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A note on the amino acid sequence of residues 381–391 of human immunoglobulin gamma chains
Moltwlar Immunology. Vol. 16, pp. 923-925. Pergamon Press Ltd 1979 Printed in Great Britain
A NOTE ON THE AMINO ACID SEQUENCE 381-391 OF HUMAN IMMUNOGLOBULIN CHAINS* THEO HOFMANN Department
and DOROTHY
of Biochemistry, Medical Science Building, Toronto, Canada MSS IA8
OF RESIDUES GAMMA
M. PARR University
of Toronto,
(Received 1 March 1979) Abstract-The amino acid sequence of residues 381-391 ofthe y-chain of IgG2 Zie has been determined. Trp-Glu-Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr. It is proposed that this is the typical sequence residues 381-391 of human y-chains.
INTRODUCTION
Although amino acid sequences of parts of human y chain constant regions have been known for some time (Pink et al., 1970; Press & Hogg, 1970; Wang er al., 1973; Wolfenstein-Todel et al., 1976; Pardo et al., 1978) the only complete sequences available have been those of IgGl Eu (Edelman et al., 1969) and IgGl Nie (Ponstingl & Hilschmann, 1972). These two sequences were found to be extremely similar; the only differences in the constant region which were not due to differences in allotype or to amide assignments were found between residues 385 and 3891_(Ponstingl & Hilschmann, 1972). These authors suggested that the latter differences may not be real, but may be caused by technical problems. Indeed, in other published sequences of the C,3$ domain from each of the human y chain sub-classes, amino acids following residues 383,384 or 385 were not determined directly, but were aligned with the sequence of y1 Eu by homology and amino acid composition. Thus, this appears to be a difficult section to analyse. In our own studies of the sequences of the three constant region domains of IgG Zie, a myeloma protein belonging to the IgG2 sub-class (Connell et al., 1979) we, too, initially were unable to obtain an unambiguous sequence for residues * This work was supported by grant No. MA-694 from the Medical Research Council of Canada. i Eu numbering is used throughout. 1 Abbreviations used: CNBr, cyanogen bromide; y chain, heavy chain of IgG; fragment F(ab’),, both light chains and the N-terminal halves of both heavy ‘chains, v&h disulphide bonds intact; fragment pFc’, residues 334-446 of the heavy chain; C,l, CR2, CR3, constant homology regions of immunoglobulin heavy chains.
It is for
385-389. We have now succeeded in resolving the ambiguities and present a sequence of these residues which, apart from amide assignments, is identical with that of IgGl Nie (Ponstingl & Hilschmann, 1972).
MATERIALS
AND METHODS
IgG Zie was isolated from plasma by cryoprecipitation as described previously (Connell et al., 1979). Peptide CNBr-I (residues 359-397) was prepared as follows: (i) IgG Zie was digested with pepsin to give F(ab’), and pFc’ fragments, (ii) pFc’ fragmen)ts in 70% formic acid were treated with CNBr, and the C-terminal octadecapeptide (peptide CNBr-III) was removed by gel filtration, (iii) the remainder of the CNBr-treated pFc’ fragments were reduced and alkylated, and peptide CNBr-II (residues 398-428) and peptide CNBr-I were separated by gel filtration. Complete details of the preparation of peptide CNBr-I are given in Connell et al., 1979. Automated sequencing was carried out as described previously (Connell et al., 1979). The identity of every residue in one preparation was confirmed by amino acid analysis of the phenylthiohydantoin derivative (Smithies et al., 1971; Parr et al., 1976).
RESULTS
AND DISCUSSION
Three preparations of peptide CNBr-I, which is obtained by CNBr cleavage at Met-358 and Met-397, were sequenced automatically. The result of one run, for which 5.6 mg (1.4 pmole) of peptide of high purity were available, is shown in 923
924
THEO Table
Cycle I 1 i
4
5 6
7 8 9 I0
II I? I3 14 15 I6 17 I8 19 20 21 22 23 24 25 26 27 2x 29 30 31 32 33 34 35 36 37 38
I. Analysts
Positton in chain 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 314 37.5 376 317 378 379 3x0 381 3x2 3x3 384 385 386 387 388 389 390 391 392 393 394 395 396
of pepttde CNBr-I sequencing
HOFMANN by automated
Amino acid identified and yield Gas chromatoAmino actd graphy fimole analysis Thr Lys Asn Gin Val Ser Leu Thr ii-Se? Leu Val
400
240 450
LYS Gly Phe Tyr Pro (Ser] Asp Ile Ala Va] Glu
400 400 400 350
340 400 363 580 280 290 156 246 181 400 290 319 285 225
Asp Ile Ala Val Glu _r Glu Ala’ Asnh
I45
140 60 80 85 45 50 50 55 40 30 35
G]Y Glnb Pro Glu AStlh Asnh
80 35 25 35 40
Tyr (Lys?) Thr
755 670 420 800 508 312 500
G]Y Phe Tyr Pro Ala’
355 295 280 200 I60 150
Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn
flmole
r-AB” Lys Asnh G]nh Val Ala’ Leu (lost) Ala” Leu Va] *Lys
(200) (200) 730
and DOROTHY
Tyr Lys r-AB”
Table 1. Unambiguous identification of the first 34 residues of the 39-residue peptidc was possible; evidence for threonine in cycles 35 and 36 was also obtained. In another analysis the presence of proline in cycles 37 and 3X was established. A repetitive yield of 94”,, was obtained as far as cycle 24 or 25. where it dropped to around X6”,,, probably because of the Asn-26-Gly-27 bond, which is known to cause reduced yields (Kolb er al., 1974). and the diminishing size of the remaining peptide. Asparagine and glutamine were identified unambiguously both by gas chromatography and by amino acid analysis. The sequence deduced from these results is shown in Table 2 where it is compared with the sequences of the corresponding stretches of the 7 chains of Eu (Edelman et ul., 1969) and Nie (Ponstingl & Hilschmann, 1972). Apart from amide assignments, the sequence of Zie is identical with that of Nie but differs from that of Eu in residues 38.5-389. The differences (again apart from amide assignments) can be eliminated, however, if Asp-385 of Eu is moved to position 3X9. Ponstingl and Hilschmann (1972) had suggested that the differences in residues 385-3X9 between Eu and Nie required further investigation. The results presented here appear to clarify the ambiguities and suggest that the typical sequence for residues 381-391 of human ;’ chains is -TrpGlu-Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-. ,4ckno~M~rmmts~ We should
like to thank Mrs. A. Cunningham and Mrs. A. Leung for excellent technical assistance, Mr. S. Rhee for the automated sequence analyses, and Mr. C. Yu for amino acid analyses.
23
- -__
” G(-AB: z-amino-butyric acid derived from PTHthreonine. ‘Identified as amides because of concomitant rise in ammonia. ’ Alanine arising from PTH-serine. ’ d-serine arising from PTH-carboxymethyl cysteine. “Alanine arising from PTH-carboxymethyl cysteine. ‘Tryptophan lost in acid hydrolysis. Table 2. Amino acid sequence
M. PARR
REFERENCES Connell G. E., Parr D. M. & Hofmann T. (1979) The amino acid sequences of the three heavy chain constant region domains of a human IgG2 myeloma protein. C‘un. J Biochcm. 57, 158-767.
of peptide CNBr-1 from ‘;2 chain Zie compared Eu
with the sequences
of71 chains Nie and
360 Zic Nie Eu ZlC Nte EU
cys
Leu
Val
370 LYS
Gly
Phe
Zie Nte EU indicates
identity
with ;’ chatn Zie
Tyr
Thr
Lys
Asn
Gln
Val
Set
Lcu
Thr
Pro
Set
Asp
Be
Ala
Val
380 Glu
7rp
Residues
381-391
of Human
Edelman G. M., Cunningham B. A., Gall W. E., Gottlieb P. D., Rutishauser U. & Waxdal M. J. (1969) The covalent structure of an entire yG immunoglobulin molecule. Proc. natn. Acad. Sci. U.S.A. 63, 78-85. Kolb E., Harris J. I. & Bridgen J. (1974) Triose phosphate isomerase from the coelacanth. An approach to the rapid determination of an amino acid sequence with small amounts of material. Biochem. J. 137, 185-197. Pardo A. G., Rosenwasser E. S. & Frangione B. (1978) The primary structure of a human immunoglobulin G2 (lgG2) pFc’ fragment. J. Immun. 121, 1040-1044. Parr D. M., Hofmann T. & Connell G. E. (1976) Characterization of upFc, a fragment of human immunoglobulin Gl produced by pepsin in urea. Biochem. J. 157, 535-540. Pink J. R. L., Buttery S. H., de Vries G. M. & Milstein C. (1970) Partial amino acid sequence of the constant region of a y4 chain. Biochem. J. 117, 3347. Ponstingl H. & Hilschmann N. (1972) The primary structure of a monoclonal IgGl immunoglobulin (myeloma protein
Immunoglobulin
Gamma
Chains
925
Nie), II: amino acid sequence of the constant part of the Hchain, localization of genetic factors. Hoppe-Seyler’s Z. physiol. Chem. 353, 1369-I 372. Press E. M. & Hogg N. M. (1970) The amino acid sequences of the Fd fragments of two human yl heavy chains. Biochem. J. 117, 641-660. Smithies O., Gibson D., Fanning E. M., Goodfliesh R. M., Gilman J. G. & Ballantyne D. L. (1971) Quantitative procedures for use with the Edman-Begg sequenator. Partial sequences of two unusual light chains, Rzf and Sac. Biochemistry 10, 49 12-492 1. Wang A. C., Gergely J. & Fudenberg H. H. (1973) Amino acid sequences at constant and variable regions of heavy chains of monotypic immunoglobuhns G and M of a single patient. Biochemistry 12, 528-534. Wolfenstein-Todel C., Frangione B., Prelli F. & Franklin E. C. (1976) The amino acid sequence of “heavy chain disease” protein Zuc. Structure of the Fc fragment of immunoglobulin G3. Biochem. biophys. Res. Commun. 71, 907-914.
A NOTE ON THE AMINO ACID SEQUENCE 381-391 OF HUMAN IMMUNOGLOBULIN CHAINS* THEO HOFMANN Department
and DOROTHY
of Biochemistry, Medical Science Building, Toronto, Canada MSS IA8
OF RESIDUES GAMMA
M. PARR University
of Toronto,
(Received 1 March 1979) Abstract-The amino acid sequence of residues 381-391 ofthe y-chain of IgG2 Zie has been determined. Trp-Glu-Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr. It is proposed that this is the typical sequence residues 381-391 of human y-chains.
INTRODUCTION
Although amino acid sequences of parts of human y chain constant regions have been known for some time (Pink et al., 1970; Press & Hogg, 1970; Wang er al., 1973; Wolfenstein-Todel et al., 1976; Pardo et al., 1978) the only complete sequences available have been those of IgGl Eu (Edelman et al., 1969) and IgGl Nie (Ponstingl & Hilschmann, 1972). These two sequences were found to be extremely similar; the only differences in the constant region which were not due to differences in allotype or to amide assignments were found between residues 385 and 3891_(Ponstingl & Hilschmann, 1972). These authors suggested that the latter differences may not be real, but may be caused by technical problems. Indeed, in other published sequences of the C,3$ domain from each of the human y chain sub-classes, amino acids following residues 383,384 or 385 were not determined directly, but were aligned with the sequence of y1 Eu by homology and amino acid composition. Thus, this appears to be a difficult section to analyse. In our own studies of the sequences of the three constant region domains of IgG Zie, a myeloma protein belonging to the IgG2 sub-class (Connell et al., 1979) we, too, initially were unable to obtain an unambiguous sequence for residues * This work was supported by grant No. MA-694 from the Medical Research Council of Canada. i Eu numbering is used throughout. 1 Abbreviations used: CNBr, cyanogen bromide; y chain, heavy chain of IgG; fragment F(ab’),, both light chains and the N-terminal halves of both heavy ‘chains, v&h disulphide bonds intact; fragment pFc’, residues 334-446 of the heavy chain; C,l, CR2, CR3, constant homology regions of immunoglobulin heavy chains.
It is for
385-389. We have now succeeded in resolving the ambiguities and present a sequence of these residues which, apart from amide assignments, is identical with that of IgGl Nie (Ponstingl & Hilschmann, 1972).
MATERIALS
AND METHODS
IgG Zie was isolated from plasma by cryoprecipitation as described previously (Connell et al., 1979). Peptide CNBr-I (residues 359-397) was prepared as follows: (i) IgG Zie was digested with pepsin to give F(ab’), and pFc’ fragments, (ii) pFc’ fragmen)ts in 70% formic acid were treated with CNBr, and the C-terminal octadecapeptide (peptide CNBr-III) was removed by gel filtration, (iii) the remainder of the CNBr-treated pFc’ fragments were reduced and alkylated, and peptide CNBr-II (residues 398-428) and peptide CNBr-I were separated by gel filtration. Complete details of the preparation of peptide CNBr-I are given in Connell et al., 1979. Automated sequencing was carried out as described previously (Connell et al., 1979). The identity of every residue in one preparation was confirmed by amino acid analysis of the phenylthiohydantoin derivative (Smithies et al., 1971; Parr et al., 1976).
RESULTS
AND DISCUSSION
Three preparations of peptide CNBr-I, which is obtained by CNBr cleavage at Met-358 and Met-397, were sequenced automatically. The result of one run, for which 5.6 mg (1.4 pmole) of peptide of high purity were available, is shown in 923
924
THEO Table
Cycle I 1 i
4
5 6
7 8 9 I0
II I? I3 14 15 I6 17 I8 19 20 21 22 23 24 25 26 27 2x 29 30 31 32 33 34 35 36 37 38
I. Analysts
Positton in chain 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 314 37.5 376 317 378 379 3x0 381 3x2 3x3 384 385 386 387 388 389 390 391 392 393 394 395 396
of pepttde CNBr-I sequencing
HOFMANN by automated
Amino acid identified and yield Gas chromatoAmino actd graphy fimole analysis Thr Lys Asn Gin Val Ser Leu Thr ii-Se? Leu Val
400
240 450
LYS Gly Phe Tyr Pro (Ser] Asp Ile Ala Va] Glu
400 400 400 350
340 400 363 580 280 290 156 246 181 400 290 319 285 225
Asp Ile Ala Val Glu _r Glu Ala’ Asnh
I45
140 60 80 85 45 50 50 55 40 30 35
G]Y Glnb Pro Glu AStlh Asnh
80 35 25 35 40
Tyr (Lys?) Thr
755 670 420 800 508 312 500
G]Y Phe Tyr Pro Ala’
355 295 280 200 I60 150
Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn
flmole
r-AB” Lys Asnh G]nh Val Ala’ Leu (lost) Ala” Leu Va] *Lys
(200) (200) 730
and DOROTHY
Tyr Lys r-AB”
Table 1. Unambiguous identification of the first 34 residues of the 39-residue peptidc was possible; evidence for threonine in cycles 35 and 36 was also obtained. In another analysis the presence of proline in cycles 37 and 3X was established. A repetitive yield of 94”,, was obtained as far as cycle 24 or 25. where it dropped to around X6”,,, probably because of the Asn-26-Gly-27 bond, which is known to cause reduced yields (Kolb er al., 1974). and the diminishing size of the remaining peptide. Asparagine and glutamine were identified unambiguously both by gas chromatography and by amino acid analysis. The sequence deduced from these results is shown in Table 2 where it is compared with the sequences of the corresponding stretches of the 7 chains of Eu (Edelman et ul., 1969) and Nie (Ponstingl & Hilschmann, 1972). Apart from amide assignments, the sequence of Zie is identical with that of Nie but differs from that of Eu in residues 38.5-389. The differences (again apart from amide assignments) can be eliminated, however, if Asp-385 of Eu is moved to position 3X9. Ponstingl and Hilschmann (1972) had suggested that the differences in residues 385-3X9 between Eu and Nie required further investigation. The results presented here appear to clarify the ambiguities and suggest that the typical sequence for residues 381-391 of human ;’ chains is -TrpGlu-Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-. ,4ckno~M~rmmts~ We should
like to thank Mrs. A. Cunningham and Mrs. A. Leung for excellent technical assistance, Mr. S. Rhee for the automated sequence analyses, and Mr. C. Yu for amino acid analyses.
23
- -__
” G(-AB: z-amino-butyric acid derived from PTHthreonine. ‘Identified as amides because of concomitant rise in ammonia. ’ Alanine arising from PTH-serine. ’ d-serine arising from PTH-carboxymethyl cysteine. “Alanine arising from PTH-carboxymethyl cysteine. ‘Tryptophan lost in acid hydrolysis. Table 2. Amino acid sequence
M. PARR
REFERENCES Connell G. E., Parr D. M. & Hofmann T. (1979) The amino acid sequences of the three heavy chain constant region domains of a human IgG2 myeloma protein. C‘un. J Biochcm. 57, 158-767.
of peptide CNBr-1 from ‘;2 chain Zie compared Eu
with the sequences
of71 chains Nie and
360 Zic Nie Eu ZlC Nte EU
cys
Leu
Val
370 LYS
Gly
Phe
Zie Nte EU indicates
identity
with ;’ chatn Zie
Tyr
Thr
Lys
Asn
Gln
Val
Set
Lcu
Thr
Pro
Set
Asp
Be
Ala
Val
380 Glu
7rp
Residues
381-391
of Human
Edelman G. M., Cunningham B. A., Gall W. E., Gottlieb P. D., Rutishauser U. & Waxdal M. J. (1969) The covalent structure of an entire yG immunoglobulin molecule. Proc. natn. Acad. Sci. U.S.A. 63, 78-85. Kolb E., Harris J. I. & Bridgen J. (1974) Triose phosphate isomerase from the coelacanth. An approach to the rapid determination of an amino acid sequence with small amounts of material. Biochem. J. 137, 185-197. Pardo A. G., Rosenwasser E. S. & Frangione B. (1978) The primary structure of a human immunoglobulin G2 (lgG2) pFc’ fragment. J. Immun. 121, 1040-1044. Parr D. M., Hofmann T. & Connell G. E. (1976) Characterization of upFc, a fragment of human immunoglobulin Gl produced by pepsin in urea. Biochem. J. 157, 535-540. Pink J. R. L., Buttery S. H., de Vries G. M. & Milstein C. (1970) Partial amino acid sequence of the constant region of a y4 chain. Biochem. J. 117, 3347. Ponstingl H. & Hilschmann N. (1972) The primary structure of a monoclonal IgGl immunoglobulin (myeloma protein
Immunoglobulin
Gamma
Chains
925
Nie), II: amino acid sequence of the constant part of the Hchain, localization of genetic factors. Hoppe-Seyler’s Z. physiol. Chem. 353, 1369-I 372. Press E. M. & Hogg N. M. (1970) The amino acid sequences of the Fd fragments of two human yl heavy chains. Biochem. J. 117, 641-660. Smithies O., Gibson D., Fanning E. M., Goodfliesh R. M., Gilman J. G. & Ballantyne D. L. (1971) Quantitative procedures for use with the Edman-Begg sequenator. Partial sequences of two unusual light chains, Rzf and Sac. Biochemistry 10, 49 12-492 1. Wang A. C., Gergely J. & Fudenberg H. H. (1973) Amino acid sequences at constant and variable regions of heavy chains of monotypic immunoglobuhns G and M of a single patient. Biochemistry 12, 528-534. Wolfenstein-Todel C., Frangione B., Prelli F. & Franklin E. C. (1976) The amino acid sequence of “heavy chain disease” protein Zuc. Structure of the Fc fragment of immunoglobulin G3. Biochem. biophys. Res. Commun. 71, 907-914.