Free Thiol of Transthyretin in Human Plasma Most Accessible to

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The Free Thiol of Transthyretin in Human Plasma is Most Accessible to Modification/Oxidation Caroline Donzeli Pereira, Naoto Minamino, and Toshifumi Takao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03431 • Publication Date (Web): 06 Oct 2015 Downloaded from http://pubs.acs.org on October 6, 2015

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Analytical Chemistry

The Free Thiol of Transthyretin in Human Plasma is Most Accessible to Modification/Oxidation

Caroline Donzeli Pereira∆, Naoto Minamino†, and Toshifumi Takao∆* ∆

Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

†

National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita,

Osaka 565-8565, Japan.

ABSTRACT Free-thiol(s) in proteins, especially, when located on the surface of the molecule, are susceptible to oxidation/modification, which may cause loss of function or an alteration in the ternary structure. This suggests that the status of thiol group(s) of cysteine residue(s) in a protein, i.e. free-thiol versus an oxidized/modified form, in vivo, could reflect the physiological state of the molecule with respect to susceptibility to oxidative stress. To address this issue, we established an efficient method for isolating proteins that contain free thiol groups from a complex mixture, which permits the amount of free-thiol form(s) to modified/oxidized forms to be estimated. Albumin, which accounts for 55% of the total plasma proteins and has such a free thiol and has been reported to scavenge various reactive oxygen species (ROS) in vivo. The developed method was used to isolate the free form of albumin from fresh plasma. However, contrary to our expectations, transthyretin (TTR), which also has a single free thiol, was found to be the major protein that was the most susceptible to modification/oxidation. In addition, the free-thiol form could be separated from oxidized or modified molecules, permitting the relative abundance of the free-thiol form to be estimated. The findings show that the levels of the free-thiol form of TTR in plasma was significantly lowered after a hydrogen peroxide treatment, even at low concentrations (0.1 mM), suggesting that TTR could be a useful biomarker for monitoring a ROS imbalance in relation to various oxidative stress conditions. Key words: transthyretin, oxidation, modification, free-thiol, human plasma

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INTRODUCTION In an organism, the global thiol-disulfide redox status is defined as the total distribution of thiols and disulfides in diverse cellular pools. The activity and stability of many enzymes, chaperones and transcription factors are dependent on the maintenance of this status inside the various cellular compartments.1 Prokaryotes and eukaryotes have a thiol-mediated reversible switching mechanism that controls many cellular processes.2 In eukaryotes, 2-3% of the total proteins contain free-thiol groups and these residues are involved in substrate binding or catalytic processes.3 Moreover, these residues may be subject to posttranslational modification, acting as mediators of redox signaling and regulation due to the high reactivity of the thiol group;4 these groups are mostly observed in proteins, not in peptides sequences,1 and are amenable to various types of modifications, which raises the question as to whether the free and modified forms of a protein might have functions. It is, thus, important to have information on the types of and degree of modification of such molecules in relation to physiological or pathological events. There have been many reports on the modifications of Cys residues that are involved the functions of a protein. However, methods for specifying the relative ratio of a free-thiol to a modified or oxidized form in a sample derived from a living organism are few in number. Such information is critical in terms of evaluating the physiological state of a protein with respect to susceptibility to oxidative stress. In order to determine the fraction of a free-thiol-containing protein versus its modified forms in a biological sample, we established a method that permits the specific isolation of thiol-containing compounds from a complex mixture, and applied the methodology to human plasma. About 95% of the mass of proteins in human plasma are made up of only 22 proteins,5 among which the most abundant protein, albumin (3.5 g/mL), contains a single free-Cys, and would be expected to be isolated as a major free thiolcontaining protein by the present method. In fact, modifications at Cys34 of albumin have been extensively studied in relation to several physiological states rationalized in terms of oxidative stress.6,7 However, the findings of our study, unexpectedly, showed that transthyretin (TTR) was the major trappable protein containing a free-thiol group using the current method. Transthyretin (TTR), formerly called prealbumin, is a protein that is produced by the liver and secreted into the plasma and is also found in cerebrospinal fluid. It was sequenced for the first time by Kanda and coworkers in 1974.8 The levels of TTR in adult human plasma are 20‒40 mg/dL, but the expression rate decreases over the age of fifty. TTR can bind to thyroid hormones and retinol binding proteins. It is composed of 127 amino acid residues, which are organized in a β-sheet


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structure with a small α-helix, formed from seven amino acids. The overall protein has a homotetrameric structure with a molecular mass of 55 kDa, and each monomer is associated with another monomer, forming an αβ dimer which interact with each other via strong hydrogen bonding in the beta-sheet region.9,10 TTR has a short half-life ‒ around two days ‒ in the blood stream and is excreted by the kidneys after being metabolized.11 The level of TTR in the serum is correlated with nutritional status and health conditions: e.g. the level of transient TTR decreases during an inflammatory process and postoperative states; conversely, alcohol intoxication, prednisone therapy, and metabolites produced during pregnancy, increase the level of this protein.12 Studies conducted by Gaudiani and coworkers12 reported that TTR can be used as a biomarker in cases of severe illness and in malnourished patients. The TTR sequence contains a single cysteine residue per monomer (Cys10). The Cys10 can form a disulfide bond with various reactive thiol molecules, forming adducts such as sulfonate, cysteine, homocysteine, etc., while 5‒20% of the Cys10 is known to exist in the reduced form. Several studies have reported that this residue plays an important role in maintaining the stability of the protein. In amyloidosis patients, the TTR tetramer is composed of a mixture of Cys10-modified species, and the ratio of TTR with modified Cys to that with a free Cys has been reported to be high in patients with familial amyloidotic polyneuropathy (FAP),13,14 which could be rationalized by destabilization of the TTR structure caused by Cys modification, followed by the abnormal assembly of a monomer into fibrils. TTR is also known to be the most abundant protein in fibrils in elderly individuals who are suffering from systemic amyloidosis (SSA),10 which affects ~25% of the population over the age of eighty.15

According to Giustarini and coworkers16 the thiol

composition in proteins decreases with age; in the case of TTR, the decrease in the free-Cys form may contribute to SSA development as in the case of FAP.14 In addition, some other diseases such as hyperhomocysteinemia or homocystinuria have been reported to be associated with an increase in the extent of modification of Cys10 of this molecule. Lim and colleagues17 reported that for patients suffering from end-stage renal disease, the level of TTR modified with homocysteine at Cys10 increased, suggesting that the modified TTR could be used as a surrogate marker for monitoring the pathophysiological conditions of these diseases. The method, utilized in the present study, is based on the capture of proteins containing free Cys residue(s) by a resin modified with a hetero-bifunctional cross-linker, 6-[3’-(2-pyridyldithio)propionamido] hexanoate, followed by recovery of the proteins from the resin by treatment with a reducing agent. It allows for the rapid separation of free thiol-containing proteins, with TTR being



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the major component in the case of plasma, from other components including Cysmodified/oxidized proteins, thus enabling the relative abundance of the free-thiol form versus modified/oxidized forms, which could correlate with the physiological states in relation to various oxidative stress conditions, to be estimated.

EXPERIMENTAL SECTION Materials

A sample frozen fresh plasma from healthy volunteers was purchased from the Tennessee Blood Service Corporation (Memphis, TN); each sample was obtained from a single donor (Table S1, Supporting Information). 6-[3’-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP), the resin for perfusion chromatography, Poros 20 NH Amine were purchased from Applied Biosystems (Foster City, CA). Other materials are described in Supporting Information.

Preparation of Pyridyldithio-coupled Resin

A sample of Poros 20 NH resin (100 mg) was washed with 100% ACN, 50% aqueous ACN, water, and then equilibrated with ethanol (EtOH). The process was repeated twice with each solvent. LC-SPDP (20 mmol) was then dissolved in ethanol (EtOH), mixed with the resin, and the resulting preparation was then incubated overnight at room temperature. The excess LC-SPDP was removed by washing the resin twice with 100% EtOH, 70%, and 50% aqueous EtOH. The linkage yield of LC-SPDP to the resin was estimated from the amount of pyridine 2-thoine, which has a molar extinction coefficient (8.08 × 103 M-1 cm-1 at 343 nm), liberated from the resin upon reduction (Figure S1, Supporting Information). Prior to incubation with a sample, the LC-SPDP-coupled resin was washed three times with the working solution (50 mM NH4CO3H pH 8, 25% EtOH, 100 mM NaCl, 1mM EDTA).


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Sample preparation

Samples obtained from healthy donors were treated according to the standard operating procedures provided by the Tennessee Blood Service Corporation; the anticoagulant added to the sample was EDTA (Na2) and the plasma was stored at -80 °C until used. Fifteen microliters of each plasma were mixed with the working solution, and the resulting solution was directly applied to the Poros 20 NH LC-SPDP resin (50 mg).

Isolation of free thiol-containing proteins using LC-SPDP resin

The samples added to the Poros 20 NH LC-SPDP resin were incubated for 12 h at 4 °C under a nitrogen atmosphere. After spinning down the resin (2000 g for 15 s), the supernatant was retained as the flow-through (F-) fraction. The resulting resin was exhaustively washed with the working solution, and then incubated in the same solution containing 50 mM DTT for 1 h at room temperature to give the eluted (E-) fraction. Both fractions were desalted using an ultra-0.5 centrifugal filter (cutoff 10 kDa).

Matrix-assisted laser desorption ionization mass spectrometry

Mass spectra were acquired on a 4800 MALDI-TOFMS instrument (AB Sciex, Framingham, MA). Sinapinic acid at a concentration of 10 mg mL-1 in 30% ACN/0.1% TFA was used as the matrix. The sample solution (5 µL) was desalted using ZipTip µC4 (Millipore, Billerica, MA), spotted onto the MALDI plate, mixed with the matrix (0.5 µL), and allowed to dry at room temperature. The instrument was operated in the positive linear mode, and mass spectra were acquired over the entire sample surface using an average of 3000 laser shots. Mass calibration was accomplished using the [M +H]+ ions of insulin (m/z 5733.4), ubiquitin (m/z 8565.9), cytochrome C (m/z 12361.2), myoglobin (m/z 16952.6), carbonic anhydrase II (m/z 29025.8), and bovine serum albumin (BSA ‒ m/z 66434.5).



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Western blotting

The protein bands in a SDS-PAGE gel were transferred to a nitrocellulose membrane using a transfer buffer containing 0.1% SDS, 380 mM glycine, 20% methanol and 50 mM Tris. The process was carried out at a constant current of 150 mA and 20 V for 120 min. The membrane was then washed with washing buffer (Tween/TBS-20 (Tween-20 (0.4%) in 50 mM Tris-HCl, 150 mM NaCl pH 7.4), and, to avoid non-specific binding, the membrane was incubated with a Tween/TBS solution containing 1% BSA at room temperature with agitation overnight. After this blocking step, the nitrocellulose membrane was washed twice for 5 min using TBS/Tween-20, and the primary antibody against TTR (rabbit monoclonal anti-prealbumin – 1:5000 dilution) was then applied to the membrane, which was incubated for a minimum of 1 h, and washed three times with TBS/Tween-20 to remove excess primary antibody. The secondary antibody, a rabbit anti-IgG (1:2000 dilution), was added to the membrane, incubated for at least 1 h at room temperature, and washed three times with TBS/Tween-20. The proteins that were specifically recognized by the primary antibody were visualized using enhanced chemiluminescence reagents in a dark chamber. The blot images were processed in TIFF format. The images were analyzed using the software ImageJ (National Institute of Health, Maryland), which allowed for calculation of the areas, pixel values, statistics, and intensities of protein bands.

Modification of free thiols by N-ethylmaleimide (NEM)

The free thiols in the plasma sample, were alkylated using N-ethylmaleimide (NEM) prior to separation with the Poros 20 NH LC-SPDP resin. Fifteen microliters of plasma were diluted in phosphate buffered saline (PBS – 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4, pH 7.4) containing 50 mM NEM and incubated at 37 °C for 2 h. The reaction was quenched by adding 25 mM cysteine to the solution; the sample was incubated for an additional 30 min at room temperature. The excess NEM and cysteine were removed from the solution by centrifugation using an ultra-0.5 centrifugal filter (cutoff 10 kDa).


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Treatment with hydrogen peroxide

The effect of hydrogen peroxide (H2O2), a major in vivo reactive oxygen species (ROS), on the free-form of TTR was tested using frozen fresh plasma. Based on the protocol proposed by Kocha and coworkers,18 the plasma was diluted in PBS and treated with 0.1‒5 mM of H2O2 for 18 h at 37 °C under a nitrogen atmosphere. The excess H2O2 was then removed by centrifugation using an ultra-0.5 centrifugal filter (cutoff 10 kDa). The phosphate buffer was replaced by the working solution before applying the sample to the Poros 20 NH LC-SPDP resin.

RESULTS In regard to the specificity and recovery for the free-thiol-containing proteins, we first compared present resin with results for Thiopropyl SepharoseTM 6B, one of the most frequently utilized chromatography systems for isolating free-thiol containing peptides and proteins. We found that the POROS 20 NH LC-SPDP resin was advantageous for the specific isolation of free-thiolcontaining proteins, compared to Thiopropyl SepharoseTM 6B (see Figure S2, Supporting Information) and also applicable to other crude mixtures derived from Carica papaya latex, which is known to contain a thiol-containing enzyme, papain (see Figure S3, Supporting Information). To analyze the proteins containing free thiols in plasma, fresh frozen plasma, obtained from a healthy single donor (R01 in Table S1, Supporting Information), was used. The sample was directly applied to the Poros 20 NH LC-SPDP resin. The two fractions, flow-through (F) and eluted (E) fractions obtained from the resin (see Experimental section), were subjected to SDS-PAGE; in the F-fraction, numerous proteins could be seen, meanwhile, a few proteins were recovered in the E-fraction, one of which with a molecular mass ~14 kDa was the most abundant (Figure 1A). This protein was subjected to in-gel digestion and the resulting peptides were then analyzed by nano-flow LC linear ion-trap time-of-flight (TOF) mass spectrometry and identified with Mascot as being assigned to TTR (Figure S4, Supporting Information). TTR has been reported in many studies to be a homotetramer; however, it is well established that the dimer is the most stable form. Nonetheless, on SDS-PAGE only a monomer was observed. In order to examine whether or not the recovered TTR is a dimeric form, the E-fraction from the plasma was not heated at 95 °C before being used in



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the electrophoresis procedure (semi-native PAGE); as a result, the dimer was exclusively observed in the SDS-PAGE (E2, Figure 1A). The protein bands obtained for both the F- and E-fractions in Figure 1A were further confirmed by western blot analysis, in which the proteins were transferred to a nitrocellulose membrane and treated with the monoclonal anti-TTR antibody (Figure 1B). The results clearly show that the monomer of TTR as a major band and the dimer is located, not only in the E-fraction but also in the F-fraction, suggesting that two forms of TTR with (E-fraction) and without (F-fraction) a free thiol are present, the latter of which would become attached to the LCSPDP resin (see below).

Figure 1. Identification of transthyretin in human plasma. (A) SDS-PAGE (12.5%) of human frozen fresh plasma: lane M, molecular mass standard; lane F, flow-through fraction (F-fraction) from the Poros 20 NH LC-SPDP resin; lane E, the fraction eluted with DTT (E-fraction) and lane E2 without boiling at 95 °C before electrophoresis. The protein band, indicated by the arrow in lane E1, was identified by nanoLC/ESI-MS/MS (see Figure S4). (B) Western blot analysis of the F(lane F) and E-fraction (lane E), obtained in a manner similar to those in lane F and E1 in A, with anti-TTR antibody (rabbit monoclonal antibody).

To confirm that the TTR recovered from the E-fraction contained a free thiol group and the TTR from the F-fraction did not, the plasma sample was pre-treated with N-ethylmaleimide (NEM), a reagent that reacts rapidly with sulfhydryl groups, and the sample was then applied to the Poros 20 NH LC-SPDP resin. As a result, a protein band corresponding to TTR was not present in the E-


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fraction (Figure 2), implying that TTR was bound to the LC-SPDP resin via the single sulfhydryl group at Cys10 of TTR. On the other hand, the TTR recovered from the F-fraction, as a result of the absence of proteins being linked to the resin, can be attributed to the fraction of TTR in which the sulfhydryl group had been modified or oxidized. In order to further confirm the sulfhydryl modifications of TTR in plasma, the F-fraction was subjected to MALDI-MS (see below).

Figure 2. Thiol-containing proteins sensitive to N-ethylmaleimide in human plasma. SDS-PAGE (12.5%) of F- (lane F) and E-fraction (lane E1) were obtained similarly to lanes F and E in Figure 1A; the E-fraction (lane E2) was obtained from the same plasma sample but was treated with 50 mM NEM (see Experimental section) prior to separation by Poros 20 NH LC-SPDP resin.

The concentrations of TTR in the plasma used in the present experiment, as determined by ELISA (see Experimental section, Supporting Information), were determined to be 28‒39 mg/dL (Table S1, Supporting Information), which is within the reported values for plasma from a healthy volunteer (20‒40 mg/dL).9,12 In each experiment, 15 µL of plasma, which contain 4.32‒5.81 µg of TTR on the basis of the above concentration, were applied to the Poros 20 NH LC-SPDP resin. The amounts of TTR recovered from the F- and E-fractions were obtained as in Table S1: The recovery of the TTR by the present separation method was 79.1 ± 5.2%, based on the sum of the recovered TTR (F + E-TTR in Table S1) over the total TTR. It is noteworthy that the relative abundance of the free-form TTR (E-fraction TTR in Table S1) over the sum of the recovered TTR (F + E-TTR), was calculated to be 34.1 ± 5.4%, the value of which is significantly higher than the reported values for the free form (5‒20%) in normal plasma.13,14 This can be attributed to individual differences or by



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the fact that the free thiol in a protein is susceptible to oxidation/modification during sample preparation/handling. Different from previous methods, the present method allows for the prompt separation of the free-thiol proteins from other forms by applying fresh plasma onto the resin in a direct manner. In order to obtain direct evidence for the modifications of TTR, both the F- and E-fractions were analyzed by MALDI-TOFMS. As a result, the E-fraction predominantly gave a molecular mass at m/z 13,762.8, which corresponds to the unmodified TTR monomer (MH+theoretical: 13,762.5, Figure 3A), whereas the F-fraction produced several ion peaks, among which the peaks at m/z 66,552.8 (MH+) and 33,377.3 ([M+2H]2+) were the most intense and were assigned to albumin, based on the fact that it is the most abundant protein in plasma (Figure 3B). In addition, several ion peaks were observed around m/z 13,882.6, among which those at m/z 13,882.6 and 13,939.2 were larger in mass by 119.8 and 176.4 Da, respectively, than the intact TTR (m/z 13,762.8), and corresponds to a cysteine (+119 Da) and a cysteinylglycine (+176 Da) adduct. These adducts turned out to be linked to TTR via disulfide bonds between their Cys and Cys10 of TTR based on the fact that these ion peaks were shifted to m/z 13,762.4, which corresponds to the intact TTR with a free thiol, after treatment with 5 mM DTT at 60 °C for 1 h (Figure 3C). It is noteworthy that these modifications of TTR have been reported previously as the major species found in plasma.19,20


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Figure 3. MALDI-TOFMS spectra of the E- (A), F-fractions (B), obtained from the Poros 20 NH LC-SPDP resin, and F-fraction treated with 5 mM DTT for 60 min at 60 °C (C). The inset in B shows the expanded region indicated by the bracket.

The presence of free thiol groups in proteins has been proposed as a mechanism for maintaining homeostasis in the case of oxidative stress, because this group is sensitive to free radicals or reactive oxygen and nitrogen species. In order to determine whether or not the free thiol of TTR was also sensitive to oxidizing species, the effect of H2O2 on TTR and its dose response on the suppression of the free form of TTR was examined using the Poros 20 NH LC-SPDP resin,



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which enables the prompt and efficient separation of the free form from the modified forms. The frozen fresh plasma (R01 in Table S1, Supporting Information) was treated with 0, 0.1, 1, 2, and 5 mM of H2O2 at 37 °C for 18 h, and subjected to the Poros 20 NH LC-SPDP resin, followed by western blot analysis (Figure 4A). The amount of TTR with a free thiol at Cys10, which was recovered in the E-fraction (Figure 1), was significantly reduced as the result of the 0.1 mM H2O2 treatment, and was nearly completely suppressed when more than 2 mM H2O2 was used (Figure 4A). The relative quantities of TTR in the F- and E-fractions, as measured by ELISA, clearly showed that the ratio of the free form of TTR to the oxidized forms decreased upon treatment with 0.1, 1, 2 and 5 mM H2O2 (Figure 4B). It should be noted that the amounts of TTR recovered in F(3.260 µg) and E-fractions (0.763 µg) obtained at 0 mM H2O2 in the table of Figure 4B were shifted from those (F-fraction: 3.041 µg; E-fraction: 1.861 µg) obtained from a fresh plasma sample (R01 in Table S1) since during the incubation of the plasma under the above conditions, the free form could have been converted into oxidized/modified forms, even in the absence of H2O2. These results strongly suggest that the free thiol at Cys10 can be oxidized with a relatively low concentration of H2O2, to give the modified forms of TTR that were recovered in the F-fraction and increased upon treatment with H2O2 (Figure 4B). In addition, when the F-fraction, obtained by the treatment with 5 mM H2O2, was analyzed by MALDI-TOFMS, new ion peaks corresponding to the TTR molecule were observed at m/z 13,826.6 and 13,897.3. The former value was larger in mass by 63.8 Da than the intact TTR (m/z 13,762.8 in Figure 3A), which can be attributed to the oxidized TTR, originating from the free-thiol form, containing a cysteine sulfonic acid (+48 Da) and a methionine sulfoxide (+16 Da). The latter value was larger in mass by 134.5 Da than the intact one, which could be ascribed to TTR modified with a cysteine (+119 Da), which is abundant in plasma (see Figure 3B), and further modified with one oxygen (+16 Da). This was proved by its conversion to the free form, upon reduction with DTT, but with one oxygen (m/z 13,778.5 in Figure S5, Supporting Information). They were, most probably, produced as the result of oxidation at the single Cys at position 10 and/or the single Met at position 13 of TTR (Cys and Met are both known to be very sensitive to oxidation).19,20


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Figure 4. Effect of H2O2 treatment on the recovery of the free-Cys form of TTR. (A) Western blot analysis with anti-TTR monoclonal antibody of the F- and E- fractions prepared from the human plasma treated with 0, 0.1, 1, 2 and 5 mM H2O2 at 37 °C for 18 h. (B) Quantification of the TTR in the F- and E-fractions by ELISA (see Experimental section, Supporting information). The ratios of the free-form to modified/oxidized forms of TTR were plotted as a function of concentration of H2O2. The error bars denote the standard deviation for three experiments. (C) MALDI-TOFMS spectrum of the F-fraction obtained in 5 mM H2O2 treatment in A. The inset shows the expanded region indicated by the bracket.



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DISCUSSION The heterobifunctional linker LC-SPDP can be used to isolate specific proteins because it is capable of forming disulfide bonds with any free thiol group present in a sample.21 The reaction occurs through the interchange between the 2-pyridylthio moiety of LC-SPDP and a sulfhydryl group, and proceeds even in acidic media; this specificity allows distinct thiol groups to be selectively modified.22 Many plasma proteins contain cysteine residues, most of which form disulfide bonds, which play an important role in stabilizing protein structures.23 Meanwhile, a few proteins retain free-thiols at Cys residues, which are often involved in the function of the protein and regulation.24 In this regard, since the present method permits the rapid isolation of these freethiol-containing proteins, profiling of the free form versus oxidized/modified forms of thiolcontaining proteins in a complex biological sample such as plasma becomes feasible. Such a profiling would give insights into the functional roles of those proteins or reflect the physiological states in response to some internal environment in vivo. When the frozen fresh plasma sample was analyzed by the present method, TTR, which has a single Cys at position 10, was the major isolated product, and only a small amount of albumin was recovered in the E-fraction, although albumin has been reported to be the major plasma protein that contains a free thiol at Cys34, and makes up 80% of the free thiol content in plasma.25 This could be ascribed to the accessibility of Cys10 of TTR, which is located in the N-terminal tail (the helical region near strand A in Figure 5A) and is exposed to the exterior in solution.26 Note that while the thiol of Cys10 could form a sulfur-hydrogen bond with the carbonyl oxygen of Gly57 in the β-strand D27, this linkage is much weaker than the oxygen-hydrogen bond.


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Figure 5. Three dimensional structures of TTR and albumin. (A) TTR dimer (PDB code: 3W3B): the eight strands are indicated by the letters A‒H. Also, an enlarged image of the region where the Cys10 is located is shown in the inset. The Gly57 and Cys10 are highlighted in the structure. (B) Human serum albumin (PDB code: 1AO6): an enlarged image of the region where the Cys34 is located is shown in the inset. The Cys34, Pro35, Asp38, His39, Val77 and Tyr84 are highlighted in the structure. The figures were obtained using the PyMOL software.

The cysteine residue on TTR has been reported to be highly reactive with respect to ROS and carbon centered free-radicals.28 Meanwhile, Cys34 in albumin is located in domain I (Figure 5B), and is surrounded by the side chains of Pro35, Asp38, His39, Val77 and Tyr84. Moreover, the sulfur atom of Cys34 is oriented toward the interior of the protein and the sulfhydryl group of Cys34 interacts with the His39 and Tyr84 side chains,29,30 diminishing the possible interactions between the sulfhydryl group and the other compounds.30 Such an environment renders the sulfhydryl group of Cys less active. Meanwhile, Cys10 of TTR, located in the proximal portion of protein surface, may act as a nucleophilic agent, which, as a result, would be susceptible to modification.31 Many authors have reported that albumin, which is the most concentrated protein in plasma (0.6‒0.8 mM), has a scavenger role in plasma: the Cys34 contributes to approximately 40% of the antioxidant activity of the plasma proteins.

6,32

Anraku and coworkers32 estimated the levels of the

scavenger property of the Cys34 against H2O2 and observed that the Cys34 contributes to 68% of the H2O2 that reacts, which is consistent with the fact that the thiols in proteins act as a nonenzymatic defense against ROS; about 70 of the ROS in the environment could be scavenged and covalently linked with thiols.

3,25

However, the structural circumstances at Cys10 of TTR and at

Cys34 of albumin, as described above, should significantly affect the initial accessibility of ROS to those residues; that is, it would not be surprising to find that Cys10 of TTR is more susceptible to oxidation or modification compared to the free thiol group in albumin. It has been reported that the protein-SH levels in plasma could be used as a biomarker for oxidative stress in patients with chronic renal disorders, because the level of free thiol groups in plasma is significantly reduced in end stage patients.33 The usage of some drugs, inflammatory



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diseases and cancers could also be associated with oxidative modifications of albumin and other proteins containing free thiols; in fact, in lung cancer, variations in the modified and free forms of TTR have been observed, in which the free form was reduced in patients with lung cancer.34,35 The fact that the level of H2O2 in normal blood has been reported to range from 13 to 57 µM

36

and is

deeply involved with ROS, prompted us to speculate that the ratio between the modified/oxidized forms and free-thiol forms may reflect the status of oxidative stress caused by endogenous ROS or related diseases, which could allow for the development of a standard for changes in redox state in relative to diseases. Anraku and coworkers32 demonstrated that the ratio between the oxidized and reduced forms of albumin could be a useful biomarker for oxidative stress and as a predictor of certain types of medical treatments. The high susceptibility of the free-form of TTR to oxidation was demonstrated by the significant decrease in the free form of TTR with a relatively low concentration (0.1 mM) of H2O2 and almost complete suppression when 2 mM H2O2 was used (Figure 4A). Despite the fact that the relative abundance of the free form versus modified/oxidizedforms could be changed individually- or age-dependently, the ratio of the free form to the modified/oxidized form of TTR, which was decreased upon incubation with H2O2 (Figure 4B), could reflect the physiological state of an individual and could be useful as a surrogate marker for monitoring a ROS imbalance. It is also noteworthy that TTR is one of most abundant proteins in cerebrospinal fluid (CSF) and is responsible for hormone transport in the brain; taking into account the fact that ROS and oxidative stress could occur during the evolution of Alzheimer’s disease, the ratio of the free form to modified/oxidized form of TTR could also be used as a marker for the diagnosis or stage of the disease; it has been recently reported that Alzheimer’s patients displayed higher concentrations of the oxidized form of TTR in their CSF.37

CONCLUSIONS Free thiol(s) in a protein, exposed to the environment, can be easily modified/oxidized by electrophilic substances such as various ROS; thus, the relatively high reactivity of a thiol should be taken into consideration when they are subjected to structural analysis or biochemical or biological assays, because the thiol(s) on a Cys residue is often involved in protein function. This property of


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thiol groups, in turn, could be useful for monitoring oxidative status in vivo by directly examining the composition of a biological fluid, such as plasma or CSF, in which thiol-containing proteins are more or less exposed to various ROS, and as a result, could be modified/oxidized. The resulting molecular forms and the relative ratios among the modified species and the free form may be correlated with a subject’s physiological state, could be used as an index of susceptibility to oxidative stress, or the propensity to develop some diseases as a disease-specific signature. Since the present method permits the efficient and rapid isolation of free-thiol-containing proteins from the others, it allows, for the first time, the identification of a thiol-containing protein that is most accessible to oxidation in human plasma, namely, TTR, which has a single Cys in its sequence that has been reported to be susceptible to oxidation/modification. It could also permit differences in the amount of free-form of TTR versus oxidized/modified ones to be pinpointed, which might be involved with physiological changes or health disorders.

ASSOCIATED CONTENT

Supporting Information Other information on EXPERIMENTAL SECTION and RESULTS as indicated in the text.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: +81-06-6879-4312 Author Contributions Caroline Donzeli Pereira and Toshifumi Takao designed and conducted all experiments, and Naoto Minamino provided the samples and discussed on sample preparation. Notes The authors declare no competing financial interests.



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For TOC only


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Free Thiol of Transthyretin in Human Plasma Most Accessible to

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