, the nonredundant NCBI database was searched using Mascot 2.1 (www.matrixscience.com) through the BioTools 2.1 interface provided by Bruker Daltonics. Search parameters were set as follows: carbamidomethyl cystein as fixed modification, oxidized methionine as variable modification, peptide mass tolerance 80 ppm and 1 missed trypsin cleavage site. In all protein identifications, the probability scores were greater than the score fixed as significant with a p-value minor than 0.05. Protein spot numbers 3, 4, 5, and 6 were subjected to MS/ MS sequencing analysis using the MALDI-TOF/TOF mass spectrometer 4700 Proteomics analyzer (Applied Biosystems, Framingham, MA). Spectra were processed using 4000 series explorer v.3.0 software, the acquisition method was MS/MS1Kv in reflector positive mode with CID for fragmentation, the


collision gas was atmospheric, and the precursor mass window was (10 Da. The plate model and default calibration were optimized for the MS/MS spectra processing. The parameters used to analyze the data were: signal-to-noise of 20, and resolution >6000. For protein identification, Global Protein Server V3.5 (Applied Biosystem) was used for automatic search using a local license of MASCOT 1.9. MS/MS search parameters were set as follows: taxonomy: all entries; database: NCBI nr; enzyme: trypsin; maximum mixed cleavages: 1; precursor tolerance: 100 ppm; MS/MS fragment tolerance: 0.3 Da; fixed modification: carbamidomethyl cysteine; variable modification: oxidized methionine; minimum ion score C.I.% (peptide): 95. In all cases, the probability scores were greater than the score fixed as significant with a p value minor than 0.05. Western Blotting. Control and MMA cultured patients fibroblasts were lysed by freezing and thawing in 100 µL of 10 mM sodium phosphate pH 7, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol and protease inhibitors (Protease inhibitor cocktail tables, Roche Diagnostics). After centrifugation, protein content from supernatants was determined using the Bradford method (Bio-Rad) with BSA as standard. Mitochondria from control and MMA patients fibroblasts were isolated according to Rabilloud, T.19 The final mitochondria pellet was resuspended in the same lysis buffer described above, and assayed for protein concentration using the Bradford method. Samples were subjected to 15% (cytochrome c: cyt c) and 12% (manganese superoxide dismutase: MnSOD) SDS-PAGE gels and blotted onto nitrocellulose transfer membranes. Poinceau staining was used to exhibit equal amount of protein loaded. Western blotting was carried out using anti-cytochrome c monoclonal Ab (clone 7H8.2C12, Neomarkers) and antimanganese superoxide dismutase polyclonal Ab (Santa Cruz Biotechnology, Inc) as primary antibodies diluted 1:500 and 1:100, respectively. 1:10 000 dilutions of goat anti-mouse (cyt c) and rabbit anti-goat (MnSOD) IgG-horseradish peroxidase conjugated (Santa Cruz Biotechnology, Inc) were used as second antibodies, and were detected with the Enhanced Chemiluminescence System (GE Healthcare).

Results Identification of Mitochondrial Proteins by 2-DE/MS. In this study, two-dimensional electrophoresis was applied: (i) to determine the sample quality and composition in mitochondria preparation from human cultured fibroblasts, and (ii) to analyze the pH range in which the majority of mitochondrial proteins were present. Several protein spots detected by silver staining in 2-D gels were excised and identified by MALDITOF. Subproteomic analysis resulted in the identification of 100 different gene products in the 3-10, 6-11, and 5-6 2-D gel reference maps (Supplementary figure and supplementary table). On the basis of subcellular location, 53 proteins were annotated as mitochondrial, 17 as cytosolic, 5 as cell membrane components, 2 as nuclear and 6 as endoplasmic reticulum proteins. 17 poorly characterized, hypothetical or unknown proteins were detected. 2-DE results indicated that our preparation is enriched in mitochondrial proteins (65%), and about 83% of these proteins are enzymes or enzymatic subunits with a wide spectrum of catalytic activities. Comparative Analysis of Mitochondrial Proteome by 2-D DIGE. To detect and identify differentially expressed proteins between control and methylmalonic acidemia patients, we used the novel fluorescence technique two-dimensional difference gel electrophoresis (2-D DIGE), in which protein expression

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Figure 2. 2-D DIGE analysis of mitochondrial proteome of a patient with cblH/cblD disorder. Differentially expressed proteins are indicated on a silver restained 2-D DIGE gel using a 18 cm pH 3-11NL IPG strip for the first dimension. Overexpressed and under-expressed protein spots in cblH/cblD patient are indicated in red and green, respectively. Numbers indicate the identified proteins listed in Tables 2 and 3.

differences can be detected and identified with an increased confidence in comparison with 2-DE technique. On the basis of the 2-DE results that indicated a high number of mitochondrial proteins in the pH ranges used, we decided to use a pH range of 3-11 and 12.5% SDS-PAGE gels to detect also small molecular weight proteins in 2-D DIGE analysis. As indicated in Table 1, two different extracts enriched in mitochondrial proteins of control and cblH/cblD patient were labeled alternatively with Cy3 and Cy5, mixed and co-resolved on four different 2-D gels covering the pH range of 3-11 NL. Resulting gel images were analyzed by DeCyder differential analysis software. Comparison of control/patient relative amount of 3616 detected spots, allowed us to select 27 spots with significant variation. These protein spots exhibited differences in standardized average spot volume ratios g 1.7 and a t-test e 0.03. Among 27 spots, 9 spots were more abundantly expressed, whereas 18 were expressed at lower levels in cblH/ cblD patient fibroblasts. These differentially expressed protein spots were subjected to mass spectrometry (MALDI-TOF and MALDI-TOF/TOF), and a total of 12 spots were successfully identified, corresponding to 10 different proteins. The differentially expressed gene products are shown in Figure 2 and the proteins identified are summarized in Tables 2 and 3. Three of the identified proteins were substantially overexpressed in patient with cblH/cblD disorder: a protein spot of R-2 type VI collagen, glycerol-3P-dehydrogenase mitochondrial precursor (mGPDH) and superoxide dismutase (Mn) mitochondrial precursor (MnSOD). Among the seven under-expressed proteins, succinyl-CoA ligase (GDP-forming) β chain mitochondrial precursor, cytochrome c and KIAA0158 protein were included. Analysis using BLASTP software showed that GenBank currently Journal of Proteome Research • Vol. 5, No. 7, 2006 1605

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Table 2. Differentially Expressed Protein Spots Identified in Control and cblH/cblD Patient Fibroblasts by 2-D DIGE/MS spot no.a

accession no.b

1 2 3 4

gi|41350923 gi|1169999 P04179 P04179

5 6 7 8

P29373 P99999 gi|42734430 gi|52788292

9 10 11 12

gi|10645186 gi|8922712 gi|8922712 gi|40788885

Mr/pIc

protein name

t-test

proteins that are overexpressed in cblH/cblD patient R-2 type VI collagen 109736/5.85 glycerol-3P-dehydrogenase, mitochondrial precursor 80814/6.98 superoxide dismutase (Mn) mitochondrial 24722/8.35 superoxide dismutase (Mn) mitochondrial 24722/8.35 proteins that are under-expressed in cblH/cblD patient cellular retinoic acid binding protein 2 15561/5.43 cytochrome c 11617/9.59 polymerase I and transcript release factor 43450/5.51 succinyl-CoA ligase [GDP-forming] β-chain, 46510/6.15 mitochondrial precursor meningioma-expressed antigen 5s 76829/4.78 septin 11 49652/6.36 septin 11 49652/6.36 KIAA0158 42348/6.05

average vol ratiod

0.000 16 0.030 0.00065 0.0023

2.54 2.21 1.96 1.84

0.0020 2.4e-005 0.0015 0.0074

-3.58 -3.34 -2.56 -2.21

0.000 12 2.5e-005 0.000 11 0.000 58

-2.08 -1.72 -1.72 -1.70

a Numbers refer to spots numbers as given in Figure 2. b SWISS-PROT or NCBI accession no. c Theoretical molecular mass and pI values were determined using SWISS-PROT database. d A positive value signifies overexpression and a negative value signifies under-expression in terms of fold-differences.

Table 3. Mass Spectrometry Data of the Differentially Expressed Protein Spots Detected by 2-D DIGE spot no.a

1 2 3 4

5 6 7 8 9 10 11 12

no. masses searched

protein name

sequence coverage %

MASCOT score

precursor mass (M/Z)

MS/MS peptide sequence

individual ion scoreb

proteins that are overexpressed in cblH/cblD patient 21 12 13 109 15 8 13 81

alpha 2 type VI collagen glycerol-3P-dehydrogenase, mitochondrial precursor superoxide dismutase (Mn) mitochondrial superoxide dismutase (Mn) mitochondrial cellular retinoic acid binding protein 2 cytochrome C

PMF no. masses matched

7

5

27

164

1743.89

AIWNVINWENVTER

82

6

5

20

116

1743.89

AIWNVINWENVTER

52

VGEEFEEQTVDGRPCK

49

TGPNLHGLFGR TGQAPGFSYTDANK

73 49

proteins that are under-expressed in cblH/cblD patient 11 5 31 107 1879.85

polymerase I and transcript release factor succinyl-CoA ligase [GDP-forming] β-chain, mitochondrial precursor meningioma-expressed antigen 5s septin 11 septin 11 KIAA0158

24(MS/MS)

-

15

8

20

146

6

5

14

72

21 24 21 24

7 10 9 6

13 21 22 24

122 101 94 69

1168.71 1456.75

a Numbers refer to spots numbers as given in Figure 2. All spots were identified by MALDI-TOF, except spot number 6 that was identified by MS/MS with a MALDI-TOF TOF mass spectrometer. Spot numbers 3, 4, and 5 were further confirmed by MS/MS. bIndividual ion scores >45 indicate identity or extensive homology (p < 0.05).

associates KIAA0158 protein with septin 2. None of the identified proteins apparently could correspond to cblH or cblD variant 2 gene products. In this study, we have identified differentially expressed proteins involved in a variety of biological functions. The most notable proteins are related to apoptosis (cytochrome c), oxidative stress (MnSOD) and metabolism (mGPDH, and succinyl-CoA ligase (GDP-forming) β-chain mitochondrial precursor) (Figure 3). Differential Expression of Cytochrome c and Manganese Superoxide Dismutase Proteins in Multiple MMA Patients by Western Blot. To further validate the differences in protein expression observed using 2-D DIGE, we examined the expression levels of cytochrome c (cyt c, 11 kDa) and manganese superoxide dismutase (MnSOD, 25 kDa) in fibroblast and mitochondria extracts from cblH/cblD patient and control individual by immunoblotting. These proteins were selected because antibodies were commercially available, and they might be functionally relevant in MMA disease, as it has been 1606


observed in other disorders.25,26 Total cell and mitochondria extracts from fibroblasts derived from cblH/cblD patient and control individual were prepared as indicated in materials and methods, and equal amount of lysates were loaded. Western blot results confirmed our previous findings and indicated that cytochrome c protein level in cblH/cblD patient was clearly reduced than that found in control sample (Figure 4A). Densitometric analysis of the immunoblots revealed that cytochrome c expression in cblH/cblD patient was 2-fold lower than normal. Next, we examined the protein levels of mitochondrial enzyme manganese superoxide dismutase by immunoblotting in the patient with cblH/cblD disorder. Mitochondria extracts could not be tested because the amount of protein in these lysates was too low for the experiments. The results validated our findings by 2-D DIGE study, and showed an increased expression of MnSOD in whole cell extracts of cblH/cblD patient

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Figure 4. Western Blot analysis for expression of cytochrome c (A and B) and manganese superoxide dismutase (C) in MMA patients. (A) 1, GM9503 control cell line; 2, 10657 cblH/cblD cell line. Equal amounts of control and cblH/cblD patient extract protein were loaded: 50 µg of whole-cell lysates and 20 µg of mitochondria lysates. This result is representative of three independent experiments. (B) 1, GM9503 control cell line; 2, GM595 cblA cell line; 3, GM1674 cblB cell line; 4, GM306 cblA cell line; 5, 17547 cblB cell line; 6, 17017 cblC cell line. Equal amounts of control, cblA, cblB and cblC patient extract protein were loaded: 50 µg of whole-cell lysates and 40 µg of mitochondria lysates. (C) 1, GM9503 control cell line; 2, 10657 cblH/cblD cell line; 3, GM306 cblA cell line; 4, GM595 cblA cell line; 5, GM1674 cblB cell line; 6, 17017 cblC cell line. 200 µg of wholecell lysates of control, cblH/cblD, cblA, cblB, and cblC patients were loaded. The results obtained with cblH/cblD and cblA patients are representative of two independent experiments.

Figure 3. 2-D DIGE Image analysis of several relevant differentially expressed proteins. (A) Post-silver stained image, (B) image view, (C) 3-D view of DeCyder analysis, and (D) ImageQuant view of Cydye image. In B and C, left panel corresponds to control sample, and right panel corresponds to cblH/cblD patient. Numbers correspond to proteins indicated in Tables 2 and 3 and Figure 2: 2, Glycerol-3P-dehydrogenase, mitochondrial precursor; 3, Superoxide dismutase (Mn) mitochondrial; 6, Cytochrome c and 8, Succinyl-CoA ligase [GDP-forming] β-chain, mitochondrial precursor.

cytochrome c levels were decreased (Figure 4B), whereas MnSOD protein levels are increased compared to control sample (Figure 4C). It is worth noting that MnSOD protein level was especially increased in a patient with cblA disorder (Figure 4C). In contrast, cytochrome c and MnSOD protein levels in cblC patient and control individual are similar (Figure 4B and 4C). Taken together, these results emphasizes the high sensitivity of the DIGE technique, and show that the differences in protein expression identified in this study, are a common effect in MMA patients with defects in mitochondrial formation of Adocbl.

(Figure 4C). Expression of MnSOD in cblH/cblD patient was 2-fold higher than normal by densitometric analysis.

Discussion

To analyze if the differential expression of cytochrome c and MnSOD proteins was a general effect in MMA disorder related to defects in mitochondrial formation of Adocbl, we next examined the expression of these proteins in fibroblast and mitochondria extracts from four methylmalonic acidemia patients classified into cblA and cblB complementation groups. We also used lysates from a cblC patient with methylmalonic aciduria and homocystinuria, which has a defect in the recently identified gene MMACHC.27 MMACHC encodes a cytoplasmic protein whose function is unknown at this time. Immunoblot experiments in cblA and cblB patients, clearly indicated that

Methylmalonic acidurias are a group of inborn errors of metabolism that are biochemically characterized by an accumulation of large amounts of methylmalonic acid, and secondarily of propionic acid. Neurological symptoms and brain abnormalities are characteristic of MMA patients, and it has been suggested that these pathological changes are caused by the accumulation of the toxic organic acids.3 The increase of methylmalonic acid leads to impairment of mitochondria function, generation of reactive oxygen species and some others secondary metabolic perturbations.28,29 The mechanisms underlying the neurophathology of MMA disorder are far from Journal of Proteome Research • Vol. 5, No. 7, 2006 1607

research articles understood. To better understand the secondary metabolic effects in MMA mitochondria, we investigated changes in expression of mitochondrial proteins in a MMA patient with cblH/cblD disorder by 2-D DIGE technology. We detected 27 protein spots that displayed difference in quantity between control and cblH/cblD samples, and 10 of these proteins have been identified. A closed examination of the list of differentially expressed proteins showed that the most noteworthy proteins are related to apoptosis (cytochrome c), oxidative stress (manganese superoxide dismutase) and metabolism (succinyl-CoA ligase and mitochondrial glycerophosphate dehydrogenase). We found that cytochrome c and succinyl-CoA ligase proteins were under-expressed. In contrast, manganese superoxide dismutase and mitochondrial glycerophosphate dehydrogenase were found to be overexpressed in MMA patient with cblH/ cblD disorder. 2-D DIGE technique allowed us to identify several differentially expressed proteins in MMA patient, and the results obtained by this proteomic approach were further validated by a complementary technique, such as immunoblotting. Apoptosis is an important mechanism of maintenance of tissue homeostasis during development, characterized by membrane blebbing, nuclear breakdown, cell shrinkage, and DNA fragmentation.30,31 Abnormalities in cell death control can lead to a variety of human diseases such as cancer, neurodegenerative disease and ischemic stroke.32 Mitochondria play a central role in initiation of this multistep process and control cell apoptosis by cytochrome c release. Cytochrome c, an electron transporter of the mitochondrial respiratory chain localized in the outer membrane, is known to be released from mitochondria to the cytosol during apoptosis.33 It has been reported that cytochrome c release and ROS overproduction are closely associated and are important in the development of muscular damage in mitochondrial diseases, such as CPEO, KSS, and MELAS.25 In contrast, in maple syrup urine disease (MSUD), an inborn error of metabolism caused by a deficiency in branched chain R-keto acid dehydrogenase, increased concentrations of MSUD metabolites induced apoptosis through a cytochrome c-independent pathway.34 In vitro studies have revealed that striatal neurons exposed to methylmalonate underwent apoptotic death.35 Our study by 2-D DIGE showed that cytochrome c was under-expressed in patient with cblH/ cblD disorder. Immunoblotting analysis validated our previous findings and showed a reduced cytochrome c band in quantity in whole cell extracts and a cytochrome c protein almost undetectable in mitochondrial fractions of MMA patients. These findings permit two speculations: (a) that synthesis of cytochrome c might be reduced or that this protein might be more unstable in MMA fibroblasts than in normal individual, and (b) that apoptosis might occurr by cytochrome c release from mitochondria in MMA fibroblasts. The present study suggests that cell death may occur by apoptosis through a cytochrome c-dependent pathway in patients with methylmalonic acidemia. Another remarkable protein that we identified is mitochondrial FAD-dependent glycerophosphate dehydrogenase (mGPDH), which was overexpressed in patient with cblH/cblD disorder. mGPDH is located in the inner mitochondrial membrane, and acts in concert with the cytoplasmic NAD-linked glycerophosphate dehydrogenase to form the glycerophosphate shuttle. This shuttle interconverts glycerol-3-phosphate and dihydroxyacetone phosphate, transferring reducing equivalents into the electron transport chain, to reoxidize cytosolic NADH 1608


Richard et al.

produced by glycolysis. This enzyme is essential for operation of glycerophosphate shuttle, but on the other hand, it participates significantly in ROS production in mitochondria.36 Furthermore, it is thought that mGPDH represents a source of reactive oxygen species participating in induction of oxidative stress in placenta.37 A recent study showed high expression of mGPDH in prostate cancer cells and demonstrated that this protein is an important source of ROS in these cell lines.38 There is good evidence that methylmalonic acid generates free radicals in the MMA brain, suggesting a possible role of these radicals in the pathophysiology of the neurological dysfunction characteristic of MMA disorder.28 On the bases of the previous observations and our finding, we suggest that mGPDH overexpressed in fibroblast cells of MMA patients might act as a ROS generator in mitochondria, and together with methylmalonic acid, participates in pathologic processes connected with oxidative stress. The increased generation of ROS may enhance the cell damage already caused by the basic biochemical defect. This significant increase of free radical production may be mostly related to the neurological damage characteristic of MMA patients. In addition to these two significant proteins, we identified manganese superoxide dismutase, which is involved in oxidative stress. MnSOD is a nuclear-encoded mitochondrial enzyme that scavenges superoxide radicals in the mitochondrial matrix. We detected an increased expression of MnSOD in multiple MMA patients. Oxidative stress was observed in some inborn errors of metabolism owing to the accumulation of toxic metabolites leading to excessive free radical production.39 It has been reported that oxidative stress contributes at least in part to the severe neurological dysfunction found in phenylketonuria.40 Moreover, oxidative stress is specially associated with mitochondrial damage in mitochondrial diseases,41 and it has been described an increase of MnSOD in skeletal muscles from patients with mitochondrial encephalomyopathies.26 As indicated above, there might be a ROS generation in MMA mitochondria due to the accumulation of methylmalonic acid and the overexpression of mGPDH. In this regard, our observation has special relevance. This overproduction of reactive oxygen species in fibroblasts from patients with MMA disorder might induce the expression of MnSOD and this protein can protect against oxidative damage. Succinyl-CoA ligase [GDP-forming] β-chain, mitochondrial precursor was found in this study to be decreased in cblH/cblD patient. Succinyl-CoA ligase mitochondrial enzyme catalyzes the interconversion of succinyl-CoA to succinate, which is part of Krebs cycle. This enzyme consists of two types of subunits and there is evidence that there are two ligases: one specific for ADP and the other for GDP, and that the latter catalyzes the synthesis of succinyl-CoA during ketone body formation.42,43 The under-expression of succinyl-CoA ligase observed in this study might be caused by: (i) a reduced formation of succinylCoA because of the deficiency of MCM in MMA disease (Figure 1), and (ii) a secondarily accumulation of propionic acid in MMA patients, since it has been described that propionyl-CoA is a potent inhibitor of this protein.44 Proteomics is a powerful technique to identify new proteins and one goal of our work was to identify the genes responsible for the cblH or cblD variant 2 complementation groups. 2-D DIGE allowed us to identify some overexpressed and underexpressed proteins in patient with cblH/cblD disorder, however none of these proteins apparently are involved in cobalamin metabolism. The identified proteins were differentially ex-


pressed in more MMA patients, suggesting a general effect in MMA disease. Further genetic and proteomic studies will help us to identify the primary defect in cblH and cblD variant 2 disorders. cblH/cblD patient is alive and exhibited an infantile form of the disease. It is worth noting that a cblA patient showed more reduced expression of cytochrome c (Figure 4B, lane 4) and more increased expression of MnSOD (Figure 4C, lane 3) that than found in cblH/cblD sample. The phenotype of this cblA patient has not been described, but the majority of the described patients with cblA disorder have a B12-responsive MMA and exhibited the infantile form of the disease.1,10,45 On the basis of the results indicated above, it would be interesting to study the genotype and phenotype correlation relative to these findings described here. These results may help us to understand the cell phenotype in these two different groups of isolated MMA. In summary, we have used 2-D DIGE to analyze the changes in protein expression between a control individual and a MMA patient with cblH/cblD disorder. Proteins that are involved in apoptosis, oxidative stress and metabolism showed different expression levels in MMA patients. Cytochrome c release and ROS overproduction may be closely associated and be important in the pathophysiology of methylmalonic acidemia. As far as we are aware, this is the first study at protein level of patients with isolated methylmalonic aciduria. The present study clearly shows that the proteomic approach that we used is useful for understanding some secondary metabolic abnormalities in this disorder. 2-D DIGE analysis platform establishes a powerful experimental approach to study global protein expression, and might be applied to other metabolic diseases. Abbreviations. 2-D, two-dimensional; 2-DE, two-dimensional gel electrophoresis; 2-D DIGE, two-dimensional difference gel electrophoresis; PBS, phosphate buffered saline; BSA, bovine serum albumin; Ab, antibody; MS, mass spectrometry; ROS, reactive oxygen species; CPEO, chronic progressive external ophthalmoplegia; KSS, Kearns-Sayer syndrome; MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes.

Acknowledgment. This work was supported by grants from Ministerio de Educacioń y Ciencia (E. Richard), and from Fundacioń Ramoń Areces, and by a grant REDEMETH (G03/ 054) from Fondo de Investigacioń Sanitaria, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo (M. Ugarte). E. Richard was supported by a research contract from “Ramoń y Cajal” program, Ministerio de Educacioń y Ciencia, Spain. We thank Carmen Hernańdez for her excellent technical assistance in cell culture, and Ma Dolores Gutie´rrez (from Genomics and Proteomics Center, Complutense University of Madrid, Spain) for assistance in the identification of some proteins by MALDITOF/TOF mass spectrometry. Supporting Information Available: Two-dimensional gel electrophoresis method, list of mitochondrial proteins from human fibroblasts identified by 2-DE/MS, and 2-DE gel images of the identified proteins. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Fenton, W. A.; Gravel, R. A.; Rosenberg, L. E. In The Metabolic and Molecular Bases of Inherited Disease, 8th ed.; Scriver, C. R., Beaudet, A. L., Sly, W., Valle, D., Eds.; McGraw-Hill: New York, 2001, pp 2165-2190.

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PR050481R

Quantitative Analysis of Mitochondrial Protein Expression in

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