# Partial amino acid sequence of gamma-46 gliadin

T.A. Egorov, T.I. Odintsova, A.K. Musolyamov, S.F. Barbashov, V.N.

Pustobaev, J. Andersen, P. Roepstorff, Yves Popineau

► To cite this version:

T.A. Egorov, T.I. Odintsova, A.K. Musolyamov, S.F. Barbashov, V.N. Pustobaev, et al.. Partial amino acid sequence of gamma-46 gliadin. 1998, 63 (9), pp.1061-1067. [hal-02694565](https://hal.inrae.fr/hal-02694565)

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# Partial Amino Acid Sequence of γ-46 Gliadin

T. A. Egorov¹*, T. I. Odintsova², A. K. Musolyamov¹, S. F. Barbashov¹, V. N. Pustobaev¹, J. Andersen³, P. Roepstorff³, and Y. Popineau⁴

¹Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 117984 Russia; fax: (095) 135-1405; E-mail: egorov@imb.imb.ac.ru

²Vavilov Institute of General Genetics, Russian Academy of Sciences, ul. Gubkina 3, Moscow, 117809 Russia

³Department of Molecular Biology, Odense University, DK-5230 Odense M, Denmark

⁴Laboratory of Technology and Biochemistry of Proteins, INRA, 44026 Nantes Cedex, France

Received December 4, 1997

Revision received May 6, 1998

**Abstract**—We have determined the partial amino acid sequence (207 amino acids) of γ-46 gliadin isolated from wheat cultivar Hardi. The molecular mass of the protein (M~r~) estimated by electrospray mass spectrometry is 35191.3. The number of cysteine residues in γ-46 gliadin was determined as a mass difference of the protein before and after reduction and alkylation with 4-vinylpyridine. It was shown that the protein has no free SH-groups, and all cysteine residues are involved in the formation of four disulfide bonds. The partial structure of γ-46 gliadin was determined by N-terminal sequencing and sequencing of tryptic and chymotryptic peptides. The tryptic peptides were obtained by enzymatic hydrolysis of the protein, which was preliminarily reduced and immobilized at free SH-groups on thiopropyl-Sepharose 6B. The chymotryptic peptides were isolated by limited digestion of the native protein. The positions of cysteine residues, as well as surrounding amino acid sequences, are conserved in γ-46 gliadin; this is typical of gliadins.

**Key words:** storage proteins, prolamins, gliadins, cysteine residues, cysteine-containing peptides, mass spectrometry

Wheat gluten proteins, the main constituents of endosperm of dormant grain, belong to a special group of cereal storage proteins called prolamins. These proteins are rich in proline and glutamine (about 70% in wheat); however, they are poor in essential amino acids, lysine in particular. Gluten proteins are extremely heterogeneous. About 50 polypeptides are revealed by two-dimensional electrophoresis of prolamins [1]. They are subdivided into three main groups: the high-molecular-weight prolamins (high-molecular-weight glutenin subunits), S-poor prolamins (ω-gliadins), and S-rich prolamins [2]. The latter group includes gliadins and low-molecular-weight prolamins (low-molecular-weight glutenin subunits). The gliadins are a mixture of monomeric proteins, which are readily soluble in aqueous alcohol solutions. They are further subdivided into α-, β-, γ, and ω-gliadins differing in molecular mass (M~r~ = 31,000-42,000) and amino acid sequences. The low-molecular-weight glu-

tenin subunits (M~r~ = 33,000-44,000) are homologous to gliadins; however, in contrast to gliadins, they are associated with high-molecular-weight glutenin subunits (M~r~ = 64,000-70,000) by disulfide bonds. The high-molecular-weight glutenin subunits have been isolated and well characterized including the determination of complete amino acid sequences deduced from corresponding cDNA or genes (see reviews [3, 4]). Similarly, the complete amino acid sequences of five α- and γ-gliadins have been determined [5-9]. In addition, three γ-gliadins, γ₁ [10], γ₂ and γ₃ [11], were isolated and their N-terminal sequences determined. However, the available data are insufficient, especially for location of disulfide bonds in gliadins, which play a key role in gluten formation and properties. Complete or partial amino acid sequences, particularly around cysteine residues, are necessary for location of disulfide bonds in proteins.

The objective of this work was to determine the partial amino acid sequence of the γ-46 gliadin, which is isolated rather easily in a pure form, for subsequent location of disulfide bonds in this protein.

<footnote>* To whom correspondence should be addressed.</footnote>
## MATERIALS AND METHODS

Trypsin treated with tosylphenylalanine chloromethyl ketone, guanidine hydrochloride, Tris-HCl, dithiothreitol (DTT), and ammonium bicarbonate were obtained from Sigma (USA); 5 M CNBr in acetonitrile was from Aldrich (USA). Chymotrypsin was obtained from Boehringer Mannheim (Germany). Thiopropyl-Sepharose 6B was produced by Pharmacia (Sweden). Trifluoroacetic acid (TFA) and all other reagents and solvents for the sequencer were obtained from Knauer (Germany) and Applied Biosystems (USA). 4-Vinylpyridine was distilled under argon and stored in sealed vials. Deionized water was obtained using the Milli-Q System (Millipore, USA). The ultra-high-quality water for mass spectrometry was from an Elgastat UHQ (Elga, UK). All other chemicals obtained from manufacturers were of analytical grade.

**Protein isolation.** The γ-46 gliadin was isolated from the wheat cultivar Hardi and purified by different chromatographic procedures [12]. The protein was additionally purified by RP-HPLC on a 4.6 × 220 mm Aquapore RP-300 column (C-8, pore diameter 300 nm, particle size 7 µm) with an acetonitrile gradient from 20 to 50% B for 40 min at a flow rate of 0.5 ml/min and room temperature. Solvent A was 0.1% (v/v) TFA solution in water, and solvent B was acetonitrile containing 0.08% (v/v) TFA. Protein fractions were freeze-dried on a SpeedVac (Savant, USA) concentrator.

**Limited hydrolysis of the native γ-46 gliadin with chymotrypsin.** Limited hydrolysis of the intact protein with chymotrypsin was described earlier [13]. For analytical purposes, about 100 µg of lyophilized protein were resuspended in 100 µl of 0.05 M ammonium bicarbonate buffer, pH 8.0, containing 0.002 M $CaCl_2$ and 10% dioxane for complete solubilization of the gliadin. Hydrolysis with chymotrypsin was conducted at an enzyme/substrate ratio of 1:1000 at 20°C. Enzymatic hydrolysis was monitored by SDS-PAGE. Aliquots were taken from the reaction mixture at certain intervals (from 10 min to 22 h) and heated at 95°C in the electrophoretic sample buffer (pH 6.8) for 3 min in the presence or without 2-mercaptoethanol. For preparative purposes, 0.5 mg of protein was digested. The reaction was terminated by the addition of TFA to pH 2.5. The peptides were separated by RP-HPLC.

**Reduction and alkylation of the γ-46 gliadin.** The protein was alkylated with 4-vinylpyridine after reduction according to [14]. About 50 µg of lyophilized protein were dissolved in 40 µl of the alkylating buffer (0.1 M Tris-HCl buffer, pH 7.6, containing 5 mM EDTA and 5 M guanidine hydrochloride). DTT solution (1 µl, 1.4 µmoles/µl) was added to the solution,

and the reaction mixture was incubated under argon at 37°C for 4 h. A 2-µl sample of freshly distilled 4-vinylpyridine was added to the reaction mixture. The reaction was stopped in 10 min by applying the mixture to the Aquapore RP-300 column (4.6 × 100 mm) equilibrated with 20% of solvent B with subsequent desalting of the protein by RP-HPLC. After washing salts and by-products of the reaction, the protein was eluted with an acetonitrile gradient from 20 to 50% B for 30 min. Solvent A was 0.1% (v/v) TFA, and solvent B was acetonitrile in 0.08% (v/v) TFA.

**Selective isolation of tryptic cysteine-containing peptides.** The immobilization and enzymatic hydrolysis of the immobilized γ-46 gliadin was performed according to Egorov et al. [15] with some modifications [16]. About 0.5 mg of protein was reduced and desalted as described above. The protein fraction was immediately dried in a SpeedVac concentrator. Lyophilized protein was dissolved in 0.5 ml of buffer A (0.1 M Tris-HCl, pH 8.0, containing 3 mM EDTA and 5 M guanidine hydrochloride) and added to 0.5 ml of thiopropyl-Sepharose 6B equilibrated with buffer A. The suspension was stirred under argon at room temperature for 6 h using a RK12 Rotamixer (Heto, Denmark) in a 4-ml flat-bottom vial for proper mixing of the suspension. After immobilization, the suspension was transferred to a special column. A tip provided with glass cotton as a filter was used for preparation of such a column. A syringe (2-5 ml) for washing, equilibration, and elution of proteins and peptides was attached to the column. The column was packed, and the eluate was collected. Then the column was washed with five volumes of buffer A. The bulked eluate was desalted by RP-HPLC for determination of protein, which was not bound to the support. The column was equilibrated with 10 volumes of buffer B (0.1 M Tris-HCl, pH 8.0), then the conjugate was transferred to the vial. After Sepharose sedimented, the excess buffer was removed, so that the gel/buffer ratio became 1:2. Trypsin was added to the suspension at an enzyme/substrate ratio of 1:100 (w/w). The suspension was stirred at 37°C for 4 h, as described above. The reaction was stopped by the addition of 10 µl of TFA, then the suspension was transferred to the column. The eluate was collected, and the column was washed with five volumes of buffer B. The bulked eluate (fraction I – noncysteine-containing peptide residues) was kept for further analysis. The column was additionally washed with five volumes of buffer A, the eluate was discarded. Then the column was filled with 0.5 M DTT in buffer A (reducing buffer), and the reaction was allowed to proceed for 30 min at room temperature. The cysteine-containing peptides were eluted with a reducing buffer (fraction II). Fraction II was immediately applied to
an RP-column. Peptides were separated with an acetonitrile gradient after washing excess reagents and by-products of the reaction.

**N-Terminal sequencing.** N-Terminal amino acid sequences of proteins and peptides were determined using a model 816 protein sequencer (Knauer, Germany) equipped with a model 120A PTH Analyzer (Applied Biosystems, USA). Protein and peptide samples were dissolved in 10-20 µl of 30-50% acetonitrile containing 0.1% TFA and applied gradually (by 5 µl) to the Immobilone P membrane (Millipore, USA) for sequencing.

**Determination of M~r~, and the number of cysteine residues in γ-46 gliadin by mass spectrometry.** For determination of the number of cysteine residues, the mass spectra of several samples were measured. They were: 1) intact protein; 2) intact protein alkylated with 4-vinylpyridine; and 3) reduced and alkylated protein. Reduction, alkylation, and desalting of these samples were described above. Mass spectra were recorded using a Vestec model 201 single-quadrupole electrospray mass spectrometer (Vestec Corp., USA) as described earlier [17]. Protein samples were dissolved in 1% (v/v) acetic acid, 50% (v/v) methanol to a concentration of 5-25 pmoles/µl, and 5 µl were infused by a syringe pump (Orion Research, USA) at a flow rate of 0.3 µl/min. Several scans were averaged in order to obtain the best signal-to-noise ratio. The instrument was calibrated independently using the multiply charged envelope of β-lactoglobulin. Plasma desorption spectra of peptides were acquired on a time-of-flight mass spectrometer as described earlier [18].

**RP-HPLC separation of peptides.** Peptides were separated by RP-HPLC on a 4.6 × 220 mm Aquapore RP-300 column (C-8, 300 nm, 7 µm) supplied with a precolumn with an acetonitrile gradient at a flow rate of 0.5 ml/min and room temperature. Solvent A was 0.1% (v/v) TFA, and solvent B was acetonitrile in 0.1% TFA. A Gilson chromatographic system (Gilson, France) consisting of two model 302 pumps, model 812 dynamic mixer, and two model 111B and model UV-1/214 detectors (Pharmacia) for protein detection at 254 and 214 nm, respectively, was used.

**Gel electrophoresis.** SDS-PAGE in a 12.5% gel was performed according to Laemmli [19]. Low-molecular-weight protein kit (Pharmacia) was used as markers. Gels were stained with 0.1% Coomassie R-250 in isopropanol-acetic acid-water (3:1:6).

## RESULTS AND DISCUSSION

Molecular mass (M~r~), the number of cysteine residues, and N-terminal sequence of γ-46 gliadin. To locate disulfide bonds in proteins, it is necessary

first to determine the exact number of cysteine residues and the presence of free sulfhydryl groups. Several procedures for determination of cysteine residues in proteins have been proposed [20]. Alkylation of cysteine after protein reduction with iodoacetamide or 4-vinylpyridine are most common. Of them, alkylation of proteins with 4-vinylpyridine has several advantages, compared to other methods, because the derivative formed—S-β-(4-pyridylethyl)cysteine—is stable to acid hydrolysis and during protein and peptide sequencing by the Edman procedure. In addition, the introduction of a reporter group into cysteine residues allows one to detect effectively cysteine-containing peptides during HPLC separation [21]. We suggested to use mass spectrometry for determination of cysteine residues in proteins by estimating a mass difference before and after alkylation with 4-vinylpyridine [17]. The advantages of mass spectrometry, accuracy and sensitivity, compared to chromatographic methods are evident. First of all, there is no need to subject the protein to acid hydrolysis. The accuracy of chromatographic methods of amino acid analysis does not exceed 3%, while that of mass spectrometric determination of cysteine residues is much higher: it averaged 0.4% for avenins (M~r~ = 20200-28700). Mass spectrometry was used in this work for determination of the number of cysteine residues in γ-46 gliadin. The molecular mass M~r~ of γ-46 gliadin estimated by mass spectrometry was 35191.3. After alkylation of the intact protein with 4-vinylpyridine, the mass of the protein was 35192.6, namely it changed by 1.3. However, after reduction and alkylation, the mass of the protein increased by 855.5 and became equal to 36046.8 that corresponds to the mass of 8.06 pyridylethyl groups (M~r~ = 106.15). Thus, we determined M~r~ of γ-46 gliadin and the number of cysteine residues in this protein. We also showed that the protein has no free sulfhydryl groups, and all eight cysteine residues are involved in four disulfide bonds.

By sequencing of the reduced and alkylated γ-46 gliadin, we determined its N-terminal amino acid sequence (29 amino acid residues). It was identical to the N-terminal sequences of the clone pW1621 and γ-2 gliadins; however, it differed in several amino acids from other gliadins (Table 1).

**Cysteine-containing tryptic peptides of γ-46 gliadin.** By tryptic digestion of γ-46 gliadin immobilized on thiopropyl-Sepharose 6B, we obtained two fractions: the first fraction consisted of cysteine-containing peptides (fraction I), and the second one (II) consisted of noncysteine-containing peptides (this fraction was not analyzed in detail in this work). The separation of fraction I peptides by RP-HPLC is shown in Fig. 1. Complete or partial structures of γ-46 gliadin tryptic peptides (T1-T7) are given in Table 2. It is seen from
<table><caption>**Table 1.** N-Terminal amino acid sequences, the number of cysteine residues, and $M_r$ of gliadins</caption><thead><tr><th>Protein</th><th>N-Terminal amino acid sequence*</th><th>Number of cysteine residues</th><th><i>M</i><sub>r</sub>*** (calculated)</th><th>Reference</th></tr></thead><tbody><tr><td>Gliadin γ-46</td><td>NIQVDPSGQVQWLQQQLVPQLQQPLSQQP</td><td>8</td><td>35199.5 (measured)</td><td>this work</td></tr><tr><td>Clone pW1621</td><td>NIQVDPSGQVQWLQQQLVPQLQQPLSQQP</td><td>8**</td><td>35159.7</td><td>[8]</td></tr><tr><td>Gliadin γ-1</td><td>NIQVDPSGQVQWPQQQPFPQ</td><td>—</td><td>—</td><td>[10]</td></tr><tr><td>Gliadin γ-2</td><td>NIQVDPSGQVQWLQQQLVPQ</td><td>—</td><td>—</td><td>[11]</td></tr><tr><td>Gliadin γ-3</td><td>NMQVDPSGQVQWPQQQLVPQPQQPLQQQQ</td><td>—</td><td>—</td><td>[11]</td></tr><tr><td>Gene A clone</td><td>NMQVDPSDQVQWPQQQPVLLPQQPFSQQP</td><td>8</td><td>35328.7</td><td>[7]</td></tr><tr><td>Clone pW1020</td><td>NMQVDPSGQVQWPQQQPFPQPQQPFSQQP</td><td>9</td><td>32318.4</td><td>[9]</td></tr><tr><td>Clone pTag1436</td><td>NMQVDPSSQVQWPQQQPVPQPHQPFSQQP</td><td>8</td><td>31610.7</td><td>[6]</td></tr><tr><td>Gene B clone</td><td>NMQADPSGQVQWPQQQPFLQPHQPFSQQP</td><td>8</td><td>30974.1</td><td>[7]</td></tr></tbody></table>

* Amino acids residues different from those in the γ-46 gliadin are given in bold.

** In [8], seven cysteine residues were determined. Later eight cysteine residues were identified (S. Masci, personal communication).

*** In calculations of $M_r$ (except γ-46 gliadin), disulfide bonds were not taken into consideration (dash, not determined).

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** RP-HPLC separation of the γ-46 gliadin cysteine-containing peptides with an acetonitrile gradient: 5-40% B for 60 min and 40-60% B for 20 min at a flow rate of 0.5 ml/min.</figcaption></figure>
<table><caption>**Table 2.** Tryptic peptides of γ-46 gliadin</caption><thead><tr><th>Position*</th><th>Peptide</th><th>Amino acid sequence**</th><th>M<sub>r</sub><br>(calculated)</th><th>M<sub>r</sub><br>(measured)<br>of an intact<br>peptide</th><th>M<sub>r</sub><br>(measured)<br>of an<br>alkylated<br>peptide</th><th>Δu*** and<br>the number<br>of cysteine<br>residues (in<br>parentheses)</th></tr></thead><tbody><tr><td>1-151</td><td>T1</td><td>NIQVDPSGQVQWLQQQLVPQL...R/K</td><td>17825.6</td><td>—</td><td>—</td><td>—</td></tr><tr><td>152-166</td><td>T2</td><td>PFIQPSLQQQLNPCK</td><td>1740.0</td><td>1741.3</td><td>1847.9</td><td>106.6 (1.03)</td></tr><tr><td>167-174</td><td>T3</td><td>NILLQQCK</td><td>958.2</td><td>—</td><td>—</td><td>—</td></tr><tr><td>167-196</td><td>T3+T4</td><td>NILLQQCKPASLVSSLWSIIWPQSDCQVMR****</td><td>3443.1</td><td>3442.3</td><td>3653.2</td><td>210.9 (1.99)</td></tr><tr><td>197-272</td><td>T5</td><td>QQCCOQLAQIPQQLOCAAIHSVVHSL...R/K</td><td>8474.6</td><td>—</td><td>—</td><td>(3)*****</td></tr><tr><td>273-295</td><td>T6</td><td>SLVLQTLPSMCNVYVPPECSIMR</td><td>2579.1</td><td>2581.8</td><td>2790.1</td><td>208.3 (1.96)</td></tr><tr><td>296-308</td><td>T7</td><td>APFASIVAGIGGO</td><td>1187.4</td><td>1188.3</td><td>1187.1</td><td>1.2 (0)</td></tr></tbody></table>

* The position of peptides is given as in the clone pW1621 gliadin [8].

** Amino acid sequences determined by sequencing are underlined.

*** Δu, the difference between M, of alkylated and nonalkylated peptides. The number of cysteine residues is calculated by dividing Δu by M, of a pyridylethyl group (106.15).

**** C-Terminal sequence of peptide T3 + T4 (SII...R) coincides with the structure of peptide C10-2 (Fig. 3).

***** Determined from the sequence of peptide T5.

these data that all eight cysteine residues are found in peptides T2-T6, and the isolation of cysteine-containing peptides was rather selective. However, since the cleavage of Arg/Lys-Pro bonds by trypsin is never complete, the peptide fragment appeared in fraction I, which consisted of T1 and T2 peptides, as judged by N-terminal sequencing (a cysteine-containing fragment). The appearance of a peptide fragment consisting of peptides T3 and T4, which was formed during incom-

plete hydrolysis of the Arg-151 (or Lys-151)-Pro-152 bond (amino acids are numbered as in the clone pW1621 gliadin), may be explained by the same reason. The peptides T1 (N-terminal) and T7 (C-terminal) free of cysteine residues were isolated by HPLC of fraction II (data not shown). Accordingly, all eight cysteine residues in γ-46 gliadin were located and the surrounding amino acid sequences determined.

**Limited hydrolysis of γ-46 gliadin with chymotrypsin.** Figure 2 shows electrophoretic separation of the reaction products formed during limited hydrolysis with chymotrypsin. It is seen from this figure, that after 10 min, rapid degradation of the intact protein and accumulation of the degradation products occur. For preparative experiments, the protein was incubated with chymotrypsin for 22 h, when the original protein completely disappeared. The peptides were separated by RP-HPLC (Fig. 3). Five main fractions (2, 7, 8, 10, and 11) were obtained; of them, three fractions (2, 7, and 8) containing peptides C1, C2, and C2', respectively, were the fragments of the N-terminal region, as judged by N-terminal sequencing, and did not contain cysteine residues (Fig. 3). Peptides C2 and C2' had identical N-terminal sequences, but probably differed in length. Direct sequencing of fractions 10 and 11 demonstrated that they consisted of three disulfide-containing fragments. This was easily shown due to the unique amino acid composition of gliadins. The analysis of these fractions indicated that they consisted of the same three disulfide-bonded fragments. However, fraction 10 was more pure and, therefore, this fraction was used for

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** SDS-PAGE of the products of limited hydrolysis of γ-46 gliadin with chymotrypsin (dynamics): 1) 10; 2) 20 min; 3) 1; 4) 4; 5) 22 h. The samples for electrophoresis were not reduced; 6, 7) fractions 10 and 11 obtained by HPLC-separation of the products of 22-h hydrolysis (Fig. 3); 8) protein markers: phosphorylase b (94 kD), BSA (67 kD), ovalbumin (45 kD), carboanhydrase (30 kD), trypsin inhibitor (20 kD), α-lactalbumin (14 kD). Molecular masses of protein markers are shown on the right.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 3. RP-HPLC separation (90 min) of the products of limited 22-h hydrolysis of γ-46 gliadin with an acetonitrile gradient (0-50% B) at a flow rate of 0.5 ml/min.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 4. Partial amino acid sequence of γ-46 gliadin (lower line) in comparison with the clone pW1621 γ-gliadin [8]. Variant amino acid sequences in the compared proteins are shown in bold. Cysteine residues are given in boxes.</figcaption></figure>
further analysis. Fraction 10 was reduced and alkylated with 4-vinylpyridine, then the mixture was separated by RP-HPLC (data not shown). As a result, three cysteine-containing peptides were obtained (C10-1, C10-2, and C10-3), their partial structure is shown in Fig. 4. Thus, fraction 10 containing disulfide-bonded fragments may be used in future for determination of disulfide bridges in γ-46 gliadin.

**Partial amino acid sequence of γ-46 gliadin.** In this work, we determined $M_r$ and the partial amino acid sequence of γ-46 (207 amino acid residues) (Fig. 4) and showed that the protein has no free sulfhydryl groups, and all eight cysteine residues are involved in four disulfide bonds. The comparison of the available data on $M_r$, N-terminal sequences, the number of cysteine residues, and the structure of peptides with amino acid sequences of the γ-type gliadins provides evidence that γ-46 gliadin is closer to the γ-gliadins, whose primary structure was deduced from the nucleotide sequence of the pW1621 clone (308 amino acid residues) [8]. The differences between the two proteins are insignificant. Only three amino acid residues were variable in the sequences studied. The position of cysteine residues in both proteins, as well as amino acid sequences around them, are conserved; this is typical of S-rich prolamins [3, 4].

This work was supported in part by the Russian Foundation for Basic Research (grant No. 96-04-49850) and the Danish Committee for Science and Technology.

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<figure><img src="image_7.png" /></figure>