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Novak, R. L., and Dohnal, J. ( 974a), Nucleic Acids Res. I , 753. Novak, R. L., and Dohnal, J. ( 974b), Nucleic Acids Res. 1, 761.
Pisetsky, D., Berkower, I., Wickner, R., and Hurwitz, J . (1972), J . Mol. Biol. 71, 557. Reusser, F. (1973), Biochemistry 12, 1136. Staudenbauer, W. L. (1975), J . Mol. Biol. 96, 201.
Amino Acid Sequence of the VH Region of a Human Myeloma Immunoglobulin (IgG New)? R. J. Poljak,t Y. Nakashima, B. L. Chen, and W. Konigsberg*
ABSTRACT: The amino acid sequence of the heavy-chain variable region of the human immunoglobulin. New has been determined. Since the amino terminus of the heavy chain was blocked, the sequence of residues 1-69 was established by digesting the appropriate CNBr fragment separately with trypsin, chymotrypsin, and thermolysin and sequencing the resulting peptides. The region from residues 70 to 120 was present in another C N B r fragment which was submitted directly to automatic Edman degradation. The result of this
experiment extended the sequence to residue 100. The primary structure of the remaining portion of the V H region was determined by automatic Edman degradation of a lysine-blocked tryptic peptide derived from this region which included residues 98-214. The sequence of the V H region of New corresponds most closely to V H sequences of proteins in the V H I1 subgroup. This primary structure makes it possible to construct a model from the high-resolution electron-density map of protein New.
chemical degradation provided the required information to T h e three-dimensional structure of the Fab' fragment of the specify its sequence. The region from 70 to 120 was obtained human immunoglobulin New (IgG New) has been determined via CNBr cleavage of partially reduced, alkylated heavy chain to a nominal resolution of 2 8, (Poljak et al., 1974). The light followed by separation of the fragments using gel filtration. (L, A) chain of IgG New has been sequenced and reported Automatic sequenator runs on a CRBr fragment (residues (Chen and Poljak, 1974). The study of the amino acid sequence 70-249) and on a tryptic peptide derived from this fragment of the heavy (H) chain was undertaken to obtain the necessary (residues 98-214) gave results which allowed us to formulate information for a complete interpretation of the 2.0-A Fourier a sequence for the entire heavy-chain variable region of protein map of Fab' New. New. This paper represents the evidence for the proposed seThe amino acid sequence of IgG immunoglobulin H chains is usually divided into four homology regions, V H , CH 1, C H ~ , quence, gives a comparison of this sequence with other VHII sequences, and discusses the relationship of this sequence with and cH3, consisting of approximately 1 10 to 1 15 amino acids the electron-density map of protein New. (Gally and Edelman, 1972). With the exception of wellcharacterized allotypic variants, human y chains show seExperimental Section quence identity in their CHregions as well as partial identity Materials. Human myeloma immunoglobulin New (IgG,, and strong homology in their variable V H regions (Capra and Gm( 1 3- 4- 5 - ) , A) was purified from serum which had Kehoe, 1975). been kept frozen at -20 "C. Diethylaminoethylcellulose (0.9 Two approaches were used to obtain peptides from the V H mequiv/g) and carboxymethylcellulose (0.8 mequiv/g) were region of New. In one case, Fab' fragments were cleaved with purchased from Serva. Sephadex G-100 and (3-25 were obC N B r and separated by gel filtration. This gave a fragment tained from Pharmacia. Tos-PheCHzCI trypsin, chymotrypsin, comprising residues 1-69. A combination of enzymatic and carboxypeptidase A (treated with DEF), carboxypeptidase B, and pepsin were purchased from Worthington Biochemical +From the Department of Molecular Biophysics and Biochemistry. Yale University, New Haven, Connecticut (Y.N. and W . K . ) . and from thc Corp. Thermolysin was obtained from Calbiochem. DiDepartment of Biophysics, Johns Hopkins University School of Mcdicinc. thiothreitiol, ethylenimine, cyanogen bromide, iodoacetic acid, Baltimore, Maryland 21205 (R.J.P. and B.L.C.). Rrcriced March I I . dansyl chloride, and sequencing reagents were obtained either 1977. This work was supported by research Grants AI-OX202 and AIfrom Pierce Chemical Co. or from Beckman and were used 08614 from the National Institutes of Health, Grant GM-I 2607 from thc fresh without further purification. Polyamide thin-layer sheets American Cancer Society, and Grant GB-43482 from the National Science Foundation. were purchased from Gallard-Schlesinger. Dansyl amino acid *Research Career Development Awardee of the National Institutes of standards were prepared as described by Gray (l967a). Health (AI-70091). Preparation of H Chain from Protein New. IgG New was 'Abbreviations used for immunoglobulins, their chains, and fragments purified by precipitation with sodium sulfate followed by are as recommended in Bull. W.H.O.30, 447 (1964). Other abbreviations used are: dansyl, 1-dimethylaminonaphthalene-5-sulfonyl; DFP. diisochromatography on diethylaminoethylcellulose and carpropyl fluorophosphate; NaZEDTA, disodium ethylenedinitrilotetraboxymethylcellulose as described before (Rossi and Nisonoff, acetate; PhNCS, phenyl isothiocyanate; Pca, pyrrolidonecarboxylic acid: 1968). Mildly reduced and alkylated H chain was prepared by Tos-PheCHzCI, 1-tosylamido-2-phenylethyl chloromethyl ketone: Tris, reducing a 20 mg/mL solution of IgG New in 0.2 M Tris-HCI 2-amino-2-hydroxymethyl- I,3-propanediol; EDTA, (ethylenedinitril0)(pH 8.5)-0.005 M EDTA-0.02 M dithiothreitol for 2 h at tetraacetic acid.
+
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room temperature, followed by alkylation with iodoacetic acid which was added to the reaction mixture to give a final concentration of 0.06 M. Alkylation was continued for 20 rnin in the dark with rapid stirring and was followed by dialysis (2-4 h) against 1 M propionic acid also in the dark. Preparative separation of H and L chains was accomplished using 5 X 100 cm Sephadex (3-100 columns equilibrated with 1 M propionic acid and fitted with upflow adaptors. The H - and L-chain fractions were pooled, lyophilized, and stored as dried powders. Totally reduced and alkylated H and L chains were prepared by reduction of the polypeptides ( I O mg/mL) in a solution containing 8 M guanidine hydrochloride, 0.2 M Tris-HCI (pH 8.5), 0.005 M EDTA, and 0.1 M dithiothreitol for 2 h at 40 OC. After alkylation with iodoacetic acid (added to a final concentration of 0.3 M), the sample was dialyzed against freshly prepared 8 M urea-1 M acetic acid buffer and filtered through a 5 X 100 cm Sephadex G-100 column equilibrated with the same buffer. The H and L chains thus obtained were extensively dialyzed against distilled water, lyophilized, and stored as dry powders. Preparation of Fab' Fragments. Purified IgG New was digested with papain as described before (Rossi and Nisonoff, 1968) using a papain/IgG New ratio of 1 : 100.The digest was dialyzed against 0.01 M phosphate buffer, pH 8.0, and chromatographed on a diethylaminoethylcellulose column equilibrated with the same buffer. The eluted Fab' fragment was concentrated by precipitation with 75% saturated ammonium sulfate, centrifuged, redissolved, extensively dialyzed against distilled water, and lyophilized. CNBr Cleavage of Fab' and H Chains:Separation of CNBr Fragments. Lyophilized Fab' fragments or H chains were dissolved in 70% formic acid and CNBr was added to give final concentrations of 20 mg/mL protein, 60 mg/mL CNBr. Digestions were carried out for 18 h at room temperature, terminated by the addition of 5 volumes of ice-cold distilled water, and immediately lyophilized. The freeze-dried digest of Fab' New was dissolved in 1 M acetic acid and separated into three fractions by gel filtration on Sephadex G-100 columns, as shown in Figure IA. Peaks I , 11, and I11 (Figure 1A) obtained after gel filtration were totally reduced and aminoethylated or were alternatively oxidized with performic acid, following the procedures of Raftery and Cole ( I 966) and Hirs ( 1967), respectively, and again chromatographed on a Sephadex G-100 column. After this treatment, peaks I and I1 gave an essentially identical pattern of the type shown in Figure 1 B; the elution position of the minor peak 111 remained as it had been in the first chromatographic separation (Figure 1 A). Similar optical-density profiles and separations were obtained using Sephadex (3-100 columns equilibrated with 6 M guanidine hydrochloride, 0.02 M Tris-HCI, pH 8.0. The relative proportions of peaks I and I1 varied in the different runs. Since, after reduction and aminoethylation, peak I (Figure 1A) gave the same components as did peak 11, peak I was considered to consist of aggregated material. CNBr-cleaved H chains were fractionated by gel filtration in 1 .O M propionic acid on 2.5 X 100 columns of Sephadex (3-75. Reduction and Alkylation of the CNBr Fragments of Protein New Heavy Chain. The CNBr fragment (cut 1, Figure 2A) (200 mg) was dissolved in 6 M guanidine hydrochloride, containing 50 mM Tris and 5 m M EDTA, pH 8.5, and the solution was kept at 50 "C for 30 min. Dithiothreitol (40 mM) was added, the solution flushed with Nz, and the reduction allowed to proceed at 50 "C. The solution was cooled to 25 "C and 50 pCi of [3H]iodoacetic acid was added and allowed to
stand in the dark a t 25 OC for 15 min. The solution was then dialyzed first against distilled water and then against 6 M urea containing 0.1 M glycine and 1.O M propionic acid. Finally, the solution was passed through a column (4.5 X 100 cm) of Sephadex G-100 and eluted with 6 M urea containing I .O M propionic acid and 0.1 M glycine. Radiosuccinylation of a CNBr Fragment (Fraction A , Figure I B ) from the Heavy Chain. The carboxymethylated CNBr fragment (fraction A, Figure 1B) (400 mg) was dissolved in 6 M guanidine hydrochloride and the pH of the solution was adjusted to 8.5 with NaOH. ['4C]Succinic anhydride (50 pCi) (New England Nuclear) was added and the pH of the reaction mixture kept at 8.5 with 0.1 M NaOH. The reaction was carried out for 15 min and then IO0 mg of cold succinic anhydride was added and, after another 15 rnin at pH 8.5, the reaction mixture was dialyzed against distilled water, frozen, and lyophilized. Enzymatic Digestion of CNBr Fragments from the Heauy Chain. Aminoethylated or performic acid oxidized H chain CNBr fragments were digested with Tos-PhCHzCI trypsin, chymotrypsin, or thermolysin for 4 h at room temperature in 0.1 M NH4HCO3 using an enzyme:substrate ratio of 1 : 100. Digestions with pepsin were carried out in 0.1 M formic acid under similar conditions. Digestion of purified peptides with chymotrypsin, trypsin, or pepsin was performed as proposed by Smyth (1967). A similar procedure was used for therrnolysin digestions which were conducted a t 45 "C. Digestions with carboxypeptidases A and B were performed as described by Ambler (1967). Fractionation and Purification of Peptides. Peptides obtained by enzymatic digestion of H-chain fragments were separated by gel filtration on 2.5 X 100 cm columns of Sephadex G-25 (fine) equilibrated with 0.1 M N H I O H . The column effluents were monitored by absorption readings at 280 and 232 nm, which permitted the digests to be divided into fractions which were then lyophilized, redissolved in pH 6.5 buffer (10% pyridine, 0.6% acetic acid, 89.6% water), and submitted to high-voltage electrophoresis at pH 6.5 using gradients of 50 V/cm for periods of time ranging from 60 to 400 min in Varsol-cooled tanks. Cysteic acid, glutamic acid, aspartic acid, threonine, leucine, histidine, arginine, and lysine were used as electrophoretic markers and run side by side with the peptides under investigation. After electrophoresis, guide strips were stained with ninhydrin (0.2% in acetone), with a chlorine stain (Reindel and Hoppe, 1954), and with stains specific for arginine and tryptophan (Bennett, 1967). Subsequent purification steps included paper chromatography and high-voltage electrophoresis at different pH values. The papers used in chromatography were previously washed by descending chromatography with distilled water containing 0.1% EDTA. The chromatographic solvent consisted of 1 -butanol-acetic acid-pyridine-water (25:3: 10:12). Further peptide purification was achieved by high-voltage electrophoresis using a pH 2.0 buffer (formic acid-acetic acid-water 25:87:888), a pH 4.7 buffer (acetic acid-pyridine-water 1: 1 :78), and a pH 3.5 buffer (acetic acid-pyridine-water 10: 1 :189). When a two-dimensional or a "three-dimensional" separation procedure was required to purify a given peptide, its location on paper was established using a weak ninhydrin stain (0.2% ninhydrin in acetone). As soon as the peptide became visible, the area containing it was cut out and washed with a I : l mixture of ethanol-acetone. Peptides were eluted from paper (using 6 M HCI) into 50- or 100-pL glass capillaries which were sealed and kept a t 1 10 "C ( 1 8-24 h) prior to amino acid analysis. Alternatively, peptides were eluted with 0.1-0.3 mL of a 1:l BIOCHEMISTRY, VOL.
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T A B L E I : Amino Acid Composition of C N B r Fragments from Fab’ and the Heavy Chain of Protein New.“
06
I 04
Peakh
a 02
10
20
30
40
20
30
40
50
60
70
BO
90
Fraction Number
F I G U R E 1 : (A) Separation of fragments from CNBr-treated Fab’ New on a 2 X 90 cm column of Sephadex (3-100 in 1 M acetic acid using an upward-flow adaptor. Cuts were taken as indicated. (B) Gel filtration of cut 2 (Figure I A ) after reduction and aminoethylation or after performic acid oxidation. The separation was carried out exactly as described for Figure 1.4 and the cuts indicated were taken for further work.
mixture of water-pyridine and reserved for sequence determination. Amino Acid Analyses. Quantitative amino acid analyses were performed by the method of Moore et al. (1968) using a Durrum D-500 or a Beckman 121 M amino acid analyzer. Sequencing Techniques. Sequential degradation of purified peptides was performed using the Edman reaction (Edman, 1956). After each degradation step, aliquots of peptides were removed to determine the newly exposed “2-terminal residue, as proposed by Hartley (1970) and Gray (1967b). The dansyl derivatives of amino acids were identified by chromatography using the procedure of Woods and Wang (1967) on 4.5 X 7.5 cm polyamide sheets. Chromatography was performed using 1.5% formic acid in the first dimension, benzene-glacial acetic acid (9:l) in the second dimension, and ethyl acetate-methanol-acetic acid (20:l:l) in the third dimension. Automatic sequencing was performed by using a J E O L JAS-47K sequence analyzer. A standard protein program with a quadrol buffer system and a peptide program with dimethylallylamine buffer system was employed according to the procedure of Hermodson et al. (1973). The double-cleavage program was used during acid cleavage. The thiazolidine derivatives released were converted in l N HCI a t 80 OC for 10 min to phenylthiohydantoin (Pth) derivatives which were identified by gas chromatography (Pisano et al., 1972) and by thin-layer chromatography (Summers et al., 1973). Finally, a portion of the Pth derivatives was hydrolyzed in 57% HI at 130 OC for 18 h and the resulting amino acids were determined by amino acid analysis (Smithies et al., 1971). Results Two approaches were used to obtain variable-region fragments of protein New heavy chains. In one approach, the Fab’ portion of New was prepared and cleaved with CNBr, and the fragments were isolated by gel filtration. The heavy-chain region, from residue 1 to 69, was obtained in fairly good yield this way, but difficulties were encountered in isolating the region from residue 70 through the start of the constant region in pure form and in high enough yield. To circumvent this difficulty, a complementary strategy, which involved CNBr cleavage of isolated heavy chains, was employed. In this case, a fragment spanning residue 70 to 249 was obtained pure and in high yield, whereas the NH2-terminal CNBr fragment was not pure enough for further use. Tryptic peptides were obtained, however, from this region and these peptides also served as a check on the validity of the results obtained for the sequence of residues 1-69 using the Fab’ portion as mentioned above. To facilitate the presentation of the results, the isolation and characterization of the C N B r fragments, using each approach, will be discussed first. This will be followed by the
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Res 1-69
Aminoacid
111
112
113
LYS
9.7 4.2
6.8 2.3
0.1 1.1
0
4.7 Ndf 15.2 21.3 30.3 22.9 17.1 17.1 21.5 3.0 19.1
14.6 Ndf 12.5 15.3 22.5 10.1 10.8 12.6 13.8
4.9 2.0
5 2 4 9 8 7 5 7
His N H3 A rg TrP Asp Thrd Ser“
Glu Pro GlY Ala l/~-Cystine’ Val Met
