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The amino acid sequence of the ‘Fast’ avenin component (Avena sativa L.)
Journal of Cereal Science 8 (\988) 289-292
The Amino Acid Sequence of the 'Fast' Avenin Component (Avena sativa L.)* Ts. A. EGOROV N. 1. Vavilov Institute of General Genetics, Academy of Sciences of the USSR, Uf. Gubkina 3, Moscow B-333, USSR Received 15 June 1988
Remarkable progress has been made recently in elucidating the structure of prolamins. The amino acid sequences ofa number of prolamins, which were obtained by sequencing of the corresponding cDNAs and genes, are known 1 . However, little is known about the structure of oat prolamins (avenins). There are only a few papers concerning the isolation, characterization and N-terminal amino acid sequence analysis of some of the avenins 2 - 4 • Their simple compositions and low Mrs make avenins convenient for the study of the physico-chemical properties of prolamins. Thcy are also of interest in relation to the structure and evolution of this group of proteins. In the present communication I report the first complete protein sequence of one of the avenin components (designated N9), which migrates faster than the others (except one minor component) when examined by SL-PAGE and which is present in the majority of oat varieties. Total avenins were isolated from the oat variety Narymsky 943. Following extraction with 70 % (vjv) ethanol and precipitation of the protein with 4 % (wjv) NaCI, the residue was washed with water and acetone. The dried residue was redissolved in 5 M guanidinium chloride and desalted by RP-HPLC. This step is extremely important for the subsequent separation of avenins by ion-exchange chromatography. The protein fraction was dried on a rotary evaporator, redissolved in 70 % (v jv) ethanol containing 0·1 % (vjv) TFA and injected on to a Mono S 5j5 or a Mono S 10j10 column (Pharrnacia, Uppsala) for analytical or preparative separations respectively. Proteins were eluted with a salt gradient in urea at pH 3·5. Peak VII comprised a single N9 component as determined by SL-PAGE (results not shown) and SDS-PAGE analysis (Fig. I). The avenin structure was determined as follows. First, use of N-terminal sequence analysis of reduced and vinylpyridylated protein made it possible to identify 47 amino acid residues. The alkylated avenin was then subjected to tryptic digestion. It is well known that prolamins are difficult to digest with enzymes in aqueous solution, but when
* Presented in part at the Third International Workshop on Gluten Proteins, 6-9 May, 1987. Budapest, Hungary. Abbreviations used: SL-PAGE = sodium lactate polyacrylamide gel electrophoresis; SDS-PAGE = sodium dodecyl sulphate polyacrylamide gel electrophoresis RP-HPLC = reversed-phase high-performance liquid chromatography; TFA = trifluoroacetic acid; T = tryptic peptides; CB = cyanogen bromide peptides. 0733-5210/88/060289+04
$03.00/0
© I988 Academic Press Limited
Ts. A. EGOROV
290
, a 10-3
n
Ano
94 -I
67
-z
43
-3 -4 -5
Io<>z A.U
30
-6
...
-7
-0
ffI
-9
Zo-t
,,,...
-10 20
40
E~hoo
tlllla (mol
60
10\
(b)
(el
Figure 1. lon-exchange high-performance liquid chromatography of total alcoholsoluble avenin on a Mono S 5/5 column (Pharmacia, Uppsala). Proteins were eluted with a linear gradient from 0 to 100 % B over a 120 min period at 1·0 ml/min using an FPLC System (Pharmacia, Uppsala). Solvent A, 4 M urea pH 3'5 (adjusted with TFA); solvent B, solvent A+0·2 M NaCI. On the right: (a) SL-PAGE (pH 3'1) of alcohol-soluble avenin; the components are numbered in order of increasing electrophoretic mobility; (b) SDS-PAGE (pH 3·1) of the peak VII (avenin N9); (c) protein relative molecular mass (M r) standards (Pharmacia, Uppsala).
they are immobilized enzymic hydrolysis becomes easy. The general procedure for the immobilization of gliadins on Thiopropyl-Sepharose 6B (Pharmacia, Uppsala) and the enzymic hydrolysis of the immobilized proteins has been published previously6, 7. This method seems to be convenient for studying various prolamins and will be described in detail in a future paper. The peptides were recovered, purified by RP-HPLC and sequenced partially (TI, 38 amino add residues) or completely, including one peptide (T7) consisting of 52 residues. To obtain overlapping peptides the reduced and alkylated avenin was cleaved with cyanogen bromide (CNBr). As a result all four expected fragments were recovered and sequenced completely (CB3, CB4) or partially (CBl, 36 residues; CB2, 51 residues). To complete the sequencing of the fragment CB2 it was digested with trypsin. All the peptides generated were isolated and characterized, including the C-terminal peptide: The protein and peptides were sequenced on a model 470A gas-phase protein sequencer equipped with model 120A PTH analyzer (Applied Biosystems, Foster City, CA). Thus the sequence analysis of tryptic and CNBr peptides, as well as determination
AVENIN PRIMARY STRUCTURE 10
291
20
30
40
T T T V Q Y N P SEQ Y Q P Y P E Q Q E P F V Q Q Q P F V Q Q Q Q Q P f V Q Q Q I· TI I" CBI - - - - - - - - - - - - - 50
70
60
80
Q MIF L Q P L L Q Q Q L N P C K Q f L V Q Q C S P V Alv V P F L R S Q I L R Q A -I" T2 0-1- T3---1---
4------------
-+1....
90
CB2 - - - - - - - - - - - - - 100
110
120
I C Q V AIR Q Q C C R Q L A Q I P E Q L R CPA I H S V V Q A I I L Q Q Q Q Q Q
-
T4 - t - - T5
-I'
T6
.. I •
T7
- - - - - - - - - - - - - - CB2 - - - - - - - - - - - - - 130
140
150
160
"§lFFQPQLQQQVFQPQ L Q Q V F N Q P Q Q Q A Q FIE G M R A FA L Q A L - - - - - - - - - - - - T7 • I' T8 - - - - - - - - - - - - - CB2 -I" CB3-170
180
P-A-M-C-D-V-Y-V-P-P-Q-c-p~vIATAPLGGF T8 • I CB4
-I
FIGURE 2. The amino acid sequence of the' fast' avenin component (avenin N9). Arrows show the alignment of tryptic and CNBr peptides. Conserved regions A (residues 43-67), B (86-121) and C (150-174) are in boxes; repetitive sequences are underlined by double lines. The single-letter code for amino acids is used.
of N-terminal amino acid sequence of the whole protein, made it possible to reconstruct the complete primary structure of avenin N9 (Fig. 2). Avenin N9 consists of 182 amino acid residues. Its calculated M r of 21000 is in good agreement with its amino acid composition (data not shown) and mobility on SDS-PAGE (Fig. I). Its amino acid composition and sequence are characteristic of prolamins. It contains 30·7 mol % of glutamine, 11·5 mol % proline, 4 mol % cysteine, 1·6 mol % methionine and 60 mol % hydrophobic amino acids. It has a unique N-terminal amino acid sequence (residues 1-10) and contains three conserved regions: A (residues 43-67), B (residues 86-121) and C (residues 150-174), which are present in all the S-rich and HMW prolamins that have been studied so farl,5. Unlike the S-rich (1.gliadins of wheat, it does not contain polyglutamine regions. It has three sequence repeats of the type PFVQll' where n = 3, 4 or 5. The author thanks A. N. Musolyamov for his technical assistance.
References 1. Kreis, M., Shewry, P. R., Forde, B. G., Forde, J. and Millin, B. J. in 'Oxford Surveys of Plant Molecular and Cell Biology' Vol. 2 (B. J. Miflin, ed.), Oxford University Press, Oxford (1985) pp 253-317. 2. Kim, S. I., Charbonnier, L. and Mosse, J. Biochim. Biophys. Acta 537 (1978) 22-30.
292
Ts. A. EGOROV
3. Robert, L. S., Nozzolio, C. and Altosaar, I. Cereal Chem. 60 (1983) 438--442. 4. Pernollet, I.-C., Huet, J.-c., Galle A-M and SaUantin, M. Biochimie 69 (1987) 683-690. 5. Shewry, P. R., Tatham, A. S., Forde, J., Kreis, M. and Miflin, B. J. J. Cereal Sci. 4 (1986) 97-106. 6. Egorov, Ts. A, Odintsova, T. I. and Sozinov, A A Bioorganicheskaya Khimiya 12 (1986) 595--605. 7. Egorov, Ts. A., Svenson, A., Ryden, L. and Carlsson, J. Proc. Nat. Acad. Sci. U.S.A. 72 (1985) 3029-3033.
The Amino Acid Sequence of the 'Fast' Avenin Component (Avena sativa L.)* Ts. A. EGOROV N. 1. Vavilov Institute of General Genetics, Academy of Sciences of the USSR, Uf. Gubkina 3, Moscow B-333, USSR Received 15 June 1988
Remarkable progress has been made recently in elucidating the structure of prolamins. The amino acid sequences ofa number of prolamins, which were obtained by sequencing of the corresponding cDNAs and genes, are known 1 . However, little is known about the structure of oat prolamins (avenins). There are only a few papers concerning the isolation, characterization and N-terminal amino acid sequence analysis of some of the avenins 2 - 4 • Their simple compositions and low Mrs make avenins convenient for the study of the physico-chemical properties of prolamins. Thcy are also of interest in relation to the structure and evolution of this group of proteins. In the present communication I report the first complete protein sequence of one of the avenin components (designated N9), which migrates faster than the others (except one minor component) when examined by SL-PAGE and which is present in the majority of oat varieties. Total avenins were isolated from the oat variety Narymsky 943. Following extraction with 70 % (vjv) ethanol and precipitation of the protein with 4 % (wjv) NaCI, the residue was washed with water and acetone. The dried residue was redissolved in 5 M guanidinium chloride and desalted by RP-HPLC. This step is extremely important for the subsequent separation of avenins by ion-exchange chromatography. The protein fraction was dried on a rotary evaporator, redissolved in 70 % (v jv) ethanol containing 0·1 % (vjv) TFA and injected on to a Mono S 5j5 or a Mono S 10j10 column (Pharrnacia, Uppsala) for analytical or preparative separations respectively. Proteins were eluted with a salt gradient in urea at pH 3·5. Peak VII comprised a single N9 component as determined by SL-PAGE (results not shown) and SDS-PAGE analysis (Fig. I). The avenin structure was determined as follows. First, use of N-terminal sequence analysis of reduced and vinylpyridylated protein made it possible to identify 47 amino acid residues. The alkylated avenin was then subjected to tryptic digestion. It is well known that prolamins are difficult to digest with enzymes in aqueous solution, but when
* Presented in part at the Third International Workshop on Gluten Proteins, 6-9 May, 1987. Budapest, Hungary. Abbreviations used: SL-PAGE = sodium lactate polyacrylamide gel electrophoresis; SDS-PAGE = sodium dodecyl sulphate polyacrylamide gel electrophoresis RP-HPLC = reversed-phase high-performance liquid chromatography; TFA = trifluoroacetic acid; T = tryptic peptides; CB = cyanogen bromide peptides. 0733-5210/88/060289+04
$03.00/0
© I988 Academic Press Limited
Ts. A. EGOROV
290
, a 10-3
n
Ano
94 -I
67
-z
43
-3 -4 -5
Io<>z A.U
30
-6
...
-7
-0
ffI
-9
Zo-t
,,,...
-10 20
40
E~hoo
tlllla (mol
60
10\
(b)
(el
Figure 1. lon-exchange high-performance liquid chromatography of total alcoholsoluble avenin on a Mono S 5/5 column (Pharmacia, Uppsala). Proteins were eluted with a linear gradient from 0 to 100 % B over a 120 min period at 1·0 ml/min using an FPLC System (Pharmacia, Uppsala). Solvent A, 4 M urea pH 3'5 (adjusted with TFA); solvent B, solvent A+0·2 M NaCI. On the right: (a) SL-PAGE (pH 3'1) of alcohol-soluble avenin; the components are numbered in order of increasing electrophoretic mobility; (b) SDS-PAGE (pH 3·1) of the peak VII (avenin N9); (c) protein relative molecular mass (M r) standards (Pharmacia, Uppsala).
they are immobilized enzymic hydrolysis becomes easy. The general procedure for the immobilization of gliadins on Thiopropyl-Sepharose 6B (Pharmacia, Uppsala) and the enzymic hydrolysis of the immobilized proteins has been published previously6, 7. This method seems to be convenient for studying various prolamins and will be described in detail in a future paper. The peptides were recovered, purified by RP-HPLC and sequenced partially (TI, 38 amino add residues) or completely, including one peptide (T7) consisting of 52 residues. To obtain overlapping peptides the reduced and alkylated avenin was cleaved with cyanogen bromide (CNBr). As a result all four expected fragments were recovered and sequenced completely (CB3, CB4) or partially (CBl, 36 residues; CB2, 51 residues). To complete the sequencing of the fragment CB2 it was digested with trypsin. All the peptides generated were isolated and characterized, including the C-terminal peptide: The protein and peptides were sequenced on a model 470A gas-phase protein sequencer equipped with model 120A PTH analyzer (Applied Biosystems, Foster City, CA). Thus the sequence analysis of tryptic and CNBr peptides, as well as determination
AVENIN PRIMARY STRUCTURE 10
291
20
30
40
T T T V Q Y N P SEQ Y Q P Y P E Q Q E P F V Q Q Q P F V Q Q Q Q Q P f V Q Q Q I· TI I" CBI - - - - - - - - - - - - - 50
70
60
80
Q MIF L Q P L L Q Q Q L N P C K Q f L V Q Q C S P V Alv V P F L R S Q I L R Q A -I" T2 0-1- T3---1---
4------------
-+1....
90
CB2 - - - - - - - - - - - - - 100
110
120
I C Q V AIR Q Q C C R Q L A Q I P E Q L R CPA I H S V V Q A I I L Q Q Q Q Q Q
-
T4 - t - - T5
-I'
T6
.. I •
T7
- - - - - - - - - - - - - - CB2 - - - - - - - - - - - - - 130
140
150
160
"§lFFQPQLQQQVFQPQ L Q Q V F N Q P Q Q Q A Q FIE G M R A FA L Q A L - - - - - - - - - - - - T7 • I' T8 - - - - - - - - - - - - - CB2 -I" CB3-170
180
P-A-M-C-D-V-Y-V-P-P-Q-c-p~vIATAPLGGF T8 • I CB4
-I
FIGURE 2. The amino acid sequence of the' fast' avenin component (avenin N9). Arrows show the alignment of tryptic and CNBr peptides. Conserved regions A (residues 43-67), B (86-121) and C (150-174) are in boxes; repetitive sequences are underlined by double lines. The single-letter code for amino acids is used.
of N-terminal amino acid sequence of the whole protein, made it possible to reconstruct the complete primary structure of avenin N9 (Fig. 2). Avenin N9 consists of 182 amino acid residues. Its calculated M r of 21000 is in good agreement with its amino acid composition (data not shown) and mobility on SDS-PAGE (Fig. I). Its amino acid composition and sequence are characteristic of prolamins. It contains 30·7 mol % of glutamine, 11·5 mol % proline, 4 mol % cysteine, 1·6 mol % methionine and 60 mol % hydrophobic amino acids. It has a unique N-terminal amino acid sequence (residues 1-10) and contains three conserved regions: A (residues 43-67), B (residues 86-121) and C (residues 150-174), which are present in all the S-rich and HMW prolamins that have been studied so farl,5. Unlike the S-rich (1.gliadins of wheat, it does not contain polyglutamine regions. It has three sequence repeats of the type PFVQll' where n = 3, 4 or 5. The author thanks A. N. Musolyamov for his technical assistance.
References 1. Kreis, M., Shewry, P. R., Forde, B. G., Forde, J. and Millin, B. J. in 'Oxford Surveys of Plant Molecular and Cell Biology' Vol. 2 (B. J. Miflin, ed.), Oxford University Press, Oxford (1985) pp 253-317. 2. Kim, S. I., Charbonnier, L. and Mosse, J. Biochim. Biophys. Acta 537 (1978) 22-30.
292
Ts. A. EGOROV
3. Robert, L. S., Nozzolio, C. and Altosaar, I. Cereal Chem. 60 (1983) 438--442. 4. Pernollet, I.-C., Huet, J.-c., Galle A-M and SaUantin, M. Biochimie 69 (1987) 683-690. 5. Shewry, P. R., Tatham, A. S., Forde, J., Kreis, M. and Miflin, B. J. J. Cereal Sci. 4 (1986) 97-106. 6. Egorov, Ts. A, Odintsova, T. I. and Sozinov, A A Bioorganicheskaya Khimiya 12 (1986) 595--605. 7. Egorov, Ts. A., Svenson, A., Ryden, L. and Carlsson, J. Proc. Nat. Acad. Sci. U.S.A. 72 (1985) 3029-3033.