BBRC Biochemical and Biophysical Research Communications 349 (2006) 668–673 www.elsevier.com/locate/ybbrc
Characterization of oxidation end product of plasma albumin ‘in vivo’ Luca Musante a,d, Maurizio Bruschi a,d, Giovanni Candiano a, Andrea Petretto c, Nazzareno Dimasi e, Piero Del Boccio f,g,h, Andrea Urbani f,g,h, Giovanni Rialdi d, Gian Marco Ghiggeri a,b,* a
g
Laboratory on Pathophysiology of Uremia, G. Gaslini Children Hospital, Genoa, Italy b Division of Nephrology, G. Gaslini Children Hospital, Genoa, Italy c Mass Spectrometry Core Facility, G. Gaslini Children Hospital, Genoa, Italy d RenalChild Foundation, Genoa, Italy e Laboratory of Molecular Medicine, G. Gaslini Children Hospital, Genoa, Italy f Department of Biomedical Science,Universita` degli Studi di Chieti e Pescara, Italy Centro Studi sull’Invecchiamento (Ce.S.I.), Fondazione Universita` ‘‘G. D’Annunzio,’’ Chieti, Italy h IRCCS-Fondazione Santa Lucia, Rome, Italy Received 8 August 2006 Available online 23 August 2006
Abstract Anti-oxidants are paradoxically much lower in plasma than inside cells even blood is comparably exposed to the oxidative stress. ‘In vitro’ models suggest a critical role of albumin as substitutive anti-oxidant in plasma but no proof for this role is available ‘in vivo.’ Herein, we demonstrate by LC/MS/MS that plasma albumin undergoes massive oxidation in primary nephrotic syndrome, involving stable sulphonation SO3 of the free SH of Cys 34 with +48 Da increase in exact mass of the protein (ESI-MS) and formation of a fast moving isoform in the pH range between 5 and 7. Physical–chemical experiments with DSC and fluorescence spectra indicate a thermal stabilization of the structure upon oxidation. This is the first demonstration of massive oxidation of albumin ‘in vivo’ that reflects a functional role of the protein. Free radicals should be implicated in the pathogenesis of proteinuria in human FSGS. 2006 Elsevier Inc. All rights reserved. Keywords: Free radicals; Plasma albumin; Nephrotic syndrome; Antioxidant functions
Albumin (alb) is the most abundant plasma protein in mammalians and represents the oldest marker of phylogeny from primates to humans. While evolutionary stability suggests a critical role of the protein, only minor functions, generally related to transport of ions and drugs, have been documented. Several ‘in vitro’ models of oxidation indicate that alb plays key anti-oxidant functions [1–6] challenging the historical idea that only the intracellular compartment needs strenuous defence against oxidants. Actually, it seems paradoxical that levels of anti-oxidant enzymes are much lower in blood than in the intracellular compartment since *
Corresponding author. Fax: +39 010 395214. E-mail address:
[email protected] (G.M. Ghiggeri).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.08.079
blood is comparably, if not more, exposed to the oxidative stress. Potential targets of oxidants are sulphydryl groups of plasma proteins and for alb, oxidation should involve the unique free sulphydryl group of Cys 34 with formation of a sulphenic group [5]. According to structural modelling, the sulphenic residue reacts with glutathione to form non-mercapto alb that represents the 5–10% of total alb [1–3,5,7]. Probably, the small fraction of protein modified by the sulphenic residue and/or the supposed rapid formation of disulphides with glutathione does not allow any direct detection of the oxidation product ‘in vivo.’ The proper way to define alb chemistry and functional roles is to study with adequate techniques pathologic conditions characterized by increased oxidation. In this paper, we present the first evidence for oxidation end products of
L. Musante et al. / Biochemical and Biophysical Research Communications 349 (2006) 668–673
albumin ‘in vivo’ in patients with nephrotic syndrome that confirm prediction based on ‘in vitro’ models. Methods Patients. Plasma were obtained from 5 patients with FSGS and nephrotic syndrome (4M, 3F, age between 6 and 15 years, mean 12). All had been previously screened for mutations inherited form of FSGS and all had a renal biopsy showing pathologic changes of the disease. In all cases steroid resistance had been previously demonstrated according to consolidate schemes [8]; five were receiving a treatment with ACE inhibitors. Two patients had undergone renal transplant and were receiving adequate pharmacologic treatment. Recurrence was defined as re-appearance of heavy proteinuria (>40 mg/h/kg) after a period in which urine was negative. The control group consisted in 8 normal donors of the lab staff or their sons (4F, 4M, mean age 18 years, range 10–34). Adults and parents (for patients under 18 years) were requested to give the informed consent for DNA analysis (that is a part of the clinical diagnosis of FSGS) and for reviewing their clinical parameters on statistical basis. Purification of alb in non-denaturing conditions. Alb was purified utilizing polyacrylamide electrophoresis (T 4–12%) in native conditions with 2 mm gel spicers. All purification steps were performed in a native condition to prevent structural modifications according to Margolis and Kenrick [9]. One milliliter of serum was applied to gel and electrophoresis was run in Tris–borate–EDTA (80/90/2.5 mM) for 12 h with 16 mA at 12 C. Albumin was desorbed from acrylamide by gentle pest and was maintained in PBS at 4 C for 24 h with two changes of the solution and further purified by gel-filtration at 4 C using a Superdex 75 HR 10/30 column (Amersham–Pharmacia Biotech) in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and at a flow rate of 0.5 ml/min. LC/MS/MS. All mass spectrometric measurements were performed using a LTQ linear trap mass spectrometer (Thermo Electron, San Jose, USA) coupled to a HPLC Surveyor (Thermo Electron) and equipped with a Jupiter C18 column 250 · 1 mm (Phenomenex). Peptides were eluted from the column using an acetonitrile gradient, 5% B for 6 min followed by 5–90% B within 109 min ( eluent A: 0.1% formic acid in water; eluent B: 0.1% formic acid in acetonitrile) at flow-rate of 50 ll/min. The column effluent was directed into the electrospray source. The spray voltage was 5.0 kV. The capillary of ion trap was kept at 200 C and the voltage at 2.85 V. Spectra were acquired in automated MS/MS mode: each full MS scan (in the range 400–1800 m/z) was followed by five MS/MS of the most abundant ions, mass that had been analysed for more than two times this way was automatically taken up into an exclusion list for 30 s. Computer analysis of peptide MS/MS spectra was performed using Bioworks software, version 3.2, from Thermo Electron (San Jose, USA) and searched against a HSA protein database. Peptide MS/MS assignments were filtered according to the following criteria: Xcorr P 1.5 for the singly charged ions, Xcorr P 2.0 for doubly charged ions, and Xcorr P 2.5 for triply charged ions, peptide probability 60.001, DCn P 0.1 and percent ions P 30%, for all protein searches two missed cleavages was allowed. ESI-MS protein analysis. Alb containing solutions were manually injected (5 ll) into an on-line flow, using a CapLC system (Micromass, Waters) coupled with a nano-ESI-Q-TOF instrument (Micromass, Waters). The sample was eluted at 1 ll/min on a C4 pre-column LCPackings, 300 lm inner diameter · 20 mm. Elution was achieved isocrytically by H2O/ACN 50/50 both with 0.1% TFA and directed into a mass spectrometer equipped with a nano-Lock-Spray source. A 2500 and 50 V tension was applied on the PicoTip capillary (New objective, PicoTip Emitter, Tip: 10 ± 1 lm) and cone voltage, respectively, and the positive ion mode for ion scan experiment was used monitoring the 700–2200 m/z range. Data analysis was performed using Masslynx version 4.0 (Micromass/Waters). The data collected were examined for multiply charged protein spectra, which were then integrated to provide a single combined spectrum for the protein injected. A maximum entropy deconvolution algorithm (MaxEnt1) was used to deconvolute multiply charged spectra and produce molecular mass spectra.
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PEO2-maleimide assay. Free SH group reactivity of alb was determined with the maleimide–PEO2–biotin (biotinyl-3-maleimidopropionamidyl-3,6-dioxactanediamine) assay (Pierce, Rockford, IL) according to the manufacturer’s instructions. After reaction with the complex maleimide–PEO2 proteins were precipitated with cold acetone for 20 min at 20 C and centrifugation at 10,000g for 20 min. Proteins were then resuspended in PBS and a part was reduced by adding 30 mM DTE in Laemmli sample buffer. Separation was done with mono-dimensional in polyacrylamide gradient (%T = 8–16; %C = 2.6) [10]. After electrophoresis, proteins were transferred to nitrocellulose and detected with neutravidin conjugated with horseradish peroxidase. Biotin incorporation was determined with [2-(4 0 -hydroxyazobenzene)-benzoic acid] utilizing the EZ Biotin Quantitation kit (Pierce) at 500 nm with correction for the amount of albumin as determined by Coomassie R-250. Differential scanning calorimetric. DSC measurements were performed with a MicroCalorimeter VP-DSC (MicroCal, Northampton, MA, USA) at 60 C/h with a concentration of 0.0081 mM. Before measurements, samples were degassed and immediately loaded into the calorimeter cell. Instrumental baselines were determined prior to scanning with 50 mM phosphate buffer at pH 7.4 that was the reference buffer utilized in the experiment. Reversibility of the thermal transition was verified by checking the reproducibility of the calorimetric trace in a second heating immediately after cooling. Fluorescence spectra. Fluorescence and ultraviolet spectra were recorded and evaluated in a LS50B Perkin-Elmer Luminescence Spectrometer (Wellesley, MA, USA). Excitation at 295 nm was utilized to minimize the contribution of tyrosine in intrinsic fluorescence. Excitation and emission slit widths were maintained at 4.5 nm and scan speed was 300 nm/min. The temperature of sample cell was maintained constant by circulating water bath (Haake, Karlsruhe-Berlin, Germany) and monitored with a teflon-coated microthermistor probe. Spectra were analysed after subtraction of baselines. The position of middle of a chord draw at the 80% level of the maximum intensity (kmax) was taken as the position of the spectrum [11]. Ultraviolet spectra. Both normal and second derivative spectra were obtained with a UV–vis Cary BIO400 (Varian, Palo Alto, CA, USA). The temperature in the sample cell was maintained constant by circulating water bath and monitored with a teflon-coated microthermistor probe as above. Dielectric microenvironment surrounding Tyr residues was calculated from the ratio (r) between differences in second derivative absorbance peak (a/b) relative at 284 and 279 nm for Tyr and 293 and 286 nm for Trp according to Ragone et al. [12] r¼
a A00 ð284 nmÞ A00 ð279 nmÞ ¼ ; b A00 ð293 nmÞ A00 ð286 nmÞ
where A00 is the second derivative absorbance at any given wavelength k. The degree of Tyr exposure, a, is obtained from the equation r n ra a¼ ; r u ra where rn and ru are the numerical values of the ratio a/b determined for native and unfolded conformers and ra corresponds to the ratio between Tyr and Trp in a model compound in presence of ethylene glycol and represents a complete burial of all aromatic residues of protein. Since human serum albumin contains 18 Try and 1 Trp residues the second derivative peak trough ratios ra and ru were obtained for the molar ratio (18/1) with the second derivative molar extinction coefficients of Ragone et al. [12]. Thus for alb, the calculated values were ra = 2.74 and ru = 17.82 for the complete burial and exposure of Try residues, respectively. Circular dichroims. The CD measurements of healthy and FSGS albumin were performed with J-810 spectropolarimeter (Jasco, Great Dunmow, Essex, UK) instruments in the range of 190–260 nm at 20 C, using a 1 mm cell at 1 nm intervals. The concentration of both macromolecule was 0.1 mg/ml in 50 mM phosphate buffer, pH 7.2. All spectra were background corrected and analysed using CDNN software. Electrophoretic titration curves with carrier ampholytes. Determination of alb charge along a stable pH gradient was done according to the procedure previously described [13,14].
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Two-dimensional electrophoresis. The technique for 2D-ele in soft gels was described in [15].
Results and discussion Using complementary biochemistry methodologies, we characterized plasma alb in five patients affected by idio-
pathic nephrotic syndrome for FSGS, two were presenting recurrence of FSGS after renal transplant. In a first approach, alb was characterized by ESI-MS that showed a modest (+48 Da), albeit constant, increment of exact mass of the protein (66.555 vs. 66.507 kDa) in patients vs. healthy people that suggested the addition of three oxygen radicals (Fig. 1a). Using LC/MS/MS it was first
Fig. 1. Mass spectrometry analyses of plasma albumin in FSGS. Exact mass (a) of alb was determined by ESI-MS protein analysis using a CapLC system (Micromass, Waters) coupled with a nano-ESI-Q-TOF instrument (Micromass, Waters). Data analysis was performed using Masslynx version 4.0 (Micromass/Waters). The data collected were examined for multiply charged protein spectra, which were then integrated to provide a single combined spectrum for the protein injected. A maximum entropy deconvolution algorithm (MaxEnt1) was used to deconvolute multiply charged spectra and produce molecular mass spectra. LC-ESI-MS/MS spectrum of the Cys 34 tryptic fragment after reductive alkylation of alb from a FSGS patient (b) and from normal control (c). Both samples were treated with dithiothreitol and iodoacetic acid. Only the case of alb purified from the patient shows, after digestion with trypsin, the parent fragments m/z 827.96 (left panel, top) and 1241.22 (left panel, bottom) in triply and double charge state, respectively. MS/MS spectrum showed the presence of 511.71 in triply charge and of 1610.5 in double charge indicates the oxidation of the SH of Cys 34 to Cysteic Acid (+48 Da). Control albumin shows two different precursor ions, m/z 831.21 (right panel, top) and 1245.67 (right panel, bottom), in triply and double charge state. MS/MS spectrum showed the presence of 516.5 in triply charge and of 1619.6 in double charge, that indicate alkylation (+57 Da) of the free SH of Cys 34.
L. Musante et al. / Biochemical and Biophysical Research Communications 349 (2006) 668–673
targeted potential sites of oxidation such as the free SH at Cys 34, Met 147, Met 353, and Met 572. Analysis by MS/ MS (Fig. 1b and c) of two trypsin digestion fragments with m/z 827.96 and 1241.22 (in triply and double charge state respectively) demonstrated the presence of m/z 511.71 (triply charge) and of 1610.5 (double charge) fragments indicating the oxidation of the SH of Cys 34 to cysteic acid. Control alb showed instead two precursor ions with m/z 831.21 and 1245.67 that presented at MS/MS higher (+57 Da) m/z compared to free SH (i.e. 516.5 in triply charge and of 1619.6 in double charge, respectively) indicating alkylation of Cys 34. Based on the findings above, a technique was devised for direct ‘in gel’ titration of free SH 34 that is based on its alkylation by maleimide–PEO2 and detection with biotin–streptavidin. Results from ‘in gel’ titration (Fig. 2a) confirmed the absence of free reactive SH groups in alb from nephrotic patients. A second approach to oxidized alb was based on the prediction that
Fig. 2. Titration of free SH groups (a) and electrical charge (b) evaluation. (a) Titration of free SH was done by an ‘in gel’ technique that utilizes PEO2–maleimide–biotin (top panel) as a specific label. According to this method, alb is first separated by mono-dimensional electrophoresis in nondenaturing conditions and it is then stained directly in gel with PEO2– maleimide–biotin, the reaction is developed with streptavidin. Five normal samples and 5 FSGS patients were analysed; alb oxidized ‘in vitro’ was shown for comparison. Alb from healthy showed PEO2–maleimide staining corresponding to alb (see arrows) whereas alb of FSGS patients had no staining indicating absence of a free SH that is unique in alb at position 34 of the sequence (Cys 34). (b) Electrophoretic titration curves. Overlap of electrophoretic titration curve of a serum sample from a patient with FSGS and for comparison of normal serum stained with Coomassie R-250. The two proteins migrate as single and homogeneous band throughout the pH range: between 4 and 4.5 and between 7 and 9 they overlap whereas in the pH range between 4.5 and 7 oxidized alb migrates with a more acid charge. According to the Lindestrom–Lang theory, this acid charge shift fits with the introduction of a sulphonic acid group in the molecule [24]. Determination of alb charge along a stable pH gradient was done according to the procedure described by Bruschi et al. [14].
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a cysteic residue in place of SH in position 34 should make the surface charge more acidic in the pH range of ionization of this chemical group. The electrical charge of oxidized vs. normal alb was then evaluated by a two-dimensional electrophoretic technique, known as electrophoretic titration curve, that allows to determine the charge of any given protein along a pH gradient between 4 and 9. As shown in Fig. 2b, it was shown a global shift in electrical charge of oxidized alb in the pH 5–7 interval that involves all the protein and fits with introduction of cysteic acid in the molecule. Interestingly, alb domain IA containing Cys 34 does not participate in the alb major transport capacity probably because it plays a structural role and likely has escaped the selection pressure to specialize as a transport protein. ‘In vitro’ models of oxidation based on sulphenic transformation of thiol groups predict formation of new hydrogen bonds with adjacent aminoacids [16]. This observation suggests a possible protein stabilization with formation a thermodynamically favour structure. Physical–chemical experiments with DSC (Fig. 3a), intrinsic Trp fluorescence (Fig. 3b), and second derivative absorption spectra (Fig. 3c) confirmed a thermal stabilization of the structure of upon oxidation. In fact, the Tm transition is shifted at higher value of temperature of 16.57 C; the hentalpic change contribution calculated from van’t Hoff procedure with optical procedure as well by direct DSC experiment correspond to DH 510.8 kJ/mol for healthy and DH 607.1 kJ/mol for FSGS. These evidences are corroborated by circular dichroism data (Table 1) and by small angle X-ray scattering (data not shown) suggesting slight changes in protein secondary structure possibly involving a-helix reorganization and subtle changes in alb domain orientation. The observation that massive oxidation of plasma alb does occur ‘in vivo’ is new and suggests two considerations. The first is about physiologic functions of alb that can be now extended to an anti-oxidation effect so far only hypothesised on the basis of ‘in vitro’ experiments. In normal conditions oxidation of alb should involve only a minor part of the protein due to limited stress. It is likely that the quota of non-mercaptoalbumin represents the end product of the reaction that involves the formation of a reactive sulphenic intermediate of the free SH 34 that reacts with free glutathione and/or with homocysteine. Extension of structural and conformational analysis of alb in pathologic conditions was critical to obtain a clear demonstration of a stable thermodynamic end product of oxidized alb. Therefore, alb may be considered the major anti-oxidant substance in plasma where, due to the high plasma levels (0.8 mM), overwhelms by an exponential factor other antioxidants such as free glutathione whose mean plasma levels are only 0.006 mM. The second point implies that in nephrotic syndrome oxidation is markedly activated and probably plays a key role in pathogenesis. This concept is supported by preliminary and indirect evidence on peroxidation of plasma
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Fig. 3. Thermal unfolding analysis. (a) Differential scanning calorimetry. Temperature dependence of the excess molar heat capacity (solid line) of healthy (top panel) and FSGS (bottom panel) in 50 mM phosphate buffer a pH 7.5. Graphics were drawn after subtraction of experimental traces for the instrument baseline and the chemical baseline. Dotted lines are the result of the experimental data on one domain with the theory of two state transitions, approximation [25]. Protein concentration was 0.0081 mM. The scan rate was 60 C/h. The analysis of the curves has been performed following the procedures reported in Origin software. The thermodynamic parameters obtained were DH = 510.8 kJ/mol and Tm = 65.61 C for healthy and DH = 607.1 kJ/mol and Tm = 65.61 C for FSGS. (b) Thermal melting profile of albumin of healthy (solid squares) and FSGS (open circles) monitored by measuring the fraction change in kmax. Dotted lines are the result of fitting of experimental data according to van’t Hoff equation [26]. Protein concentration was 0.0022 mM (c) Thermal melting profile of albumin of healthy (solid squares) and FSGS (open circles) monitored by measuring the fraction change in second derivative absorption spectra. Dotted lines are the result of fitting of experimental data according to van’t Hoff equation [26]. Protein concentration was 0.0053 mM.
Table 1 Circular dichroism of albumin purified by healthy controls and patients with FSGS k nm
Healthy 190–260 (%)
a-Helix Antiparallel Parallel b-Turn Random coil Total
FSGS 195–260 (%)
200–260 (%)
205–260 (%)
210–260 (%)
190–260 (%)
195–260 (%)
200–260 (%)
205–260 (%)
210–260 (%)
88.9 0.0 2.2 6.8 2.4
91.8 0.0 2.3 5.9 2.7
92.0 0.0 2.6 6.1 2.2
93.0 0.0 3.1 5.7 1.8
92.1 0.0 3.4 4.9 2.3
81.5 0.0 3.0 7.9 5.3
84.5 0.0 2.8 7.3 5.2
85.8 0.1 3.1 7.4 4.1
87.3 0.1 3.5 7.1 3.3
87.3 0.0 3.9 6.1 4.4
100.3
102.7
102.9
103.6
102.6
97.8
99.9
100.4
101.2
101.7
Spectra were recorded with J-810 spectropolarimeter (Jasco, Great Dunmow, Essex, UK) instruments in the range of 190–260 nm at 20 C, using a 1 mm cell at 1 nm intervals. The concentration of both macromolecule was 0.1 mg/ml in 50 mM phosphate buffer, pH 7.2. All spectra were background corrected and analysed using CDNN software.
membranes and consumption of intra-erythrocyte GSH in humans with FSGS [17–19] and on animal models of the disease that include puromycin (PAN) and adriamycin (ADR) nephrosis in rats and Mvp 17/ mice [18,20–23]. In conclusion, the stable end product of oxidation of plasma alb was for the first time demonstrated ‘in vivo.’ This suggests a key role of plasma alb as an anti-oxidant. Description of the basic chemistry of oxidized alb can also contribute to the analysis of oxidation in other diseases.
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