0161-5890:79/0801-0565SOZ.OO/O
Molecular Immunology, Vol. 16, pp. 56ir570. ~~Pergamon Press Ltd. 1979. Pm&d in Great Britain
THE COMPLETE AMINO-ACID SEQUENCE OF A CANINE MU CHAIN* LARRY
J. McCUMWER
and J. DONALD
CAPRA
Department of Microbiology, The University Southwestern
Medical
of Texas Health Science Center School. Dallas, TX 75235, U.S.A.
at Dallas,
Abstract-The amino-acid sequence of the CHl and the CH2 domains of a canine mu chain has been determined, completing the primary structural analysis of this molecule. The result extends our previous conclusions,concerning the remarkably high degree of interspecies homology to human IgM, as the canine CHl and CH2 domains are 81 and 722, homologous to their human counterparts. The overall homology of the entire constant region of the canine mu chain to the human mu chain is 81%. This study represents the first complete primary structure of a non-IgG heavy chain from a species other than man.
INTRODUCTION
sequence analyses of the constant regions of kappa, lambda and gamma chains indicate a modest degree of sequence homology between species (Gaily, 1973; Kubo et al., 1973; Novotny rt al., 1972). Most comparisons indicate approximately 6Oy, preservation of identical residues at particular positions in immunoglobulin polypeptide chains 1977). Phylogenetic, evolutionary and (Capra, immunochemical data suggests that the IgM class of immunoglobulins has been more rigidly preserved in evolution than either kappa, lambda or gamma chains (Marchalonis, 1972). A recent study from this laboratory is consistent with this suggestion as it was found that the amino-acid sequence of the Fc region of a canine mu chain was 820/ honlologous to its human counterpart (Wasserman & Capra, 1978). As a continuation of this study, the amino-acid sequence of the canine IgM Fd region has been determined to ascertain the extent of homology of the total canine mu chain to the human mu chain. Amino-acid
purposes. Peptides derived from digestion of Moo CB l-3 will be named as follows: CT. citraconylated tryptic; Chy, chymotryptic; T, tryptic. Isolation qf the citraconylated Moo CBI-3
tryptic
peptides
derived from
An aliquot of the Moo CBl-3 pool (8 mg) was reacted with citraconic anhydride (25 kiti]for 15 min) in 1.0 ml of 6.0 M guanidine-HCI-1 .O MTris (pH 9.0) and then dialyzed against I70 NH,HCO, (pH 7.8). Tryptrc digestion (TPCKWorthington Biochemical Corp.) was performed in I’;/ NH,HCO, at 37°C for I6 hr at an enzyme-to-substrate ratio of 1:lOO. The digest was decitraconylated (250,: acetic acid, 37 C, 4 hr), lyophilized, dissolved in 1.5 ml of I’YONH,HCO, and applied to a 1x lOO-cm column of Sephadex G-5OSF equilibrated with 1% NH,HCO,. As shown in Fig. 1, seven peaks were obtained and pooled as indicated. The peptides in pool 4 were further separated by high-voltage paper electrophoresis at pH 2.1, yielding CT-4A and CT-4B. Pool 5 contained the peptide CT-5. Peptides CT-6B and CT-6C were obtained by paper electrophoresis of pool 6. Isolation
ofchymotryppric
derivedfrom
peptides
Moo
CBI-3
An aliquot of the Moo CBl-3 pool (7 mg) was dissolved in I.5 ml of 1% NH, WCC?, and reacted with chymotrypsin
MATERIALS AND NIETHODS 0.3Or +.!...+&,A,
4
;:
5
,A,
7
,
0.25-
Canine IgM Moo wasisolated and the muchain purified as described previously (Wasserman & Capra, 1977). The mu chain was digested with cyanogen bromide (CB) and the resulting fragments were initially separated on a column of Sephadex G-100 equilibrated with 5.0 M guanidine-HCI as previously described (Fig. 3, Wasserman & Capra, 1977). The peptides excluded from the column (pool 1, termed CB I, Fig. 3, Wasserman & Capra, 1977) were completely reduced and carboxymethylated and recycled on the same column. The third peak eluting from the column (CBI-3) contained two protein fragments of similar size and charge, representing residues 152-23 1and 232-304 of the canine mu chain. These two fragments will be called Moo CBI-3 for discussion --
ozo-
0 I g
0.15-
0.10 -
QO5 -
20
24
28
32
36
TUBE
40
44
48
52
56
60
NUMBER
Fig. I. Chromatography of the tryptic peptides of citraconylated fragment CBI-3 of mu chain Moo. The digest was applied to a 1 x 100 cm column packed with Sephadex GSOSF equilibrated with I”/, NH,HCO,. Pool 4 contained peptides CT-4A and CT4B. Pool 5 contained peptide CT-5 Pool 6 conldined peptides CT-6B and CT-6C.
*This work was supported by grants from the N.I.H. (AI 12127) and the National Science Foundation (PCM7&22411). L.J.M. was supported by Cancer immunology Training Program GA09082. 565
566
LARRY
J. McCUMBER
and J. DONALD
1.6r
CAPRA
solvent. Five pools were made 3s shown in FIB. 7. Pool 7 contained the peptidc Chy-2. hO”,, of which ~a\ further digested with trypsin to gi,e the peptide Chy-2T-G after further purificatton by high-voltage paper electrophoresrs. The peptide Chy-3 was the m;t_tor component in pool 3. Pool 5 contained the peptide Chy-X-3 which wits puntied from the pool
by
chromatography
propionic
acid.
on
Seph:rJex
followed
by
CI-IOF
in
htgh-voltage
1.0 .21
paper
electrophoresis
30
40
50
60 TUBE
70
80
90
Electrophoresis on Whatman 3MM paper in formic acid buffer at pH 2.1 and 3000 (Crumpton & Wilkinson. 1965) under Varsol Savant apparatus. Peptides were eluted from M NH-l.
100
NUMBER
Fig. 2. Chromatogmphy of the chymotrypt~~ peptides of fragment CBl-3 of mu chain Moo. The digest was appfied to a 1.5~ 190-cm column packed with Sephadex G-50SF equilibrated with 1.0 M propionic acid. Pool 2 contained peptides Chy-2 and Chy-ZT-G. Pool 3 contained peptide Chy-3. Pool 5 contained peptide Chy-5C-3. (Worthington) at 37 C for 15 hr at an enzyme-to-substrate ratio of 1: 100. The digest was then lyophilized. dissolved in 4 ml 1.O A4 propionic acid, and applied to a 1.5 x 190-cm column of Sephadex G-50 SF equiIibrated with the same
GLY
CYS
LEU ALA
ARG
ASP
PHE LEU
After evacuation in a nitrogen atmosphere, peptides were hydrolyzed in 6 .w HCI for 20 brat I IO C. The hydrolysntes were analyzed on a Durrum D-500 ammo-acid analyzer.
The use ofthe automated protein-peptide sequencer in this laboratory has been previously described (Capra & Kunkel. 1970; Caprn et al., 1977). All peptides were sequenced with the aid of polybrene (Klapper et ul., 1978). Identification of PTH-amino acids was made by gas-liquid chromatography employing a Beckman CC-65 (P&no & Bronzert, 1969).
160 PRO GLY
SER
ILE THR
ALA
ILE ASN
SER
THR ARG c
__
Chy-3
TRP
LYS
TYR
LEU ARG
GLY
GLY
LYS TYR
190
180 GLY PHE
PRO SER VAL
4
CT-65
b
_
CT-6C+
---_-_-_--_-_-_-------4 210
200 VAL ALA
THR
SER
GLN
VAL
PHE LEU
PRO SER
VAL ASP
CYS
LYS
ILE
ILE GLN
GLY
THR ASP
GLU
HIS
VAL
Chy-2 _-_--___---_----
&-_--------____-_-____
ILE VAL
170 GLY
PHE SER
CT-4A
Molecular Immunology, Vol. 16, pp. 56ir570. ~~Pergamon Press Ltd. 1979. Pm&d in Great Britain
THE COMPLETE AMINO-ACID SEQUENCE OF A CANINE MU CHAIN* LARRY
J. McCUMWER
and J. DONALD
CAPRA
Department of Microbiology, The University Southwestern
Medical
of Texas Health Science Center School. Dallas, TX 75235, U.S.A.
at Dallas,
Abstract-The amino-acid sequence of the CHl and the CH2 domains of a canine mu chain has been determined, completing the primary structural analysis of this molecule. The result extends our previous conclusions,concerning the remarkably high degree of interspecies homology to human IgM, as the canine CHl and CH2 domains are 81 and 722, homologous to their human counterparts. The overall homology of the entire constant region of the canine mu chain to the human mu chain is 81%. This study represents the first complete primary structure of a non-IgG heavy chain from a species other than man.
INTRODUCTION
sequence analyses of the constant regions of kappa, lambda and gamma chains indicate a modest degree of sequence homology between species (Gaily, 1973; Kubo et al., 1973; Novotny rt al., 1972). Most comparisons indicate approximately 6Oy, preservation of identical residues at particular positions in immunoglobulin polypeptide chains 1977). Phylogenetic, evolutionary and (Capra, immunochemical data suggests that the IgM class of immunoglobulins has been more rigidly preserved in evolution than either kappa, lambda or gamma chains (Marchalonis, 1972). A recent study from this laboratory is consistent with this suggestion as it was found that the amino-acid sequence of the Fc region of a canine mu chain was 820/ honlologous to its human counterpart (Wasserman & Capra, 1978). As a continuation of this study, the amino-acid sequence of the canine IgM Fd region has been determined to ascertain the extent of homology of the total canine mu chain to the human mu chain. Amino-acid
purposes. Peptides derived from digestion of Moo CB l-3 will be named as follows: CT. citraconylated tryptic; Chy, chymotryptic; T, tryptic. Isolation qf the citraconylated Moo CBI-3
tryptic
peptides
derived from
An aliquot of the Moo CBl-3 pool (8 mg) was reacted with citraconic anhydride (25 kiti]for 15 min) in 1.0 ml of 6.0 M guanidine-HCI-1 .O MTris (pH 9.0) and then dialyzed against I70 NH,HCO, (pH 7.8). Tryptrc digestion (TPCKWorthington Biochemical Corp.) was performed in I’;/ NH,HCO, at 37°C for I6 hr at an enzyme-to-substrate ratio of 1:lOO. The digest was decitraconylated (250,: acetic acid, 37 C, 4 hr), lyophilized, dissolved in 1.5 ml of I’YONH,HCO, and applied to a 1x lOO-cm column of Sephadex G-5OSF equilibrated with 1% NH,HCO,. As shown in Fig. 1, seven peaks were obtained and pooled as indicated. The peptides in pool 4 were further separated by high-voltage paper electrophoresis at pH 2.1, yielding CT-4A and CT-4B. Pool 5 contained the peptide CT-5. Peptides CT-6B and CT-6C were obtained by paper electrophoresis of pool 6. Isolation
ofchymotryppric
derivedfrom
peptides
Moo
CBI-3
An aliquot of the Moo CBl-3 pool (7 mg) was dissolved in I.5 ml of 1% NH, WCC?, and reacted with chymotrypsin
MATERIALS AND NIETHODS 0.3Or +.!...+&,A,
4
;:
5
,A,
7
,
0.25-
Canine IgM Moo wasisolated and the muchain purified as described previously (Wasserman & Capra, 1977). The mu chain was digested with cyanogen bromide (CB) and the resulting fragments were initially separated on a column of Sephadex G-100 equilibrated with 5.0 M guanidine-HCI as previously described (Fig. 3, Wasserman & Capra, 1977). The peptides excluded from the column (pool 1, termed CB I, Fig. 3, Wasserman & Capra, 1977) were completely reduced and carboxymethylated and recycled on the same column. The third peak eluting from the column (CBI-3) contained two protein fragments of similar size and charge, representing residues 152-23 1and 232-304 of the canine mu chain. These two fragments will be called Moo CBI-3 for discussion --
ozo-
0 I g
0.15-
0.10 -
QO5 -
20
24
28
32
36
TUBE
40
44
48
52
56
60
NUMBER
Fig. I. Chromatography of the tryptic peptides of citraconylated fragment CBI-3 of mu chain Moo. The digest was applied to a 1 x 100 cm column packed with Sephadex GSOSF equilibrated with I”/, NH,HCO,. Pool 4 contained peptides CT-4A and CT4B. Pool 5 contained peptide CT-5 Pool 6 conldined peptides CT-6B and CT-6C.
*This work was supported by grants from the N.I.H. (AI 12127) and the National Science Foundation (PCM7&22411). L.J.M. was supported by Cancer immunology Training Program GA09082. 565
566
LARRY
J. McCUMBER
and J. DONALD
1.6r
CAPRA
solvent. Five pools were made 3s shown in FIB. 7. Pool 7 contained the peptidc Chy-2. hO”,, of which ~a\ further digested with trypsin to gi,e the peptide Chy-2T-G after further purificatton by high-voltage paper electrophoresrs. The peptide Chy-3 was the m;t_tor component in pool 3. Pool 5 contained the peptide Chy-X-3 which wits puntied from the pool
by
chromatography
propionic
acid.
on
Seph:rJex
followed
by
CI-IOF
in
htgh-voltage
1.0 .21
paper
electrophoresis
30
40
50
60 TUBE
70
80
90
Electrophoresis on Whatman 3MM paper in formic acid buffer at pH 2.1 and 3000 (Crumpton & Wilkinson. 1965) under Varsol Savant apparatus. Peptides were eluted from M NH-l.
100
NUMBER
Fig. 2. Chromatogmphy of the chymotrypt~~ peptides of fragment CBl-3 of mu chain Moo. The digest was appfied to a 1.5~ 190-cm column packed with Sephadex G-50SF equilibrated with 1.0 M propionic acid. Pool 2 contained peptides Chy-2 and Chy-ZT-G. Pool 3 contained peptide Chy-3. Pool 5 contained peptide Chy-5C-3. (Worthington) at 37 C for 15 hr at an enzyme-to-substrate ratio of 1: 100. The digest was then lyophilized. dissolved in 4 ml 1.O A4 propionic acid, and applied to a 1.5 x 190-cm column of Sephadex G-50 SF equiIibrated with the same
GLY
CYS
LEU ALA
ARG
ASP
PHE LEU
After evacuation in a nitrogen atmosphere, peptides were hydrolyzed in 6 .w HCI for 20 brat I IO C. The hydrolysntes were analyzed on a Durrum D-500 ammo-acid analyzer.
The use ofthe automated protein-peptide sequencer in this laboratory has been previously described (Capra & Kunkel. 1970; Caprn et al., 1977). All peptides were sequenced with the aid of polybrene (Klapper et ul., 1978). Identification of PTH-amino acids was made by gas-liquid chromatography employing a Beckman CC-65 (P&no & Bronzert, 1969).
160 PRO GLY
SER
ILE THR
ALA
ILE ASN
SER
THR ARG c
__
Chy-3
TRP
LYS
TYR
LEU ARG
GLY
GLY
LYS TYR
190
180 GLY PHE
PRO SER VAL
4
CT-65
b
_
CT-6C+
---_-_-_--_-_-_-------4 210
200 VAL ALA
THR
SER
GLN
VAL
PHE LEU
PRO SER
VAL ASP
CYS
LYS
ILE
ILE GLN
GLY
THR ASP
GLU
HIS
VAL
Chy-2 _-_--___---_----
&-_--------____-_-____
ILE VAL
170 GLY
PHE SER
CT-4A
