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Transition of serine residues to the d-form during the conversion of ovalbumin into heat stable S-ovalbumin Tetsuya Miyamoto a,c , Nobuyuki Takahashi b , Masae Sekine c , Tetsuhiro Ogawa a , Makoto Hidaka a , Hiroshi Homma c,∗∗ , Haruhiko Masaki a,∗ a
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan c Department of Pharmaceutical and Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan b
a r t i c l e
i n f o
Article history: Received 30 January 2015 Received in revised form 13 April 2015 Accepted 20 April 2015 Available online xxx Keywords: d-Amino acid d-Serine Ovalbumin Isomerization LC–MS/MS
a b s t r a c t Ovalbumin, a major protein in chicken egg white, is converted into a more thermostable molecular form, known as S-ovalbumin, during the storage of shell eggs. Our previous X-ray crystallographic study indicated that S-ovalbumin contains three d-Ser residues (S164, S236, and S320), which may account for its thermostability. Here, we confirmed the presence of these d-Ser residues in ovalbumin using a technique combining deuterium labeling of ␣-protons of amino acids and liquid chromatography-tandem mass spectrometry (LC–MS/MS). Ovalbumin from chicken egg white and recombinant ovalbumin were incubated for approximately 12 days at pH 9.5 and 37 ◦ C. They were then hydrolyzed in DCl/D2 O vapor, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and analyzed by LC–MS/MS. A timedependent increase in the d-Ser contents in native ovalbumin was observed over a period of 7 days, reaching approximately 8%. This corresponds to a value of three serine residues per molecule, and is consistent with the prediction based on our previous crystallographic analysis. Nearly identical results were obtained with recombinant ovalbumin. We then used this technique to investigate whether damino acid residues could arise within other proteins under mild alkaline conditions and detected small but significant amounts of d-Ala and/or d-Ser residues that increased in a time-dependent manner in some proteins. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Chicken ovalbumin comprises more than 50% of the protein in egg white. During the storage of unfertile eggs and the development of fertile eggs, ovalbumin is converted into a heat-stable form, S-ovalbumin, via an intermediary form (I-ovalbumin) [1–3]. Differential scanning calorimetry demonstrated that the denaturation temperature of S-ovalbumin is 8 ◦ C, higher than that of native ovalbumin [4,5]. The conversion of ovalbumin to S-ovalbumin under physiological conditions is associated with an increase in the pH of egg whites to 9.5, which is caused by the release of carbon dioxide through the eggshell [3]. The thermostabilization of ovalbumin has been extensively studied [6–12] and we uncovered a unique feature of S-ovalbumin using X-ray crystallographic analysis [13].
∗ Corresponding author. Tel.: +81 3 5841 3080; fax: +81 3 5841 8016. ∗∗ Corresponding author. Tel.: +81 3 5791 6229; fax: +81 3 5791 6381. E-mail addresses:
[email protected] (H. Homma),
[email protected] (H. Masaki).
S-Ovalbumin is composed of three -sheets and eleven ␣-helices, and surprisingly, our results showed that the residues Ser164, Ser236, and Ser320 appeared to be in a d-configuration. These serine residues are exposed to the molecular surface, and analysis of ovalbumin mutants suggested that the Ser164 and Ser320 residues contribute to the thermostability of the protein [14,15]. However, it was not chemically verified that S-ovalbumin contains d-Ser residues. In a previous report, we established a sensitive method for detecting innate d-amino acid residues in proteins by combining deuterium labeling of ␣-protons associated with isomerization of amino acids and liquid chromatography-tandem mass spectrometry (LC–MS/MS) [16]. We applied this method to recombinant -galactosidase, and found that the protein contains no innate damino acid residues and undergoes isomerization during a very early stage of hydrolytic incubation [17]. In the present study, we used this method to determine whether d-Ser residues are present in native and recombinant S-ovalbumin. In addition, the recombinant ovalbumin mutants S164 V, S236G, S320 V, and S164 V/S320 V were also analyzed in order to elucidate in detail the formation of
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Table 1 d-Ser contents in native ovalbumin incubated at different pH values. Incubation
D/(D + L) (%)
Temp.
Day
pH 9.5
pH 7.4
0 ◦C
10
4.0
1.4
◦
1 3 5 7 10
4.4 5.7 7.2 7.9 7.8
–a –a –a –a 1.7
37 C
a
Not determined.
Table 2 d-Ser contents in recombinant ovalbumin incubated under mild alkaline conditions (pH 9.5). Incubation Temp. ◦
D/(D + L) (%) Day
WT
S164V
S236G
S320V
S164V/S320V
0 C
12
0.7
0.8
0.2
0.5
0.6
37 ◦ C
3 6 9 12
3.3 5.6 7.8 8.4
2.8 4.2 5.8 6.9
2.3 4.4 7.0 7.9
2.8 4.8 6.5 7.2
2.0 2.9 3.5 4.5
d-Ser residues. The results described in this report indicate that the d-Ser contents in both native and recombinant ovalbumin increase in a time-dependent manner to approximately 8% under mild alkaline conditions (pH 9.5), which corresponds to the presence of three serine residues per molecule. These findings are consistent with the prediction made based on the results of our previous crystallographic analysis [13]. Furthermore, we detected the formation of small but significant amounts of d-Ala and/or d-Ser residues in other proteins under mild alkaline conditions. 2. Materials and methods 2.1. Chemicals Albumin from chicken egg white (product no. A7641), three ␣amylases from Bacillus licheniformis, Aspergillus oryzae and porcine pancreas, carbonic anhydrase from bovine erythrocytes, bovine serum albumin (BSA), and human serum albumin (HSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). ␣-Amylase from Bacillus subtilis, deuterium oxide (99.9%), and deuterium chloride (99.5%) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Lysozyme from egg white was purchased from Seikagaku Corporation (Tokyo, Japan). 4-Fluoro-7-nitro-2,1,3benzoxadiazole (NBD-F) was obtained from Dojindo Laboratories (Kumamoto, Japan). 2.2. Production and purification of recombinant ovalbumin Wild-type ovalbumin and the S164 V, S236G, S320 V, and S164 V/S320 V mutants were expressed in Escherichia coli BL21 (DE3) using the expression plasmid pET/Ova and its derivatives, and purified as described previously [18]. All mutant plasmids were prepared using the QuickChange PCR mutagenesis kit (STRATAGENE, CA, USA), as described previously [15]. 2.3. Alkaline treatment of proteins Native ovalbumin and all recombinant ovalbumins (each 100 g) were incubated in 100 l of 100 mM glycine/NaOH buffer, pH 9.5, for 1–12 days at 37 ◦ C. After incubation, samples were stored at 0 ◦ C for up to 10 (Table 1) or 12 (Table 2) days. As a control,
ovalbumins were incubated in the same buffer for 10 (Table 1) or 12 (Table 2) days at 0 ◦ C. Prior to analysis, the buffer of the incubated samples was changed to 20 mM potassium phosphate buffer, pH 7.4, using a VIVASPIN 500 MWCO 5000 (Sartorius AG, Hannover, Germany). 2.4. Hydrolysis and Sample Preparation for LC/ESI–MS/MS Each sample protein (10 g) was hydrolyzed with a Pico TagTM workstation (Waters, Milford, MA, USA) for 24 h at 110 ◦ C under 6 M DCl/D2 O vapor. The hydrolysates were dissolved in 40 l of 50 mM borate buffer (pH 9.5), and a 20 l aliquot of the sample was reacted with 10 l of 50 mM NBD-F in dry acetonitrile at 60 ◦ C for 5 min. This reaction was terminated by the addition of 270 l of 1% trifluoroacetic acid (TFA) in water. An aliquot (10 l) of this mixture was injected and separated on a TSK-gel ODS-80Ts column (250 × 4.6 mm i.d.; Tosoh, Tokyo, Japan) at 33 ◦ C by HPLC, as described previously [19,20]. Elution solvent A was 1% tetrahydrofuran (THF), 0.02% TFA in 5% acetonitrile, and solvent B was 1% THF, 0.02% TFA in acetonitrile. Elution was carried out at a flow rate of 1.0 ml/min for 15 min with solvent A, then a linear gradient was applied for 50 min from 0 to 22% solvent B. Subsequently, a linear gradient was applied for 65 min from 22 to 32% solvent B, followed by an elution for 95 min with 32% solvent B. The NBD-d/l-amino acids eluted in a reproducible manner (e.g., the retention time of 35–37 min for Ser, 51–53 min for Ala, 70–72 min for Val, 84–86 min for Leu, and 86–89 min for Phe) and the sample fractions of each NBD-amino acid were collected according to the retention time. These isolated NBD-d/l-amino acid samples were dried by rotary evaporator and then dissolved in 0.01% TFA in water and injected into the LC/ESI–MS/MS. 2.5. Detection of d/l-amino acids by LC/ESI–MS/MS The LC/ESI–MS/MS system consisted of an Agilent 1100 separation module (Agilent Technologies, Palo Alto, CA, USA) equipped with CHIROBIOTIC TAG (150 × 2.1 mm i.d.; Astec, Whippany, NJ, USA) and a quadrupole tandem mass spectrometer with an electrospray ion source (API-3000; Applied Biosystems, Foster City, CA, USA). NBD-Ser, NBD-Val, NBD-Ala, NBD-Leu, and NBD-Phe were eluted in methanol/water/TFA (80:20:0.01, 70:30:0.01, 85:15:0.01, 73:27:0.01, and 82:18:0.01, v/v/v, respectively) at a flow rate of 0.2 ml/min. The ES capillary voltage was 5.0 kV, and the source temperature was 400 ◦ C. Other parameters were optimized using spectrometer software (Applied Biosystems). The multiple reaction monitoring (MRM) mode was used in the ESI–MS/MS to detect precursor and product ions. The amounts of NBD-d/l-amino acids were determined based on the peak area in chromatograms. The m/z values of the precursor and characteristic product ions of NBD-Ser, NBD-Val, NBD-Ala, NBD-Leu, and NBD-Phe were determined as 269/252, 281/235, 253/236, 295/249, and 329/283, respectively, in the positive-ion mode. The calibration range of NBD-d-amino acids is from 5 to 100 pmol, and the LOD of NBD-d-amino acids is approximately 1 pmol (S/N = 3). The NBD-d-amino acid ratios, e.g., in the case of Ser, were calculated as (d-amino acid (m/z 269/252))/(d-amino acid (m/z 269/252 + 270/253) + l-amino acid (m/z 269/252 + 270/253)). These ratios show the innate d-amino acid contents in proteins. 3. Results 3.1. Detection of d-serine residues in native ovalbumin We investigated the generation of d-Ser residues in ovalbumin under mild alkaline conditions. Native ovalbumin, containing 38 serine residues out of 385 total residues, was incubated in 100 mM
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Fig. 1. Multiple reaction monitoring (MRM) chromatograms of NBD-d/l-Ser (m/z 269/252) isolated from native ovalbumin hydrolyzed under DCl/D2 O. (A) Sample ovalbumin was incubated at pH 9.5 for 10 days at 37 ◦ C. (B) Sample was incubated at pH 7.4 for 10 days at 37 ◦ C.
glycine/NaOH, pH 9.5, for approximately 10 days at 37 ◦ C. Thereafter, the incubated samples were hydrolyzed in DCl/D2 O vapor and the d-Ser contents determined by HPLC and LC/ESI–MS/MS. The d-Ser content (D%) of the control sample, which was incubated for 10 days at 0 ◦ C, was 4.0%, while the content of samples incubated at 37 ◦ C increased up to 7.9% by 7 days (Table 1). The d-Ser contents at days 7 and 10 (approximately 8%) corresponded to the presence of three d-Ser residues per molecule (Fig. 1A), and this is in agreement with the prediction made based on our previous X-ray crystal structure analysis [13]. The d-Ser content of the sample incubated in 50 mM potassium phosphate buffer, pH 7.4, for 10 days was 1.7%, suggesting that the isomerization of serine residues takes place under mild alkaline conditions (Table 1 and Fig. 1B). It is of note that d-Ser residues were produced by incubation for 10 days at pH 9.5 even at 0 ◦ C (4.0%, Table 1), whereas the d-Ser contents were considerably smaller following incubation for 10 days at pH 7.4 at 0 ◦ C (1.4%, Table 1). 3.2. d-Serine contents in ovalbumin mutants We then confirmed the generation of d-Ser in wild-type recombinant ovalbumin (WT) and its mutants, S164 V, S236G, S320 V, and S164 V/S320 V, following incubation under mild alkaline conditions (Table 2). Compared with the results obtained by native ovalbumin, the d-Ser content in the WT were lower following incubation for 10–12 days at 0 ◦ C (native ovalbumin, 4.0%; WT, 0.7%; Table 1 and 2). Although the WT has the same conformational properties as native ovalbumin and is transformed into S-ovalbumin in a similar manner [18,21], native ovalbumin undergoes post-translational modification (an N-glycosylated sugar chain at Asn292 and phosphoserines at residues 68 and 344 [21,22]). Such modification might facilitate the isomerization of serine residues in native ovalbumin. The d-Ser content of the WT increased to 7.8% after incubation at 37 ◦ C for 9 days at pH 9.5, which appeared to be equivalent to three d-Ser residues per molecule, and reached a value of 8.4% by 12 days of incubation (Table 2). Although the d-Ser contents in the S164 V, S236G, and S320 V mutants increased during incubation at 37 ◦ C, the contents were lower than in the WT (5.8–7.0% in the mutants vs. 7.8% in the WT; Table 2). However, the differences were less than 2.6%, which presumably correspond to one d-Ser residue per molecule. After incubation for 12 days, the contents of the S164 V, S236G, and S320 V mutants reached 6.9, 7.9, and 7.2%, respectively, nearly corresponding to a value of three d-Ser residues per molecule. In the double mutant S164 V/S320 V, the dSer content was markedly lower than both the WT and the single
mutants, but still higher than the value corresponding to only one d-Ser residue (4.5% after incubation for 12 days, which is equivalent to 1.7 residues; Table 2). Collectively, these results suggest that the serine residues in ovalbumin are susceptible to isomerization, and that the residues at 164 and 320 are more susceptible than the other serine residues. It is noteworthy that no d-Val residues were detected in the WT or any of the mutants (data not shown). 3.3. Isomerization of alanine and serine residues in various proteins under mild alkaline conditions To examine whether amino acid residues in other proteins are also isomerized under mild alkaline conditions, we examined the damino acid contents in various proteins. Both d-Ala and d-Ser were detected in bovine carbonic anhydrase, and d-Ser was detected in hen egg lysozyme (Table 3). No d-Leu, d-Phe, or d-Val residues were detected in either protein. The d-Ser content in carbonic anhydrase increased to 4.8% after incubation at pH 9.5 for 12 days at 37 ◦ C, while the d-Ser content in lysozyme appeared to be unchanged (0.6%), even after incubation for 12 days (Table 3). Conversely, no d-amino acids (Ser, Ala, Leu, Phe, and Val) were detected in bovine serum albumin (BSA) incubated for 5 days or in human serum albumin (HSA) incubated for 10 days (data not shown). Subsequently, isomerization was also analyzed in ␣-amylase from various species (Table 4). d-Ala and d-Ser were detected in ␣-amylase from B. subtilis and B. licheniformis. The d-Ala contents of B. subtilis ␣-amylase remained unchanged, even after incubation for 6 days (0.6%), while the d-Ser contents in both ␣-amylases continued to rise over the 6-day incubation period. By contrast, d-Ser in A. oryzae ␣-amylase and d-Ala and d-Ser in porcine pancreatic ␣-amylase were detected only after incubation for 6 days. Therefore, some amino acid residues, at least Ala and Ser, in proteins from various species do undergo isomerization under mild alkaline conditions. Table 3 The d-amino acid contents in carbonic anhydrase and lysozyme incubated under mild alkaline conditions (pH 9.5). Incubation
D/(D + L) (%) Carbonic anhydrase
Lysozyme
Day
Ala
Ser
Ala
Ser
0 C
12
0
0
0
0
37 ◦ C
5 12
0 0.2
0.8 4.8
0 0
0.6 0.6
Temp. ◦
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Table 4 The d-amino acid contents in ␣-amylase from various organisms incubated under mild alkaline conditions (pH 9.5). Incubation
D/(D + L) (%) B. subtilis
B. licheniformis
A. oryzae
Porcine pancreas
Temp.
Day
Ala
Ser
Ala
Ser
Ala
Ser
Ala
Ser
0 ◦C
6
0.6
0.2
0.1
0.1
0
0
0
0
37 ◦ C
3 6
0.6 0.6
0.4 0.7
0.2 0.4
0.3 0.5
0 0
0 0.6
0 0.5
0 1.6
4. Discussion
5. Conclusions
Here, we showed that time-dependent isomerization and transition from l-Ser to d-Ser residues occurs in both native and recombinant ovalbumin molecules, during incubation at 37 ◦ C under mild alkaline conditions (pH 9.5). The final d-Ser content (7.9%) in native ovalbumin observed after incubation for 7 days appears to be equivalent to the presence of three d-Ser residues per molecule. This result agrees well with the results of X-ray crystallographic study of S-ovalbumin, which predicted that the protein contains three d-Ser residues [13]. In non-fertilized eggs, the pH of egg white increases from 8.2 to 9.5 during the first 2 days of incubation at 37 ◦ C, and this value remains steady thereafter [3]. The isomerization of d-Ser residues in ovalbumin is presumed to be dependent on pH, as a d-Ser content of only 1.7% was detected in native ovalbumin incubated for 10 days at pH 7.4 (Table 1). In a previous report, the proportion of S-ovalbumin was observed to gradually increase during incubation at pH 9.5 and 37 ◦ C via the intermediary I-ovalbumin form, leaving little of the native form of ovalbumin (5.9% after incubation for 8 days and 4.0% after 10 days [3]). This observation apparently correlates well with the d-Ser content determined in this report. The d-Ser contents in the S164 V, S236G, S320 V, and S164 V/S320 V mutants were also analyzed during incubation under mild alkaline conditions (Table 2). After incubation of the single serine mutants (S164 V, S236G, or S320 V) for 9 days, the dSer contents appeared to be nearly equivalent to or slightly higher than two residues per molecule. However, the contents increased to approximately three residues per molecule after incubation for 12 days. In the double S164 V/S320 V mutant, the contents were apparently smaller than those of WT and the single mutants, but exceeded one residue per molecule after incubation for 6 days. In addition, the d-Ser content of WT after incubation for 12 days was 8.4%, which exceeds the value equivalent to three residues per molecule. Taken together, these results suggest that serine residues other than the three predicted residues [13] are also isomerized in recombinant ovalbumin. We also investigated the d-amino acid contents in other proteins after incubation at 37 ◦ C under mild alkaline conditions (pH 9.5). After incubation for 12 days, the d-Ser content was approximately 5% in carbonic anhydrase, while only 0.6% d-Ser was detected in lysozyme. Under physiological conditions, the serine residues in lysozyme might be isomerized, because lysozyme is a component in egg white and exposed to mild alkaline conditions, like ovalbumin. In the ␣-amylases from four different species (B. subtilis, B. licheniformis, A. oryzae, and porcine pancreas), d-Ala and d-Ser were detected (Table 4). The ␣-amylases from B. subtilis and B. licheniformis produced only small amounts of d-Ala and d-Ser, even after incubation for 6 days (0.4–0.7%, Table 4). d-Ala and d-Ser residues from ␣-amylases in A. oryzae and porcine pancreas were detected only after incubation for 6 days. Collectively, these results suggest that the amino acid residues in some proteins, at least serine and alanine, undergo isomerization under mild alkaline conditions. The conversion of l-Ser to d-Ser residues has been postulated to occur via a carbanion or dehydroalanine intermediate [23].
It has been suggested that the S-ovalbumin contains d-Ser residues and these residues may contribute the thermostability of the protein [14,15]. We demonstrated the presence of d-Ser residues in ovalbumin using a technique combining deuterium labeling of amino acids and LC–MS/MS. Furthermore, we demonstrated that d-Ala or d-Ser arise within various proteins under mild alkaline conditions. In the literature, d-Ser as well as d-Asp residues in proteins is suggested to be related to aging or disease [24–27]. Changes in the physical properties of proteins resulting from isomerization of Ser residues, as occurs in the case of ovalbumin, is also a very interesting phenomenon. Therefore, it is important to identify proteins that possess physiologically isomerized d-amino acid residues. The method utilized in this report will be useful for this purpose.
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