Oxidized Transthyretin in Amniotic Fluid as an Early Marker of

Researches, University of Udine, 33100 Udine, Italy. Received June 27, 2006. Preeclampsia is a pregnancy-specific hypertensive syndrome and a major ca...
0 downloads 0 Views 861KB Size
Oxidized Transthyretin in Amniotic Fluid as an Early Marker of Preeclampsia Carlo Vascotto,†,# Anna Maria Salzano,‡,# Chiara D’Ambrosio,‡,# Arrigo Fruscalzo,§,# Diego Marchesoni,§ Carla di Loreto,O Andrea Scaloni,‡ Gianluca Tell,† and Franco Quadrifoglio†,* Department of Biomedical Sciences and Technologies, University of Udine, 33100 Udine, Italy, Proteomic and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy, Institute of Obstetrics and Gynaecology, University of Udine, 33100 Udine, Italy, and Department of Medical and Morphological Researches, University of Udine, 33100 Udine, Italy Received June 27, 2006

Preeclampsia is a pregnancy-specific hypertensive syndrome and a major cause of maternal and fetal morbidity and mortality. At the present time, no reliable screening tests to identify women at risk are available. We have compared the amniotic fluids (AF) proteomic maps of five preeclamptic patients with those of five controls. The analysis was carried out by two-dimensional electrophoresis followed by peptide mapping and tandem mass spectrometric analysis. Besides the implementation of the previously published AF proteomic maps, our results show that transthyretin (TTR), the protein responsible for transporting both the thyroid hormone tyroxine and the retinol binding protein, is present in the AF of both preeclamptic and control women as a mixture of dimeric and post-translationally modified monomeric forms. Although the nature of these forms is similar in both groups, the preeclamptic women showed a significant increase in the amount of monomeric proteins with respect to the control group. Since the TTR monomeric forms are the results of different oxidizing reactions, we hypothesize that the higher oxidative stress in preeclampsia is the major destabilizing factor of the TTR functional dimeric form in the preeclamptic women. Keywords: amniotic fluid • preeclampsia • proteomics • transthyretin

Introduction Preeclampsia is a life threatening disorder of pregnancy affecting about 2.5-3% of the women, characterized by hypertension (>140/90 mmHg) and abnormal protein loss in urine (proteinuria >0.3 g/24 h).1 These clinical findings are only a small component of a multi-system disorder sometimes leading to maternal multi-organ damage and fetus-placental insufficiency. High risk conditions for both the woman and the fetus can develop at any time of the second half of pregnancy. Usually, the early onset of the disease (140 mm/Hg (systolic) or >90 mm/Hg (diastolic) on at least two occasions and at least 4-6 h apart after the 20th week of gestation in women known to be normotensive beforehand. Hypertension was regarded as severe in the presence of sustained raises in blood pressure to at least 160 mm/Hg (systolic), to at least 110 mm/Hg (diastolic) or both. Proteinuria was defined as the renal excretion of at least 300 mg of proteins in a 24 h urine sample. Preeclampsia was regarded as severe when severe hypertension was associated with proteinuria. Among the whole number, we selected 5 AF samples of patients who later developed a severe preeclampsia (study group) and 5 samples of women matching the study group with uncomplicated pregnancy (control group). All 10 women were of Caucasian race, and the clinical features regarding the patients were summarized in Table 1. Women with preexisting hypertension were excluded from the study group. Women with

gestational week at child birth (weeks + days)

38 + 6 40 + 2 40 + 5 39 + 2 39 + 4 30 + 0 28 + 1 32 + 5 33 + 6 36 + 3

type of delivery

Spontaneous Spontaneous Spontaneous Spontaneous Spontaneous Caesarean section Caesarean section Caesarean section Caesarean section Caesarean section

other pregestational diseases and pregnancy complications were also excluded from both groups. The delivered infants of all individuals investigated presented a normal fetal karyotype and were normal. Samples Preparation. To preserve protein components, AF samples were mixed with a protease inhibitor cocktail (Sigma, Milan, Italy) and then centrifuged at 3600g and 4 °C for 10 min to remove amniotic fluid cells. A Western blot analysis for a cellular high-abundance nonsecreted protein, namely, apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/ Ref-1),11 showed no signal, confirming that each sample was not contaminated by cellular components (data not shown). The samples were further centrifuged at 12 000g and 4 °C for 30 min to remove insoluble components, and the amniotic fluids were then aliquoted and frozen at -80 °C. To remove salts, proteins were precipitated overnight at -20 °C with 4 vol of acetone, and centrifuged at 12 000g and 4 °C for 30 min. Pellets were then washed with cold 20% (v/v) methanol and dissolved in lysis buffer containing 7.0 M urea, 2.0 M thiourea, 2% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 10 mM dithiothreitol (DTT), and 1% carrier ampholytes, pH 4-7 (Amersham-Pharmacia Bioscience, Milan, Italy). Protein quantification was performed by the Bradford colorimetric method12 and verified by SDS-PAGE stained with Coomassie Blue R350. Two-Dimensional Polyacrylamide Gel Electrophoresis. AF proteins (30 µg) were loaded onto 13 cm, pH 4-7 IPG strips (Amersham-Pharmacia Bioscience), and isolectric focusing (IEF) was conducted using an IPGPhor II system (AmershamPharmacia Bioscience) according to the manufacturer’s instructions. Focused strips were equilibrated with 6.0 M urea, 26 mM DTT, 4% (w/v) SDS, and 30% (v/v) glycerol in 0.1 M Tris-HCl (pH 6.8) for 15 min, followed by 6.0 M urea, 0.38 M iodoacetamide, 4% (w/v) SDS, and 30% glycerol in 0.1 M TrisHCl (pH 6.8) for 15 min. The equilibrated strips were applied directly to 10% SDS-polyacrylamide gels and separated at 130 V. Gels were fixed and stained with colloidal Coomassie Blue13 or ammoniacal silver.14 Evaluation of Differentially Represented Spots. Gels were scanned with an Image Master 2-D apparatus and analyzed by the Melanie 5 software (Amersham-Pharmacia Bioscience). For each sample, three 2-DE gels were performed, and then, a comparative analysis was conducted.15 Protein spots were detected and matched between the different samples; individual spot volume values were obtained according to the program instruction and normalized using the program’s volume normalization function. Ratio of the different samples’ Journal of Proteome Research • Vol. 6, No. 1, 2007 161

research articles

Vascotto et al.

gel, S-alkylated, and digested with trypsin or endoprotease AspN, as previously reported.16 In the case of extensive mass mapping experiments, alkylation of TTR isoforms was also performed without previous protein reduction. Samples were desalted using µZipTipC18 tips (Millipore, Bedford, MA) before MALDI-TOF-MS analysis and/or directly analyzed by µLCESI-IT-MS/MS. Peptide mixtures were loaded on the MALDI target together with CHCA as matrix, using the dried droplet technique.17 Samples were analyzed with a Voyager-DE PRO spectrometer (Applera, Foster City, CA). Peptide mass spectra for protein identification experiments were acquired in reflectron mode and in linear mode for extensive mass mapping experiments.18 Internal mass calibration was performed with peptides derived from protease autoproteolysis. Data were elaborated using the DataExplorer 5.1 software (Applera). Peptide mixtures were also analyzed using a LCQ Deca Xp Plus mass spectrometer (ThermoFinnigan, San Jose`, CA) equipped with an electrospray source connected to a Phoenix 40 pump (ThermoFinnigan).19 Peptide mixtures were separated on a capillary Hypersil-Keystone Aquasil C18 Kappa column (100 × 0.32 mm, 5 µm) using a linear gradient from 10% to 60% of acetonitrile in 0.1% formic acid, over 60 min, at a flow rate of 5 µL/min. Spectra were acquired in the range of 200-2000 m/z. Data were elaborated using the BioWorks 3.1 software provided by the manufacturer. Figure 1. SDS-PAGE analysis of AF samples from control and preeclamptic patients. (A) After precipitation and resuspension in rehydration buffer, 2 µL of each sample was analyzed on 12% SDS-PAGE, and proteins were detected by ammoniacal silver staining. (B) To evaluate high molecular weight protein species, the same samples were also analyzed on a 7% SDS-PAGE, and protein bands were evidenced by colloidal Coomassie staining. Direct comparison of electrophoretic profiles highlighted the presence of a slow-migrating protein component significantly upregulated in all preeclamptic samples, which was further identified as fibronectin.

normalized volume values for the candidate proteins were compared with each other, and a mean relative difference in spot intensity was calculated. The different samples’ normalized volume values (relative intensity values) for the candidate proteins were visualized as histograms representing the ratios of the intensities of matched spots from preeclamptic versus control samples. The mean and median of logarithmic ratios of the intensity values for each pair of matched spots were determined. These latter values clustered around 0, as expected, if the mean error of our analysis was normally distributed. The normal distribution was further demonstrated by the Kolmogorov-Smirnov test (performed by the Graph Pad computer software). Since the Kolmogorov-Smirnov parameter was close to 0, standard deviations of logarithmic ratios distributions were a valid parameter to assess the variability of our analysis and the significance of the quantitative differences measured. In our experiments, the average value of logarithmic ratios of SD was 0.237. Therefore, a logarithmic ratio value above 0.711 (over 3 SDs) was set as the cutoff point to evaluate protein spots differentially expressed between distinct samples. From a statistical point of view, values over 3 SDs from the mean have less than 1% probability of representing not differentially expressed protein species. Mass Spectrometry Analysis. Spots from 2-DE of preeclamptic and normal individual samples were excised from the 162

Journal of Proteome Research • Vol. 6, No. 1, 2007

ProFound software was used to identify spots from NCBI non-redundant database by PMF experiments.20 Candidates with ProFound’s Est’d Z-scores >2 were further evaluated by the comparison of the Mr and the pI experimental values obtained from 2-DE. Sequest software was used to identify proteins with data deriving from µLC-ESI-IT-MS/MS experiments.21 Candidates from NCBI non-redundant database with more than 3 identified CID spectra of peptides belonging to the same protein and Sequest Xcorr values >2.5 were further evaluated by the comparison with the experimental Mr values obtained from 2-DE. Assignment of peptide signals to specific protein modifications was achieved by GPMAW 4.23 software (Lighthouse Data, Odense, Denmark), which generated a mass database output based on protein sequence, protease specificity, and dynamic modification of cysteine residue. Immunohistochemical Analysis. Placental specimens from the 5 normal term pregnancies and from the 5 preeclamptic patients who delivered preterm were collected (see Table 1 for patient details). Immunoperoxidase staining for transthyretin was performed on 5 µm thick paraffin sections. After dewaxing, rehydratation, and endogenous peroxidase quenching with 3% (v/v) H2O2 in methanol for 15 min, sections were incubated with purified immunoglobulin fraction of rabbit antiserum to transthyretin (Dako A/S, Glostrup, Denmark), at 1:300 dilution, for 1 h at room temperature. Incubation with peroxidase-based EnVision (Dako A/S) for 30 min at room temperature and treatment with diaminobenzidine for 3 min followed. Sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. Liver tissue was used as positive control; negative controls were carried out replacing the primary antibody with nonimmune rabbit serum. Human liver cells showed cytoplasmic labeling with anti-transthyretin as primary antibody. When tonsillar tissue was immunostained, no labeling was observed (data not shown). Serial dilutions of transthyretin antiserum were done to check the optimal dilution.

Oxidized Transthyretin and Preeclampsia

research articles

Figure 2. Reference 2-DE proteome map of AF samples from control patients. 2-DE gel was visualized by ammoniacal silver staining. All newly identified protein species are highlighted in red. Black numbers correspond to proteins already identified but mapping at different positions of the 2-DE gel. Spots highlighted with the asterisk confirm the AF 2-DE map published by Liberatori et al.8 Spot identification numbers correspond to numbering in Table 2. All protein species were identified by MALDI-TOF peptide mass fingerprint experiments.

Results Proteome Profile of Amniotic Fluids. The quality of the protein samples from AF was checked using 12% SDS-PAGE and silver (Figure 1A) and Coomassie staining (not shown). The profile, showing clear, sharp bands, was practically the same for all of the analyzed samples; the large majority of protein species displayed a medium-low molecular weight. To better evaluate the distribution of protein species in the range of high molecular weight protein species, not easily detectable by 2-DE, 7% SDS-PAGE analysis was also performed (Figure 1B). Coomassie colloidal staining of this gel showed a protein species displaying an apparent molecular weight of about 260 kDa, specifically up-regulated in samples from preeclamptic patients. Peptide mass fingerprint analysis through MALDI-TOF-MS allowed the unambiguous identification of this protein as fibronectin (Swiss-Prot code P02751). This result confirmed previous observations on amniotic fluid samples from patients with severe preeclampsia.22,23 AF samples were subjected to 2-DE analysis in the range of pH 4-7. Staining of 2-DE allowed for the simultaneous quantitative evaluation of about 100 protein spots in each gel (Figure 2). After comparison with already published AF proteome maps,7-10 a total of 43 protein spots resulted as unknown species. MALDI-TOF peptide mass fingerprint analysis and non-redundant sequence database matching allowed the un-

ambiguous identification of all of the analyzed species. Table 2 reports the nature of each identified spot, the measured 2-DE coordinates, and relative sequence coverage, together with their known functional properties. Sixteen spots corresponded to 8 newly identified protein species (numbered in red in Figure 2). Twenty-six spots corresponded to 8 already known protein components of AF (numbered in black in Figure 2) but showing different pI/Mr coordinates with respect to the already published 2-DE map.8 Thirty-three additional spots (marked with the asterisk in Figure 2) were assigned to 10 different proteins previously identified.7,8 Altered Transthyretin Stability in AF Samples from Preeclamptic Patients. Comparative analysis of quantitative differences in protein spots intensities between healthy and preeclamptic samples showed a significant difference in the amount of the spots corresponding to the dimeric and monomeric forms of transthyretin (Figure 3A). TTR was present in the AF proteomic map as four differently focalized spots (indicated with a-d in Figure 3A, cropped images). Spot a, displaying an apparent molecular weight of about 35 kDa, corresponded to the dimeric form of the TTR protein. The occurrence of this oligomeric species was already described in a series of 2-DE maps from different biological fluids and is the result of strong noncovalent interactions between monomers, not affected by second dimension sampling conditions.24-26 Journal of Proteome Research • Vol. 6, No. 1, 2007 163

research articles

Vascotto et al.

Table 2. List of Identified Protein Species in AF Proteomic Maps, As Detected by 2-DE and Identified by MALDI-TOF Peptide Mass Fingerprint Analysisa number of matched/ measured peptides

protein no.

protein identity

no. of spots

1

Zinc-alpha-2-glycoprotein

3

P25311

45, 44, 43 (34)

4.7, 4.8, 4.9 (5.6)

43, 53, 27

14/20, 16/20, 6/9

2

Gelsolin

1

P06396

50 (86)

5.0 (5.9)

24

9/15

3

Sex hormone-binding globulin Pigment epitheliumderived factor Complement factor I

1

P04278

45 (44)

5.4 (6.2)

39

9/16

1

P36955

45 (46)

5.9 (6.0)

13

4/5

1

P05156

40 (66)

5.0 (7.7)

15

6/10

3

P20908

30, 30, 30 (183)

6.0, 6.3, 6.6 (4.9)

6, 6, 5

7/9, 6/9, 5/8

Inflammatory response Structural protein

3

P08123

31, 31, 30 (129)

5.4, 5.7, 5.7 (9.0)

6, 4, 5

5/10, 5/6, 7/9

Structural protein

3 5

P01236 P04217

1

P01024

45 (187)

4.7 (6.0)

8

4/6, 5/5, 5/7 11/12, 12/13, 12/13, 12/13, 10/11 17/19, 17/19, 17/19 12/14, 9/11, 9/11, 12/14, 7/8, 5/9, 14/30, 12/14, 13/14. 8/11, 5/7, 7/9, 7/11 8/9

Promotes lactation Not known

P02787 P02768

6.2, 6.4, 6.6 (6.5) 4.9, 5.0, 5.0, 5.1, 5.1 (5.6) 6.4, 6.5, 6.6 (6.8) 5.6, 5.7, 5.6, 5.6, 5.5, 5.4, 5.4, 5.4, 5.4, 5.5, 5.7, 5.8, 5.9 (5.9)

32, 37, 30 30, 34, 34, 34, 28

3 13

22, 22, 22, (26) 82, 81, 81, 80, 80 (55) 80 (77) 25, 35, 35, 33, 33, 45, 45, 42, 42, 42, 45, 45, 45 (69)

1

4 5 6

Collagen alpha 1 (V) chain, N-ter fragment Collagen alpha 2 (I) chain, C-ter fragment Prolactin Alpha-1B-glycoprotein

7 8 9

Swiss-Prot entry

Mr

sequence coverage %

pI

Stimulates lipid degradation Actin-modulating protein Androgen transport Neurotrophic

10 11

Serotransferrin Serum albumin

12

P08833

35 (28)

5.1 (5.1)

21

5/9

Inflammatory response IGF binding protein

1

P02452

35 (139)

5.1 (5.7)

7

7/10

Structural protein

15 16 17*

Complement C3, C-ter fragment Insulin-like growth factor binding protein 1 Collagen alpha 1 (I) chain, C-ter fragment Transthyretin dimer Apolipoprotein A-I Serotransferrin

1 1 4

P02766 P02647 P02787

6

P01011

19*

Alpha-1-antitrypsin

8

P01009

20*

Insulin-like growth factor binding protein 1 Collagen alpha 1 (III) chain, C-ter fragment Alpha-1-microglobulin Apolipoprotein A-I Ig kappa light chain Agrin fragment Transthyretin

5

P08833

1

P02461

40, 40, 46, 49, 47 8

10/14 8/12 21/25, 27/29, 31/34, 31/35 8/15, 9/13, 9/13, 9/13, 8/15, 8/15 16/19, 18/20, 18/21, 18/21, 18/21, 18/20, 16/19, 15/18 9/17, 9/17, 12/18, 14/24, 10/15 12/14

Transporter Lipids metabolism Transport of iron

Alpha-1-antichymotrypsin

5.5 (5.5) 5.0 (5.6) 6.4, 6.5, 6.6, 6.7 (6.8) 4.3, 4.3, 4.4, 4.4, 4.5, 4.5 (5.3) 4.8, 4.9, 4.9, 5.0, 5.0, 5.1, 5.1, 5.2 (5.4) 4.9, 5.0, 5.0, 5.1, 5.1 (5.1) 5.3 (6.1)

69 21 27, 39, 43, 43

18*

35 (16) 25 (31) 81, 81, 80, 80 (77) 67, 67, 66, 66, 65, 65 (48) 56, 56, 56, 55, 55, 55, 55, 55 (47) 35, 35, 35, 34, 33 (28) 32 (139)

2 1 2 1 3

P02760 P02647 gi7438711 O00468 P02766

33, 33 (39) 25 (31) 24, 25 (24) 19 (215) 15 (16)

5.0, 5.1 (6.0) 5.0 (5.6) 6.6, 6.6 (5.5) 5.2 (6.1) 5.0, 5.2, 5.4 (5.5)

23, 21 58 33, 22 4 75, 86, 86

8/12, 5/9 19/21 5/7, 5/9 5/8 6/7, 7/7, 9/9

13 14

21* 22* 23* 24* 25* 26*

24, 24, 24 20, 20, 21, 23, 15, 9, 26, 26, 22, 14, 12, 17, 16

function

21, 26, 26, 26, 21, 21 43, 47, 47, 47, 47, 47, 43, 41

Transport of iron Carrier

Not known Inhibitor of serine proteases IGF binding protein Structural protein Protease inhibitor Lipids metabolism Immune response Neurotrophic Transporter

a The spot number, protein description, accession number (Swiss-Prot entry), experimental (theoretical) molecular mass, and pI values, sequence coverage, and function are listed.

Conversely, spots b-d corresponded to monomeric transthyretin forms with different pI values, which have been previously reported but not structurally characterized.24,25 These species differed from the abundant glutathionylated, cysteinyl-glycinylated, cysteinylated, and S-sulfonated TTR forms focalizing at different pI values with respect to the unmodified protein, which are generated by disulfide exchange reactions of protein Cys10 with physiological thiols.27,28 In fact, their absence in 2-DE maps of AF (this manuscript) as well as of other biological fluids24,25 was the consequence of the strong reducing conditions used during sample preparation, as also verified by mass spectrometric analysis (see below). Thus, the occurrence of TTR forms corresponding to spots b-d was associated to other posttranslational modifications affecting pI value, nonabolished by reducing agent treatment. Quantitative evaluation of the normalized volume values for the monomeric and the dimeric protein forms (Figure 3B) revealed that the amount of the dimeric form of TTR dramatically decreased in association with preeclampsia in favor of the monomeric forms. Noteworthy, the total amount of TTR was not significantly different in healthy and preeclamptic 164

Journal of Proteome Research • Vol. 6, No. 1, 2007

samples, thus, suggesting that destabilization of the dimeric form of TTR was associated to the pathology. Post-Translational Modifications Occurring in Monomeric TTR Forms. To identify possible structural changes in TTR forms responsible for the observed dimeric-monomeric shift, spots a-c from healthy and severe preeclampsia-evolved pregnancy samples were submitted to extensive peptide mapping experiments using trypsin or endoprotease AspN as in situ hydrolyzing agents and MALDI-TOF-MS and µLC-ESI-ITMS/MS as analytical methodologies. The negligible amounts of protein recoverable from spot d prevented its detailed structural characterization (data not shown). Tryptic digests did not show significant spectral differences. In all cases, a combination of all measured signals in MALDITOF-MS and µLC-ESI-IT-MS/MS experiments, assigned to polypeptide species on the basis of their molecular mass, fragmentation spectra, and protease specificity, allowed covering 88% of the entire TTR sequence (data not shown). These measurements detected peptides corresponding to region 16127 in all analyzed spots, thus, suggesting that eventual

Oxidized Transthyretin and Preeclampsia

research articles

Figure 3. Differential proteomic analysis of AF samples from preeclamptic and control patients. (A) 2-DE proteome map of AF from preeclamptic and control patients. Croppings represent the spot positions of TTR forms. (B) Quantitation of monomeric and dimeric TTR species in control and preeclamptic AF samples, respectively. Quantitative determination was obtained from 2-DE proteome maps stained by colloidal Coomassie Blue. Statistical analysis was performed by Student’s t-test analysis.

structural modifications responsible for generation of the observed TTR forms were confined to the protein N-terminus. Spectral differences between spots were observed only in the case of the endoprotease AspN digests, which allowed covering the remaining polypeptide regions and confirmed the absence of glutathionylated, cysteinyl-glycinylated, cysteinylated, and S-sulfonated peptide adducts on Cys10 in all TTR spots occurring in our 2-DE maps. Figure 4 shows the MALDI-TOFMS analysis of spot a-c digests from a preeclamptic sample. Very similar spectra were obtained also in the case of other preeclamptic, as well as control, samples (data not shown); this finding suggests that the pathology affected the relative amount of the different TTR forms resolved on 2-DE maps (Figure 3) but not the nature of the associated structural modifications. In addition to mass peaks common to various TTR forms and relative to different protein regions, the spectrum of spots a and b showed a strong MH+ signal at m/z 1775.2 and 1775.1, respectively, which were assigned to peptide (1-17), namely, GPTGTGESKCPLMVKVL, containing Cys10 as carboxamidomethylated residue (Figure 4,A,B). Mass peaks associated to satellite oxidized products were also detected. Other mass signals related to peptide (1-17), but presenting a mass shift

of -103 Da, clearly occurred in the mass spectrum of spot b (Figure 4B). Traces of these components were observed also for spot a (Figure 4A). Tandem mass spectrometry analysis definitively demonstrated the nature of the molecular species at m/z 1775.2, 1775.1, 1672.2, and 1672.1 as peptide (1-17)CAM and its homologue bearing the amino acid replacement Cys10>Gly, namely, (1-17)Cys10>Gly, respectively (Figure 5). The occurrence of a TTR form not related to a genetic mutation but bearing this amino acid substitution has been already reported in physiological and stressed human plasma samples and was associated to oxidative stress conditions.28-30 Good fragmentation spectra were also obtained for the oxidized products indicated with asterisks in Figure 4, which were assigned to the respective (1-17) peptides bearing Met13 as sulfoxide species (data not shown). In contrast, the endoprotease AspN digest of spot c did not present peptide (1-17)CAM and (1-17)Cys10>Gly, but was characterized by a MALDI-TOF mass spectrum with a MH+ signal at m/z 1766.2 (Figure 4C). On the basis of measured mass and protease specificity, this molecular species was associated to a peptide (1-17) form in which the Cys10 thiol group was oxidized to sulfonic acid. In agreement with Journal of Proteome Research • Vol. 6, No. 1, 2007 165

research articles

Vascotto et al.

Figure 4. MALDI-TOF-MS analysis of the TTR form digests resulting from endoprotease AspN proteolysis. The spectra corresponding to spots a, b, and c (Figure 3A) are shown in panels A, B, and C, respectively. Differences between spectra were limited to peptide species associated to fragment (1-17). Peptide derivatives resulting from Met13 oxidation are indicated with an asterisk; their nature was confirmed by µLC-ESI-IT-MS/MS analysis (data not shown). Peptides generated from endoprotease AspN autoproteolysis are marked with X. Assignment of signals to modified (1-17) peptides was achieved as described in the Experimental Section, allowing a dynamic modification of the cysteine residue.

previous reports on the difficulty to detect peptides containing cysteine sulfonic acid during MALDI-TOF-MS analysis of 166

Journal of Proteome Research • Vol. 6, No. 1, 2007

whole protein digests,31 various experimental conditions were tested to finally reveal the presence of this modified species.

Oxidized Transthyretin and Preeclampsia

research articles

Figure 5. µLC-ESI-IT-MS/MS analysis of the TTR form digests resulting from endoprotease AspN proteolysis. The tandem mass spectra of the triply and doubly charged ions at m/z 592.1 and 888.1, associated to the peptide (1-17)CAM, namely, GPTGTGESKCPLMVKVL (MH+ at m/z 1775.2 in Figure 4A), from spot a (Figure 3A), are shown in panels A and B, respectively. Very similar spectra were observed also in the digest of spot b. The tandem mass spectrum of the triply and doubly charged ions at m/z 557.9 and 836.6, associated to the peptide (1-17)Cys10>Gly, namely, GPTGTGESKGPLMVKVL (MH+ at m/z 1672.1 in Figure 4B), from spot b (Figure 3A), are shown in panels C and D, respectively. Underlined is the amino acid modified with respect to TTR sequence. Very similar spectra were observed also in the digest of spot a. The tandem mass spectrum of the doubly charged ion at m/z 417.0, associated to the peptide (1-9) from spot c (Figure 3A), namely, GPTGTGESK, is shown in panel E. This fragment was generated by the endoprotease AspN hydrolysis at the additional acid site generated in the TTR sequence following Cys10 oxidation to cysteic acid. No similar spectra were observed in the digest of spots a and b.

Attempts to directly sequence this peptide by electrospray tandem mass spectrometry failed, similarly to what was observed during localization of modification sites in other overoxidized cysteine-containing proteins.31,32 However, this analysis demonstrated the unique occurrence of the peptide (1-9) in the spot c digest; this component was the result of a hydrolytic event catalyzed by endoprotease AspN at the additional acid site introduced in the TTR sequence following overoxidation of Cys10 (Figure 5E). This phenomenon was already observed in peroxiredoxin digests and was used to develop a dedicated methodology directed to the assignment

of overoxidized sites in proteins.32 The occurrence of a TTR form with cysteine thiol converted to sulfonic acid has been already reported being present under physiological and oxidatively stressed human plasma samples.27-29 This modification could explain the spot shift toward more acidic pI values observed in 2-DE for spot c with respect to spot b. In conclusion, mass mapping experiments allowed the characterization of the most abundant TTR spots in AF proteomic map and also the determination of the nature of the post-translational modifications on each analyzed species. Accordingly, spot a was associated to a dimeric TTR form Journal of Proteome Research • Vol. 6, No. 1, 2007 167

research articles

Vascotto et al.

Figure 6. Immunohistochemistry of transthyretin in human placenta samples from control (panels A-C) and from preeclamptic patients (panels D-F). Control placenta samples show normal chorionic villi and stromal fibrosis in chorionic membrane (Hematoxylin-eosin 100×) (panel A); some stromal and endothelial cells positive for transthyretin (Immunoperoxidase-hematoxylin 100×) (panel B) and some deposit of transthyretin located in the trophoblast (Immunoperoxidase-hematoxylin 200×) (panel C). In placenta samples from preeclamptic patients, villi are small, and stromal fibrosis is evident (Hematoxylin-eosin 40×) (panel D); fibrotic areas (panel E) and necrotic areas (panel F) are extensively stained for transthyretin (Immunoperoxidase-hematoxylin 100×, 40×).

mainly containing Cys10 as reduced species; spot b was assigned to a monomeric TTR species consisting of a mixture of forms either with Cys10 as reduced amino acid (more abundant) or bearing the Cys10>Gly substitution (less abundant); finally, spot c was associated to a TTR form where the Cys10 thiol group was oxidized to sulfonic acid. Thus, the dramatic quantitative decrease of dimeric TTR in favor of the monomeric isoforms, with increasing amounts of cysteic acidcontaining TTR, was related to a progressive oxidative modification of Cys10 to be associated to the pathology. Immunohistochemistry Analysis of TTR Distribution in Placenta from Healthy and Preeclamptic Patients. To evaluate a possible association between chemical alterations found in preeclamptic TTR and altered tissue distribution, immunohistochemical analysis was performed in healthy and preeclamptic placentas. Transthyretin immunoreactivity in placental specimens was found as a brown staining in trophoblast and stromal cells of villi and, occasionally, in endothelial cells (Figure 6). Fibrotic stromal material in villi and in membranes and infarction areas were intensely stained. In preeclamptic placentas, larger and more abundant infarction areas were observed than in placentas from uncomplicated pregnancy. Villi showed trophoblastic basement membrane thickening and fibrotic stroma. Fibrotic stromal material was also accumulated in chorionic membranes. Because of the prominence of these changes in preeclamptic placentas, a more extensive transthyretin accumulation was observed. However, all specimens were negative when probed for amyloid by Congo red staining.

Discussion This investigation allowed the identification of 8 new protein species in the proteome of AF, namely, zinc-R-2-glycoprotein, 168

Journal of Proteome Research • Vol. 6, No. 1, 2007

gelsolin, sex hormone-binding protein, pigment epitheliumderived factor, complement factor I, prolactin, and collagen R1 and R2 chains, which have been never detected before.7-10 More interestingly, our preliminary comparative analysis between 5 AF samples from preeclamptic and 5 samples from control women has shown a significant increase in the amount of monomeric TTR in preeclamptic patients. This preliminary result, which was evident in all 2-DE gels and has to be confirmed on a larger number of subjects, might open the possibility of using the ratio dimer/monomer as an early marker of preeclampsia. At the moment, no experimental methods are available for predicting the onset of this pathology. A direct extension of this work will be that of measuring, by a less complex approach such as Western blotting or ELISA, the amounts of the oxidized forms of TTR both in AF and in the sera of patients, with the aim of predicting the insurgence of preeclampsia. Along these lines, work is now in progress that aims at developing monoclonal antibodies that selectively recognize the distinct oxidized forms of TTR. TTR is a normal component of the amniotic fluid, and its main function is that of transporting both the thyroid hormone tyroxine and the retinol binding protein.33 TTR is a homotetramer (Mr 55 kDa); each monomer is composed of two fourstranded β-sheets and interacts very strongly with another monomer by extension of the β-sheets. In turn, a dimer interacts with another dimer, but these interactions are much lower than those stabilizing the dimer.34 When denatured, or even under physiological conditions, TTR dissociates and partially misfolds giving rise to aggregated morphologies including amyloid fibrils.35 Depending on the site of the aggregation, the process is associated to diseases as familial amyloid polyneuropathy (FAP), familial amyloid cardiopathy (FAC), and senile systemic amyloidosis (SSA).36-38 While the

research articles

Oxidized Transthyretin and Preeclampsia

former two diseases are generated by a series of point mutations occurring in the TTR molecule, the latter is sporadically caused by the wild-type protein. In all cases, under physiological conditions, the dissociation/misfolding/aggregation process is very slow, and its mechanism and dynamics are under investigation in several laboratories. In addition to specific point mutations, which increase the rate of the amyloidogenic process, several post-translational modifications of TTR, especially those modifying the cysteine located at position 10, interfere positively or negatively with the aggregation process.27,39,40 The nature of these modifications is largely dependent on the oxidative stress. Our experimental results show that the TTR monomer is present in the 2-DE gel from both control and preeclamptic patients as a mixture of different states with different pI values, although in different amounts. While the dimeric form is mainly constituted of normal TTR with only traces of oxidized forms, in the monomeric form, the oxidized species are more abundantly represented. One of these forms (corresponding to spot b) contains glycine in the place of cysteine at position 10. This change is not due to a genetic mutation but to a degradation of the TTR-mixed disulfide adducts formed with physiological thiols, as recently described by Nakanishi and co-workers.29 In fact, the β-elimination reaction of the disulfide bond may lead to the replacement of cysteine with glycine. Another of the oxidized forms is present in the spot c. Our MS analysis has shown that in this form cysteic acid occurs instead of cysteine. Unfortunately, MS analysis was not able to identify the nature of the changes present in spot d due to low abundance of this form in the 2-DE gels. What appears from the MS data obtained from AF of control and preeclamptic patients is that, although the nature of the chemical changes characterizing the monomeric TTR is similar, the amount of the monomeric form is definitely higher. Since the monomeric forms are stabilized by oxidative modifications of TTR,27,39,40 it could be hypothesized that the oxidative stress conditions, prevailing in the preeclampsia, may affect the stabilization of the dimeric functional forms of the protein, in favor of monomers. The causal or epiphenomenological role of this equilibrium alteration in the pathogenesis of preeclampsia is now to be inspected. A recent report showed that TTR aggregates may trigger ERK1/2 activation41 through RAGE receptors. It is well-settled that these pathways could lead to intracellular reactive oxygen species (ROS) production.42 Whether occurrence of TTR aggregates, associated to enhanced TTR immunoreactivity in preeclamptic placenta, would be demonstrated, it could be speculated that TTR oxidation could be causally involved in promoting an autosustaining prooxidant cellular condition, thus, primarily contributing to the pathogenesis of this disease. Future experiments on the glutathionylated, cysteinyl-glycinylated, cysteinylated, and S-sulfonated TTR forms in control and preeclamptic patients will investigate in depth this hypothesis, further exploring the possibility that preeclampsia may be directly related to protein redox modifications43 affecting TTR aggregation. Abbreviations: AF, amniotic fluid; TTR, transthyretin; APE1/ Ref-1, apurinic/apyrimidinic endonuclease 1/redox effector factor-1; DTT, dithiothreitol.

Acknowledgment. This work was partially supported by grants from MIUR (FIRB2001, RBAU01PRLA, RBAU01T97W, and PRIN 2005), from CNR (Agroalimentare, Commessa 404), and from EU (COST action CHEMBIORADICAL).

References (1) Redman, C. W.; Sargent, I. L. Latest advances in understanding preeclampsia. Science 2005, 308 (5728), 1592-1594. (2) Sibai, B.; Dekker: G.; Kupferminc, M., Pre-eclampsia. Lancet 2005, 365 (9461), 785-799. (3) Raijmakers, M. T. M.; Dechend, R.; Poston, L. Oxidative stress and preeclampsia: rationale for antioxidant clinical trials. Hypertension 2004, 44 (4), 374-380. (4) Chien, P. F.; Arnott, N.; Gordon, A.; Owen, P.; Khan, K. S. How useful is uterine artery Doppler flow velocimetry in the prediction of preeclampsia, intrauterine growth retardation and perinatal death? An overview. Br. J. Obstet. Gynecol. 2000, 107 (2), 196208. (5) Reis, F. M.; D’Antona, D.; Petraglia, F. Predictive value of hormone measurements in maternal and fetal complications of pregnancy. Endocr. Rev. 2002, 23 (2), 230-257. (6) Ruetschi, U.; Rosen, A.; Karlsson, G.; Zetterberg, H.; Rymo, L.; Hagberg, H.; Jacobsson, B. Proteomic analysis using protein chips to detect biomarkers in cervical and amniotic fluid in women with intra-amniotic inflammation. J. Proteome Res. 2005, 4 (6), 236-242. (7) Vuadens, F.; Benay, C.; Crettaz, D.; Gallot, D.; Sapin, V.; Schneider, P.; Bienvenut, W. V.; Lemery, D.; Quadroni, M.; Dastugue, B.; Tissot, J. D. Identification of biologic markers of the premature rupture of fetal membranes: proteomic approach. Proteomics 2003, 3 (8), 1521-1525. (8) Liberatori, S.; Bini, L.; De, Felice, C.; Magi, B.; Marzocchi, B.; Raggiaschi, R.; Frutiger, S.; Sanchez, J. C.; Wilkins, M. R.; Hughes, G.; Hochstrasser, D. F.; Bracci, R.; Pallini, V. A two-dimensional protein map of human amniotic fluid at 17 weeks’ gestation. Electrophoresis 1997, 18 (15), 2816-2822. (9) Nilsson, S.; Ramstrom, M.; Palmblad, M.; Axelsson, O.; Bergquist, J. Explorative study of the protein composition of amniotic fluid by liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. J. Proteome Res. 2004, 3 (4), 884-889. (10) Michel, P. E.; Crettaz, D.; Morier, P.; Heller, M.; Gallot, D.; Tissot, J. D.;Reymond, F.; Rossier, J. S. Proteome analysis of human plasma and amniotic fluid by off-gel isoelectric focusing followed by nano-LC-MS/MS. Electrophoresis 2006, 27 (5-6), 1169-1181. (11) Tell, G.; Damante, G.; Caldwell, D.; Kelley, M. R. The intracellular localization of APE1/Ref-1: more than a passive phenomenon? Antioxid. Redox Signaling 2005, 7 (3-4), 367-384. (12) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72 (1-2), 248-254. (13) Candiano, G.; Bruschi, M.; Musante, L.; Santucci, L.; Ghiggeri, G. M.; Carnemolla, B.; Orecchia, P.; Zardi, L.; Righetti, P. G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 2004, 25 (9), 1327-1333. (14) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68 (5), 850-858. (15) Vascotto, C.; Cesaratto, L.; D’Ambrosio, C.; Scaloni, A.; Avellini, C.; Paron, I.; Baccarani, U.; Adani, G. L.; Tiribelli, C.; Quadrifoglio, F.; Tell, G. Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation. Proteomics 2006, 6 (11), 3455-3465. (16) Risso, A.; Tell, G.; Vascotto, C.; Costessi, A.; Arena, S.; Scaloni, A.; Cosulich, M. E. Activation of human T lymphocytes under conditions similar to those that occur during exposure to microgravity: a proteomics study. Proteomics 2005, 5 (7), 18271837. (17) Paron, I.; D’Elia, A.; D’Ambrosio, C.; Scaloni, A.; D’Aurizio, F.; Prescott, A.; Damante, G.; Tell, G. A proteomic approach to identify early molecular targets of oxidative stress in human epithelial lens cells. Biochem. J. 2004, 378 (Pt 3), 929-937. (18) Cesaratto, L.; Vascotto, C.; D’Ambrosio, C.; Scaloni, A.; Baccarani, U.; Paron, I.; Damante, G.; Calligaris, S.; Quadrifoglio, F.; Tiribelli, C.; Tell, G. Overoxidation of peroxiredoxins as an immediate and sensitive marker of oxidative stress in HepG2 cells and its application to the redox effects induced by ischemia/reperfusion in human liver. Free Radical Res. 2005, 39 (3), 255-268. (19) D’Ambrosio, C.; Arena, S.; Fulcoli, G.; Scheinfeld, M. H.; Zhou, D.; D’Adamio, L.; Scaloni, A. Hyperphosphorylation of JNKinteracting protein 1, a protein associated with Alzheimer disease. Mol. Cell. Proteomics 2006, 5 (1), 97-113. (20) Zhang, W .Z.; Chait, B. T. ProFound: an expert system for protein identification using mass spectrometric peptide mapping information. Anal. Chem. 2000, 72 (11), 2482-2489.

Journal of Proteome Research • Vol. 6, No. 1, 2007 169

research articles (21) Qian, W. J.; Liu, T.; Monroe, M. E.; Strittmatter, E. F.; Jacobs, J. M.; Kangas, L. J.; Petritis, K.; Camp, D. G., II; Smith, R. D. Probability-based evaluation of peptide and protein identifications from tandem mass spectrometry and SEQUEST analysis: the human proteome. J. Proteome Res. 2005, 4 (1), 53-62. (22) Sakura, M.; Nakabayashi, M.; Takeda, Y.; Sato, K. Elevated fetal fibronectin in midtrimester amniotic fluid is involved with the onset of preeclampsia. J. Obstet. Gynaecol. Res. 1998, 24 (1), 7376. (23) Kupferminc, M. J.; Peaceman, A. M.; Wigton, T. R.; Rehnberg, K. A.; Socol, M. L. Fetal fibronectin levels are elevated in maternal plasma and amniotic fluid of patients with severe preeclampsia. Am. J. Obstet. Gynecol. 1995, 172 (2 Pt 1), 649-653. (24) Anderson, N. L.; Anderson, N. G. High resolution two-dimensional electrophoresis of human plasma proteins. Proc. Natl. Acad. Sci. U.S.A. 1977, 74 (12), 5421-5425. (25) Sanchez, J.-C.; Appel, R. D.; Golaz, O. G.;, Pasquali, C.; Ravier, F.; Bairoch, A.; Hochstrasser, D. F. Inside SWISS-2DPAGE database. Electrophoresis 1995, 16 (7), 1131-1151. (26) Altland, K.; Winter, P. Polyacrilamide gel electrophoresis followed by sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretin. Electrophoresis 2003, 24 (14), 2265-2271. (27) Altland, K.; Winter, P.; Sauerborn, M. K. Electrically neutral microheterogeneity of human plasma transthyretin (prealbumin) detected by isoelectric focusing in urea gradients. Electrophoresis 1999, 20 (7), 1349-1364. (28) Jin, Y.; Manabe, T. Direct targeting of human plasma for matrixassisted laser desorption/ionization and analysis of plasma proteins by time of flight-mass spectrometry. Electrophoresis 2005, 26 (14), 2823-2834. (29) Nakanishi, T.; Sato, T.; Sakoda, S.; Yoshioka, M.; Shimizu, A. Modification of cysteine residue in transthyretin and synthetic peptide: analyses by electrospray ionization mass spectrometry. Biochim. Biophys. Acta 2004, 1698 (1), 45-53. (30) Kishikawa, M.; Sass, J. O.; Sakura, N.; Nakanishi, T.; Shimizu, A.; Yoshioka, M. The peak height ratio of S-sulfonated transthyretin and other oxidized isoforms as a marker for molybdenum cofactor deficiency, measured by electrospray ionization mass spectrometry. Biochim. Biophys. Acta 2002, 1588 (2), 135138. (31) Kinumi, T.; Shimomae, Y.; Arakawa, R.; Tatsu, Y.; Shigeri, Y.; Yumoto, N.; Niki, E. Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ ionization and laser desorption/ionization on porous silicon mass spectrometry. J. Mass Spectrom. 2006, 41 (1), 103-112.

170

Journal of Proteome Research • Vol. 6, No. 1, 2007

Vascotto et al. (32) Wagner, E;, Luche, S.; Penna, L.; Chevallet, M.; Van Dorsselaer, A.; Leize-Wagner, E.; Rabilloud, T. A method for detection of overoxidation of cysteines: peroxiredoxins are oxidized in vivo at the active-site cysteine during oxidative stress. Biochem. J. 2002, 366 (Pt 3), 777-785. (33) Hamilton, J. A.; Benson, M. D. Transthyretin: a review from a structural perspective. Cell. Mol. Life Sci. 2001, 58 (10), 14911521. (34) Foss, T. R.; Wiseman, R. L.; Kelly, J. W. The pathway by which the tetrameric protein transthyretin dissociates. Biochemistry 2005, 44 (47), 15525-15533. (35) Liu, K.; Cho, H. S.; Lashuel, H. A.; Kelly, J. W.; Wemmer, D. E. A glimpse of a possible amyloidogenic intermediate of transthyretin. Nat. Struct. Biol. 2000, 7 (9), 754-757. (36) Westermark, P.; Sletten, K.; Johansson, B.; Cornwell, G. G. Fibril in senile systemic amyloidosis is derived from normal transthyretin. Proc. Natl. Acad. Sci. U.S.A. 1990, 87 (7), 2843-2845. (37) Connors, L. H.; Lim, A.; Prokaeva, T.; Roskens, V. A.; Costello, C. E. Tabulation of human transthyretin (TTR) variants. Amyloid 2003, 10 (3), 160-184. (38) Saraiva, M. J. Transthyretin mutations in health and disease. Hum. Mutat. 1995, 5 (3), 191-196. (39) Takaoka, Y.; Ohta, M.; Miyakawa, K.; Nakamura, O.; Suzuki, M.; Takahashi, K.; Yamamura, K.; Sakaki, Y. Cysteine 10 is a key residue in amyloidogenesis of human transthyretin Val30Met. Am. J. Pathol. 2004, 164 (1), 337-345. (40) Zhang, Q.; Kelly, J. W. Cys10 mixed disulfides make transthyretin more amyloidogenic under mildly acidic conditions. Biochemistry 2003, 42 (29), 8756-8761. (41) Monteiro, F. A.; Sousa, M. M.; Cardoso, I.; Barbas, do Amaral, J.; Guimaraes, A.; Saraiva, M. J. Activation of ERK1/2 Map kinases in familial amyloidotic polyneuropathy. J. Neurochem. 2006, 97 (1), 151-161. (42) Sato, T.; Iwaki, M.; Shimogaito, N.; Wu, X.; Yamagishi, S.; Takeuchi, M. TAGE (toxic AGEs) theory in diabetic complications. Curr. Mol. Med. 2006, 6 (3), 351-358. (43) Scaloni, A. Mass spectrometry approaches for the molecular characterization of oxidatively/nitrosatively modified proteins. In Redox Proteomics; from Protein Modifications to Cellular Dysfunction and Diseases; Dalle Donne, I., Scaloni, A., Butterfield, D. A., Eds.; Wiley: Hoboken, NJ, 2006; pp 59-99.

PR060315Z