MolecularImmunology,Vol. 28, No. l/2, pp. 177-182, 1991 Printedin Great Britain.
0161~5890/91 $3.00 + 0.00 Pergamon Pressplc
AMINO ACID SEQUENCES AND STRUCTURES OF CHICKEN AND TURKEY BETA2-MICROGLOBULIN KAREN G. WELINDER,* HANS M. JESPERSEN,JAN WALTHER-RASMUSSEN
and Institute of Biochemical Copenhagen K, Denmark
KAR~TEN SKJCYDT~
Genetics, University of Copenhagen, 0. Farimagsgade and TInstitute for Experimental Immunology, University Nsrre Alle 71, DK-2100, Copenhagen 0, Denmark
2A, DK-1353, of Copenhagen,
(Received 11 June 1990; accepted 23 July 1990) Abstract-The complete amino acid sequences of chicken and turkey beta2-microglobulins have been determined by analyses of tryptic, VS-proteolytic and cyanogen bromide fragments, and by N-terminal sequencing. Mass spectrometric analysis of chicken beta2-microglobulin supports the sequence-derived M, of 11,048. The higher apparent M, obtained for the avian beta2-microglobulins as compared to human beta2-microglobulin by SDS-PAGE is not understood. Chicken and turkey beta2-microglobulin consist of 98 residues and deviate at seven positions: 60, 66, 74-76, 78 and 82. The chicken and turkey sequences are identical to human beta2-microglobulin at 46 and 47 positions, respectively, and to bovine beta2-microglobulin at 47 positions, i.e. there is about 47% identity between avian and mammalian beta2-microglobulins. The known X-ray crystallographic structures of bovine beta2-microglobulin and human HLA-A2 complex suggest that the seven chicken to turkey differences are exposed to solvent in the avian MHC class I complex. The key residues of betaZmicroglobulin involved in alpha chain contacts within the MHC class I molecule are highly conserved between chicken and man. This explains that heterologous human beta2-microglobulin can substitute the chicken beta2-microglobulin in exchange studies with B-F (chicken MHC class I molecule), and suggests that the MHC class I structure is conserved over long evolutionary distances.
INTRODUCTION
is the small subunit of et al., 1973). It is presumably essential for expression of the large subunit on the cell surface. MHC class I molecules are expressed on the surface of most nucleated cell types and are in dynamic equilibrium with free b2m (Kimura et al., 1983), which is present in many physiological fluids. B2m from a variety of animals is about 11,000 in size and can be isolated from blood plasma for structural studies. The evolution of the structure of the MHC class I molecule, which is central to the cellular immune defence, is important for our understanding of the evolution of the immune system in general. Amino acid and DNA sequences have been derived for a great number of both types of subunits, in particular from mammals (Kabat et al., 1987). More recently the crystal structures of bovine b2m (Becker and Reeke, 1985) and human HLA-A2 and HLA-Aw68 (Bjorkman er al., 1987; Garret et al., 1989) have been solved. We have previously purified and characterized b2m from chicken and turkey (Skjerdt et al., 1986). Here we document the complete amino acid Beta2-microglobulin
MHC
class
(b2m)
I molecules
(Grey
sequences and discuss the results in terms of physicochemical properties and conserved MHC class I structure. MATERIALS AND METHODS
Purification of b2m
Chicken and turkey b2m were purified by monoclonal antibody affinity chromatography as previously described by Skjsdt et al. (1986), and desalted by RP-HPLC on a C4 column using a gradient of acetonitrile in 0.1% TFA. Reduction and S-carboxymethylation
B2m and disulphide-linked peptides were reduced by dithiotreitol and S-carboxymethylated by iodoacetate using standard procedures in the presence of 2% SDS as a denaturant. The use of SDS was discontinued, however, as basic groups in proteins and peptides bind SDS which retards and broadens peaks during RP-HPLC. Proteolytic cleavages
Ten nmole of a solution of desalted, native b2m was concentrated by vacuum centrifugation and neutralized with 1 M ammonium bicarbonate to a final concn of 200 mM, pH 8.1. The solution was boiled for 2-10 min to unfold the protein and chilled quickly to 20°C. TPCK-trypsin (2.4 ~1 0.1% trypsin
*To whom correspondence should be addressed. tPresent address: Institute of Medical Microbiology, University of Odense, Winslrawsvej 19, 5000 Odense C, Denmark. 177
K. G. WELINDER et al.
178
in 10 mM CaCl,) was added immediately and the reaction left for 20 hr at room temp. Digestions were stopped with 8 ~1 10% TFA and the sample diluted with an equal volume of 0.1% TFA prior to RP-HPLC. Digestion of desalted, reduced and S-carboxymethylated b2m with V8 protease was carried out similarly but in sodium phosphate buffer as described by Welinder (1988).
A227nm -T7
b2m
A. T&T9
Amino acid and sequence analyses Amino acid analyses were carried out on an instrument based on ion exchange chromatography and post-column oxidation (to analyse proline) and derivatization with ortophthaldialdehyde. The instrument and hydrolysis in gaseous or liquid acid are described in detail by Barkholt and Jensen (1989). Automatic sequencing was carried out either on a gasphase sequencer (Applied Biosystems, model 470A) followed by PTH (phenylthiohydantoin) analyses on a Waters HPLC system using a Spherisorb ODS II column developed by a methanol-ethanol gradient, or on an Applied Biosystems model 477A sequencer with on-line HPLC analysis of PTH-derivatives. Mass spectroscopy Accurate molecular mass determinations of native and reduced and S-carboxymethylated chicken b2m were carried out on 100 pmol of salt-free samples. A plasma desorption, time-of-flight mass spectrometer (Bio-Ion 20) was used. RESULTS
Tryptic peptides Experiments on b2m from chicken and turkey were run in parallel. Tryptic digestion of native b2m, preheated at 100°C for 2min, generated nearly the number of peptides expected from the amino acid composition (Skjsdt et al., 1986). The tryptic peptide maps (Fig. 1) show a peak of undigested b2m, indicating that 2 min of preheating is insufficient to Table
I. Amino Tl
Asx Thr Ser GlX Pro GUY Ala CYS Val Met Ile Lcu Tyr Phe His Lys Arg TOP Total
1.0(l) 1.0 (1) 0.9 (1)
T2
T3
0.2 1.3 (1) 1.0 (I) 0.3 0.8 0.8
0.2 0.9 (1) 1.1 (1) 0.3 0.9 (1) 1.4(l) I .9 (2)
1.0(l)
0.2
5
3.3 (3) 2.0 (2) 3.0 (3) 3.1 (3) 2.3 (2) 4.2 (4) 1.7 (2) 1.4 (2)
0.3 1.0(l)
1.0(l)
1.6 (2) 0.9 (1) 2.9 (3) \-, I .8 (2) 1.9 (2)
6
8
31
0.6(l) 0.2 (I)
T4-Tl
0.8 (I)
T5 1.0 (1) 1.0(l)
0.3
0.8(l) I .8 (2) 1.0(l)
T6 4.4 (5) 0.9 (1) 2.1 (2) 3.3 (3) 1.0(l) 2.3 (2) 1.2 (1) 1.0(l) 0.6 (2)
0.6 (1) 1.8 (2) 1.0(l)
7
0.2 1.0 (1) (1) 23
II
TIO
T5 T7
I
I
I
20
I
I
10
30
40
50
min.
Fig. 1. RP-HPLC of tryptic peptides from native b2m, (A) chicken and (B) turkey. A Cl8 column, 4.6 x 120 mm, was eluted by a linear gradient from &70% methanol in 0.1% TFA at a flow rate of 1 ml/min at room temperature. Fractions were collected manually. The peptides are numbered sequentially from the N-terminus (see Fig. 2). T8-T9 has an uncleaved Lys-Glu bond and T&T7 is disulphide
linked. The last peak constitutes undigested b2m. unfold b2m. Five min of preheating at 100°C gave complete tryptic digestion (not shown). Comparison of the HPLC profiles of tryptic peptides of chicken and turkey b2m shows that only the disulphide linked T4-T7 peptides from the two species have very different retention times. Amino acid sequence analysis (Fig. 2) verifies this difference and shows that the T7 peptides differ at 5 positions. Furthermore, sequence analyses reveal that the similar T6 peptides have a Thr to Ser substitution and T8-T9 a Glu to Asp substitution. The results of amino acid analyses (Table 1) and sequence analyses of chicken b2m (Fig. 2) are in
acid analyses of peptides derived from chicken b2m. Results are uncorrected compositions are shown in brackets
1.8 (2)
I.1
T4-T7 T6
T&T9 0.9 (1) 0.2 4.0 (4) 1.0 (1) 0.4
TlO
CmVl-V2
1.0(l)
9.4 (9) 5.9 (6) 7.7 (8) 5.8 (5) 6.2 (6) 5.1 (5) 7.1 (7) 0.5 (2) 5.6 (6) 2.6 (3) 1.9 12) 4.0 ;4j 2.6 (3) 5.9 (6) 2.1 (2) 5.0 (5) 1.9 (2) (1) 82
1.1 (1) 1.0(l)
1.8 (2)
1.0 (1) 0.7 (I) 1.0(l) 1.1 (1) I .9 (2)
13
from 20 hr hydrolysates.
0.2 (1) 5
CmV2 4.6 (5) 2.7 (3) 4.3 (4-5) 4.2 (3) 1.3 (1) 2.5 (2-3) 2.5 (3) 0.5 (1) 1.8 (2) 0.7 (1) 1.4(l) 1.8 (2) 2.7 (3) 1.1 (I) 1.4(l) 1.0(l) (1) 36
The sequence derived v3
v4
0.4
1.1(l) 1.0(l)
1.1 (1)
3.1 (3) 2.0 (2)
1.0(l)
1.0(l) 0.9(l) 0.9 (1) 0.9(l) 2.0 (2)
2
(1) 14
Structure of chicken and turkey b2m CHICKEN
CH
b2m
b2m
I 0
7 0
3 0
* 0
55555555543444344344444
Trypsln
< ,5 >< ddddddddd555455533333333331121 Cm14 > 55555555555555 CmVl-W2
0
a
0
76
R
CC)
>< T7-T4 ddddddddddddddn < ClllT7 44344434334133333
>< cmu2 555555535444233434444443433222332222
:444334443244423333233323
9 b
a
0
0
0
>< T&T9 > 4554555554432355553 > V3 >
0161~5890/91 $3.00 + 0.00 Pergamon Pressplc
AMINO ACID SEQUENCES AND STRUCTURES OF CHICKEN AND TURKEY BETA2-MICROGLOBULIN KAREN G. WELINDER,* HANS M. JESPERSEN,JAN WALTHER-RASMUSSEN
and Institute of Biochemical Copenhagen K, Denmark
KAR~TEN SKJCYDT~
Genetics, University of Copenhagen, 0. Farimagsgade and TInstitute for Experimental Immunology, University Nsrre Alle 71, DK-2100, Copenhagen 0, Denmark
2A, DK-1353, of Copenhagen,
(Received 11 June 1990; accepted 23 July 1990) Abstract-The complete amino acid sequences of chicken and turkey beta2-microglobulins have been determined by analyses of tryptic, VS-proteolytic and cyanogen bromide fragments, and by N-terminal sequencing. Mass spectrometric analysis of chicken beta2-microglobulin supports the sequence-derived M, of 11,048. The higher apparent M, obtained for the avian beta2-microglobulins as compared to human beta2-microglobulin by SDS-PAGE is not understood. Chicken and turkey beta2-microglobulin consist of 98 residues and deviate at seven positions: 60, 66, 74-76, 78 and 82. The chicken and turkey sequences are identical to human beta2-microglobulin at 46 and 47 positions, respectively, and to bovine beta2-microglobulin at 47 positions, i.e. there is about 47% identity between avian and mammalian beta2-microglobulins. The known X-ray crystallographic structures of bovine beta2-microglobulin and human HLA-A2 complex suggest that the seven chicken to turkey differences are exposed to solvent in the avian MHC class I complex. The key residues of betaZmicroglobulin involved in alpha chain contacts within the MHC class I molecule are highly conserved between chicken and man. This explains that heterologous human beta2-microglobulin can substitute the chicken beta2-microglobulin in exchange studies with B-F (chicken MHC class I molecule), and suggests that the MHC class I structure is conserved over long evolutionary distances.
INTRODUCTION
is the small subunit of et al., 1973). It is presumably essential for expression of the large subunit on the cell surface. MHC class I molecules are expressed on the surface of most nucleated cell types and are in dynamic equilibrium with free b2m (Kimura et al., 1983), which is present in many physiological fluids. B2m from a variety of animals is about 11,000 in size and can be isolated from blood plasma for structural studies. The evolution of the structure of the MHC class I molecule, which is central to the cellular immune defence, is important for our understanding of the evolution of the immune system in general. Amino acid and DNA sequences have been derived for a great number of both types of subunits, in particular from mammals (Kabat et al., 1987). More recently the crystal structures of bovine b2m (Becker and Reeke, 1985) and human HLA-A2 and HLA-Aw68 (Bjorkman er al., 1987; Garret et al., 1989) have been solved. We have previously purified and characterized b2m from chicken and turkey (Skjerdt et al., 1986). Here we document the complete amino acid Beta2-microglobulin
MHC
class
(b2m)
I molecules
(Grey
sequences and discuss the results in terms of physicochemical properties and conserved MHC class I structure. MATERIALS AND METHODS
Purification of b2m
Chicken and turkey b2m were purified by monoclonal antibody affinity chromatography as previously described by Skjsdt et al. (1986), and desalted by RP-HPLC on a C4 column using a gradient of acetonitrile in 0.1% TFA. Reduction and S-carboxymethylation
B2m and disulphide-linked peptides were reduced by dithiotreitol and S-carboxymethylated by iodoacetate using standard procedures in the presence of 2% SDS as a denaturant. The use of SDS was discontinued, however, as basic groups in proteins and peptides bind SDS which retards and broadens peaks during RP-HPLC. Proteolytic cleavages
Ten nmole of a solution of desalted, native b2m was concentrated by vacuum centrifugation and neutralized with 1 M ammonium bicarbonate to a final concn of 200 mM, pH 8.1. The solution was boiled for 2-10 min to unfold the protein and chilled quickly to 20°C. TPCK-trypsin (2.4 ~1 0.1% trypsin
*To whom correspondence should be addressed. tPresent address: Institute of Medical Microbiology, University of Odense, Winslrawsvej 19, 5000 Odense C, Denmark. 177
K. G. WELINDER et al.
178
in 10 mM CaCl,) was added immediately and the reaction left for 20 hr at room temp. Digestions were stopped with 8 ~1 10% TFA and the sample diluted with an equal volume of 0.1% TFA prior to RP-HPLC. Digestion of desalted, reduced and S-carboxymethylated b2m with V8 protease was carried out similarly but in sodium phosphate buffer as described by Welinder (1988).
A227nm -T7
b2m
A. T&T9
Amino acid and sequence analyses Amino acid analyses were carried out on an instrument based on ion exchange chromatography and post-column oxidation (to analyse proline) and derivatization with ortophthaldialdehyde. The instrument and hydrolysis in gaseous or liquid acid are described in detail by Barkholt and Jensen (1989). Automatic sequencing was carried out either on a gasphase sequencer (Applied Biosystems, model 470A) followed by PTH (phenylthiohydantoin) analyses on a Waters HPLC system using a Spherisorb ODS II column developed by a methanol-ethanol gradient, or on an Applied Biosystems model 477A sequencer with on-line HPLC analysis of PTH-derivatives. Mass spectroscopy Accurate molecular mass determinations of native and reduced and S-carboxymethylated chicken b2m were carried out on 100 pmol of salt-free samples. A plasma desorption, time-of-flight mass spectrometer (Bio-Ion 20) was used. RESULTS
Tryptic peptides Experiments on b2m from chicken and turkey were run in parallel. Tryptic digestion of native b2m, preheated at 100°C for 2min, generated nearly the number of peptides expected from the amino acid composition (Skjsdt et al., 1986). The tryptic peptide maps (Fig. 1) show a peak of undigested b2m, indicating that 2 min of preheating is insufficient to Table
I. Amino Tl
Asx Thr Ser GlX Pro GUY Ala CYS Val Met Ile Lcu Tyr Phe His Lys Arg TOP Total
1.0(l) 1.0 (1) 0.9 (1)
T2
T3
0.2 1.3 (1) 1.0 (I) 0.3 0.8 0.8
0.2 0.9 (1) 1.1 (1) 0.3 0.9 (1) 1.4(l) I .9 (2)
1.0(l)
0.2
5
3.3 (3) 2.0 (2) 3.0 (3) 3.1 (3) 2.3 (2) 4.2 (4) 1.7 (2) 1.4 (2)
0.3 1.0(l)
1.0(l)
1.6 (2) 0.9 (1) 2.9 (3) \-, I .8 (2) 1.9 (2)
6
8
31
0.6(l) 0.2 (I)
T4-Tl
0.8 (I)
T5 1.0 (1) 1.0(l)
0.3
0.8(l) I .8 (2) 1.0(l)
T6 4.4 (5) 0.9 (1) 2.1 (2) 3.3 (3) 1.0(l) 2.3 (2) 1.2 (1) 1.0(l) 0.6 (2)
0.6 (1) 1.8 (2) 1.0(l)
7
0.2 1.0 (1) (1) 23
II
TIO
T5 T7
I
I
I
20
I
I
10
30
40
50
min.
Fig. 1. RP-HPLC of tryptic peptides from native b2m, (A) chicken and (B) turkey. A Cl8 column, 4.6 x 120 mm, was eluted by a linear gradient from &70% methanol in 0.1% TFA at a flow rate of 1 ml/min at room temperature. Fractions were collected manually. The peptides are numbered sequentially from the N-terminus (see Fig. 2). T8-T9 has an uncleaved Lys-Glu bond and T&T7 is disulphide
linked. The last peak constitutes undigested b2m. unfold b2m. Five min of preheating at 100°C gave complete tryptic digestion (not shown). Comparison of the HPLC profiles of tryptic peptides of chicken and turkey b2m shows that only the disulphide linked T4-T7 peptides from the two species have very different retention times. Amino acid sequence analysis (Fig. 2) verifies this difference and shows that the T7 peptides differ at 5 positions. Furthermore, sequence analyses reveal that the similar T6 peptides have a Thr to Ser substitution and T8-T9 a Glu to Asp substitution. The results of amino acid analyses (Table 1) and sequence analyses of chicken b2m (Fig. 2) are in
acid analyses of peptides derived from chicken b2m. Results are uncorrected compositions are shown in brackets
1.8 (2)
I.1
T4-T7 T6
T&T9 0.9 (1) 0.2 4.0 (4) 1.0 (1) 0.4
TlO
CmVl-V2
1.0(l)
9.4 (9) 5.9 (6) 7.7 (8) 5.8 (5) 6.2 (6) 5.1 (5) 7.1 (7) 0.5 (2) 5.6 (6) 2.6 (3) 1.9 12) 4.0 ;4j 2.6 (3) 5.9 (6) 2.1 (2) 5.0 (5) 1.9 (2) (1) 82
1.1 (1) 1.0(l)
1.8 (2)
1.0 (1) 0.7 (I) 1.0(l) 1.1 (1) I .9 (2)
13
from 20 hr hydrolysates.
0.2 (1) 5
CmV2 4.6 (5) 2.7 (3) 4.3 (4-5) 4.2 (3) 1.3 (1) 2.5 (2-3) 2.5 (3) 0.5 (1) 1.8 (2) 0.7 (1) 1.4(l) 1.8 (2) 2.7 (3) 1.1 (I) 1.4(l) 1.0(l) (1) 36
The sequence derived v3
v4
0.4
1.1(l) 1.0(l)
1.1 (1)
3.1 (3) 2.0 (2)
1.0(l)
1.0(l) 0.9(l) 0.9 (1) 0.9(l) 2.0 (2)
2
(1) 14
Structure of chicken and turkey b2m CHICKEN
CH
b2m
b2m
I 0
7 0
3 0
* 0
55555555543444344344444
Trypsln
< ,5 >< ddddddddd555455533333333331121 Cm14 > 55555555555555 CmVl-W2
0
a
0
76
R
CC)
>< T7-T4 ddddddddddddddn < ClllT7 44344434334133333
>< cmu2 555555535444233434444443433222332222
:444334443244423333233323
9 b
a
0
0
0
>< T&T9 > 4554555554432355553 > V3 >
