Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin : Amino-Acid Sequence of the Fc Fragment

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1 Eur. J. Biochem. 43, (1974) Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin : Amino-Acid Sequence of the Fc Fragment Implications for the Evolution of Immunoglobulin Structure and Function Alain BOURGOIS, Michel FOUGEREAU, and Jose ROCCA-SERRA Centre de Biochimie et de Biologie Molkulaire, Centre National de la Recherche Scientifique, Marseille (Received October Sl/December 17, 1973) The amino-acid sequence of the Fc fragment of a mouse monoclonal IgG2a molecule is presented. With the exception of one deletion, this region possesses the same length as that of the human yl chain or that of the rabbit y chain. Identities between the Fc fragments of 3 animal species (human, mouse and rabbit) average 50 /, and are markedly more pronounced for the c ~2 domain than for the C H domain, ~ observation which is in agreement with an independent evolution of each homology region. Strikingly, the degree of conservation of the VH region is of the same osder of magnitude as that observed for the Fc fragment. This parallelism in evolution is discussed. Murine MOPC 173 immunoglobulin is a monoclonal protein of the IgG2a class [l]. Isolation and characterization of t'he CNBr fragments [2] and localization of the disulfide bridges [3,4] have led to the proposals of a topological model [4]. Cleavage of the heavy chain with CNBr allows isolation of ten fragments, which have been designated H1 to H10 [2]. Fragments HI to H3 cover residues 3 to 104, the sequence of which has been reported [5]. Fragment H4 covers the entire constant section of Fd, the hinge region and the beginning of the Fc fragment. We report in the present paper, the amino-acid sequence of the last 18 COOH-terminal residues of H4 and that of the remaining fragments H5 to H10, thus accounting for the entire Fc fragment. MATERIALS AND METHODS CNRr fragments were prepared by direct attack of the whole molecule, essentially as previously described [2], with the minor following modifications. The H4 fragment was separated from the L chain derived L2 fragment by ion-exchange chromatography on DEAE-cellnlose (Whatman DE 52), equilibrated in 6 M urea, ph 10.0, under which conditions H4 was not retained, as opposed to L2. Abbreviations. Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; Cys, cysteine; Glu, glutamic acid; Gln, glutamine; Gly, glycine; His, histidine; Ile, iaoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenyl alanine; Pro, proline; Ser, serine; Thr, threonine; Trp,tryptophan ; Tyr, tyrosine; Val, dine; Cmc, carboxamidomethylcysteine ; Hsr, homoserine. Fragments H5 and H6-7 were separated by ionexchange chromatography on a column of SE-Sephadex equilibrated in acetic acid 0.05 M made 6 M in urea (ph 5.2), and using a stepwise elution with 0.5 M NaOH containing 6 M urea (ph 13.0). Eluted material was desalted on a column of Sephadex G 25 coarse in 1.0 M propionic acid. Enzymatic hydrolyses with trypsin [treated with L-( l-tosyl-amido-2-pheny1)ethyl chloromethyl ketone (i.e. TPCK-treated, Calbiochem)], chymotrypsin and subtilisin (N.B. Co), were performed as previously described [5]. Incubation times, however, were lowered to 20 or 30 min (at 37 "C) in most instances, with the exception of H4 and H9C peptides, for which a 4-h hydrolysis was used. Occasionally, tryptic hydrolysis, was used after fragments had been citraconylated according to Gibbons and Perham [6]. For acid hydrolysis, a 1 solution of substrate (fragment HlO) was prepared in 6 N HC1. Incubation proceeded at 37 "C for 18 h. Peptides were separated by ionexchange chromatography on a column (55 x 0.9 cm) of either Dowex 50x2 (Biorad) or cation exchanger resin P (Technicon). Elution was obtained by applying a gradient of ph linear between 3.0 and 6.0 (first buffer: acetic acid-pyridine-water, 180:8:812 ph 3.0 ; second buffer : pyridine -water : 160 : 840, ph 8.9). The average flow rates were 2 ml/min and 1 ml/min for the Dowex and the resin P columns, respectively. A split-stream device was set up at the outlet of the column, so that 1/20 of the effluent was injected into an automatic peptide analyzer (Technicvn) based on ninhydrin coloration of the alkaline

2 424 Sequence of a Mouse Fc Fragment hydrolyzates [7], whereas 19/20 were harvested in a fraction collector. Synchronization between the analyand the preparative circuits was Obtained by injecting a dye solution (xylene cyanol) at the beginning and at the end of the run. In some cases the alkiline hydrolysis and the ninhydrin coloration were performed manually. Repurification of peptides by high-voltage electrophoresis on paper [8] net charge calculation [Q], detection and quantitation of tryptophan [lo] sequence determination by the Edman-dansylation method [ 111 carboxypeptidase hydrolysis [38] and technical details for amino-acid analysis were as used in the previous publication of the series [2,5]. When net charge calculation was done on peptides containing homoserine, the homoserine form was the most represented (more than 70 /,) and detected. The carboxamidomethyl cysteyl residue is neutral at ph6.5. Limited stretches of sequence have been automatically determined either with a Beckman model 890 sequencer [I21 for the H5 and H6 fragment or with the Socosi sequencer for the H9 and HI0 fragments. Phenylthiohydantoin derivatives were identified by gas chromatography, thin-layer chromatography [13], or regeneration of the amino acid from the phenylthiohydantoin derivative by hydrolysis and identification on the amino-acid analyzer Nomenclature of Peptides Peptides were termed as follows (except for the two H4-derived peptides) : the first arabic numeral indicates the CNBr fragment from which a given peptide was derived. The letters T, C, S and A refer to tryptic, chymotryptic, subtilytic and acidic cleavage, respectively. The last figure indicates the order of the given peptide in the sequence. Vlrhen citraconylation was performed before tryptic digestion, an additional c was placed after the T. RESULTS Sequence of the COOH-Terminal Section of the H4 Fragment The H4 fragment which contains the CHI domain, the hinge region and the beginning of the Fc fragment is poorly soluble. So it was citraconylated before being hydrolyzed with trypsin. The hydrolyzate was loaded onto a column of Sephadex G 75 SF equilibrated with M N-ethylmorpholine, ph 8.2. Six peaks resulted upon elution, termed A to F (Rocca-Serra and Fougereau, unpublished results). Chymotryptic digest of the peptide contained under peak D was submitted to gel-filtration on a column of Sephadex G 25 superfine in M N-ethylmorpholine, ph 8.2, which allowed further resolution into 4 peaks, termed a to d. Material of peak a was repurified Table I. Amino-acid composition of tryptic peptides Db2 and ~ ~ ~ l ~ ~ ~, " of ~ residues ~ ~ per ~ molepof ~ e ~ ~ ~ peptides. Yield is based on nanomoles of peptides isolated compared with nanomoles of fragment H4 digested. Electrophoretic mobility and net charge at ph 6.5 are given Amino acid Aspartic acid or asparagine Serine Homoserine Proline Glycine Valine Isoleucine Leucine Phenyl alanine Lysine Amount in peptide Db2 DalO residues/mol peptide Total Yield Oio 5 6 Mobility Charge +2 on Dowex 50x2 and a peptide, called Da I0 was shown to contain the homoserine COOH-terminus (see Table 1). The sequence obtained manually by the Edman-dansylation method was : Ile-Phe-Pro-Val-Lys-Ile-Lys- Asn-Pro-Leu-Hsr The lysine residue number 7 was identified by subtractive Edman procedure. The mobility of this peptide, as determined by paper electrophoresis at ph 6.5 (Table 1) dictates the aspartic acid or asparagine residue number 8 be in the amide form. Additional proof is given by the neutral charge at ph 6.5 of this peptide after seven and eight Edman degradation steps. A peptide Db 2, shown to be adjacent to the latter on the grounds of homology with the sequence of the human y l chain Eu [I51 and that of the rabbit y chain [16,17], was isolated from peak B and repurified by high voltage electrophoresis on paper, at ph 3.5 (Table 1) The sequence was directly, determined as : - Leu-Gly-Gly-Pro-Ser-Val-Phe equence of the H5 Fragment Data concerning the complete sequence of the H5 fragment is being reported separately [18], and were obtained both by automatic sequencing and by manual determination.

3 I A. Bourgois, M. Fougereau, and J. Rocca-Serra 425 Table 2. Amino-acid composition of tryptic peptides from fragment 16-7 Results are expressed in number of residues per mole of peptide.pield is based on nanomoles of peptides isolated compared with nanomoles of H6-7 fragment digested. Electrophoretic mobility and net charge at ph 6.5 are given Amino acid Amount in peptides 6'Tl 6'T2 6'T3 6'T4 6'Tcl 6'Tc2 VTc3 WTc31 6'Tc32 CTc4 Sum H6 H7 Tcl residuesjmol peptide Carboxamidomethylcysteine I ~- -- Aspartic acid or asparagine T hr e o n i n e Serine Homoserine Glutamic acid or glutamine Proline Glvcine Alanine Valine Isoleucine Leucine Tvrosine Phenyl alanine Lvsine Arginine Total "0 Yield Mobilitv Charge 0 +I l Sequence of the H6 and H7 Fragments Because of the relatively modest yield (ili 300/,) of CNBr cleavage at the methionine residue of the H6 fragment, three fragments were obtained and used for sequencing this region (Table 2) : H6 (43 residues), H7 (10 residues) and H6-7 (53 residues). Tryptic hydrolysis of fragment H6-7 yielded 4 peptides that were isolated by ion-exchange chromatography on resin P (Fig. 1) and purified by highvoltage electrophoresis on paper at ph 3.5 or 6.5 (Table 2). Because of solubility problems most peptides were lost during these purifications. Only four peptides termed 6'Tl to 6'T4 were isolated, thus accounting only partially for the H6-7 fragment. In a subsequent experiment, fragment H6-7 was thus citraconylated and then hydrolyzed with trypsin. The hydrolyzate was loaded on a Sephadex G 25 superfine column equilibrated with ammonium bicarbonate (4 g/l). Two fractions were independently pooled and further purified by ion-exchange chromatography on resin 1' (Fig.2). Six peptides were finally isolated and analyzed (Table 2). Peptides 6'Tcl to 6Tc4 accounted for the entire H6 fragment with the addition of the overlap Thr-Lys of the H7 fragment. Peptide 6Tc3 was also partially split into 6'Tc31 and 6'Tc32, the latter being a single tyrosine, which either remained

4 426 Sequence of a Mouse Fc Fragment Fig.1. Separation of tryptic peptides from HG-7 fragment by ion-exchange chromatography on a column of resin P. 30 mg H6-7 fragment was used on a 52 x 0.9-cm column. Elution was performed at 50 "C, using the gradient linear between ph 3 and ph 6 as described in Materials and Methods. Enzyme-substrate ratio was 2O/, (w/w). Incubation proceeded at 37 "C for 30 min. Absorbance at 570 nm after alkaline hydrolysis and ninhydrin coloration + c e Effluent volume (ml) < 0.10r Effluent volume (ml) I Effluent volume (ml) Fig. 2. Elution on a Sephadex G 25 superfine column in ammonium bicarbonate of a tryptic hydrolyzate of citraconylated HG-i' fragment (I). Column 200 ml, ammonium bicarbonate 4 g/l, tryptic hydrolyzate 17 mg. Enzyme-substrate ratio was lo/,, (w/w). Hydrolysis proceeded at 37 "C for 30 min. Peak A was repurified on a column (52 x 0.9 cm) of resin P (11) and peak B on the same resin, but using a column (25~0.9 cm) (111). Conditions of elution were as described under Fig. 1. Note absorbance at 570 nm is after alkaline hydrolysis and ninhydrin coloration Em. J. Biochem. 43 (1974)

5 A. Bourgois, M. Fougereau, and J. Rocca-Serra 427 attached to larger peptides through hydrophobic interaction, or is detached after the Sephadex chromatography. In spite of unexpected cleavages this region is obtained in a relatively good yield because peptides 6'Tc3 and 6'Tc31 were well separated on a short column of resin P. The complete sequence of peptides 6'T2, 6'T3, 6'T4, 6'Tc2 and 6'To3 as well as that of isolated fragment H7 was completely determined by the Edman-dansylation method. Residts are presented on Fig3 on which are also shown partial sequence of peptides 6'Tcl and 6'Tc4 and sequence data automatically obtained for the 31 NH,-terminal residues of fragment H6 on a Beckman sequencer by Dr M. Kehoe. The sequencer results make it possible to order peptides 6'Tcl, 6'Tc2 and 6'Tc3. Peptide 6'Tc4 contains the COOH-terminal portion of H6 and the overlap with the beginning of fragment H7. Peptides 6'Tl to 6'T4 were simply ordered in register with the sequence obtained automatically for fragment H6. All residues of the H6-7 fragment have thus been directly determined with the exception of the Glx residue at position 42 which is, however unequivocally deduced from the total amino-acid composition of 6Tc4. Identification of the thiohydantoins by gas chromatography allowed direct determination of the acidic or amide nature of residues 4, 10, 11, 13 and 19. Additional proof for residues 4, 10 and 11 was gained from the mobility at ph 6.5 of peptides 6'T2 and 6'T3. Net charge at ph 6.5 of peptides 6'Tc3 and that of fragment H7 implies the presence of a glutaminyl and of a glutamyl at positions 33 and 47, respectively. From mobility value at ph 6.5 of peptide B'Tc4, it can be deduced that residue 41 or 42 must be an amide, the other one being an acid. The exact relative assignment could not be made. Sequence of the H8 Fragment Fragment H8 contains only five residues : aspartic acid or asparagine (l.o), threonine (O.O), homoserine (1.O), valine (1.Q) and phenyl alanine (1.0). Neutral behaviour of this peptide at ph 6.5 implicates that the aspartic acid or asparagine residue must be in amide form. Sequence was determined directly by Edman-dansylation as follows : Val-Thr- Asn-Phe-Hsr Sequence of the H9 Fragment Fragment H9 is composed of 33 residues (Table 3). The elution patterns of tryptic and chymotryptic peptides upon ion-exchange chromatography on Dowex 50x2 are presented in Fig.4. Subtilytic peptides were separated by high-voltage electrophoresis on paper at ph 6.5 and repurified at ph 3.5. Amino-acid composition of peptides 9T1 to 9T3 accounts for

6 ~ Table 3. Amino-acid composition of tryptic, chynaotryptic and subtilitic peptides front H9 Results are expressed in number of residues per mole of peptides. Yield is based on nanomoles of peptides isolated compared with nanomoles of H, digested. Electrophoretic mobility and net charge at ph 6.5 are given ~~~~~~ ~~~ ~ Amino acid Amount in DeDtide 9T1 9T2 9T3 9T4 9C1 9C2 9C3 9C4 9C5 9C6 9S1 9x % 9S6 Sum H9 T1-T3 residues/mol peptide Aspartic acid or asparagine Threonine Serine Homoserine Glutamic acid or dutarnine Proline o Glycine Valine Isoleucine Leucine Tyrosine Phenyl alanine Lysine Tryptophan la 1.Ob la 1.20 Total Yield g a F? Mobility 4 P, s f? IP. cj Charge 0 +I I +I a Determined by fluorescence. E % b Determined by amino-acid analysis of carboxypeptidase A digest. 7 c Determined by spectrometry.. $- and - 2. d After one step of the Edman degradation the mobility and charge at ph 6.5 of peptide 953 were respectively and - 2, after three steps F 9 s 09

7 A. Bourgois, M. Fougereau, and J. Rocca-Serra I n. 2 5 ~ OO 100 Effluent volume (ml) 200 c m OI c D 0 L Fig.4. Elution patterns of tryptic and chymotryptic peptides upon ion-exchange chromatography. (I) Elution on a column of Dowex 50x2 of a tryptic hydrolyzate (enzyme-substrate ratio incubation: 20 min at 37 "C) of 35 mg fragment H9. Conditions of chromatography were indicated on Fig. 1. (11) Separation of the chymotryptic peptides of the H9 200 Effluent volume (ml) fragment on a column of Dowex 50x2 using a gradient obtained from 250 ml of 0.2 N formic pyridine buffer (ph 3.1) and 250 ml of 2 N formic pyridine buffer (ph 5.0, see [5]). The flow rate was 40 ml/h. Incubation with chymotrypsin (2 /0 w/w) was made at 37 "C for 4 h. Absorbance at 570 nm after alkaline hydrolysis and ninhydrin coloration that of the entire H9 fragment. Sequence results are presented on Fig. 5. Direct automatic sequence of the NH,-terminal residues of fragment H9 allowed location of peptide 9Tl. Peptide 9T3 contained the homoserine COOH-terminus of ragment H9, which thus imposes location of 9T2. This order is confirmed by different overlapping peptides, especially 9C5. Net charge determination for subtilytic peptides, and for 9T2,9T4 and 9C2 peptides imposes the presence of asparaginyl residues at positions 10, 11, 17 and 20, that of aspartyl residues at positions 26 and 29 and that of glutamyl and glutaminyl residues at positions 15 and 22, respectively. Identification of the phenylthiohydantoin derivatives of amino acids after automatic sequencing showed the presence of glutamyl residues at positions 2 and 7 and aspartyl residue at position 3. Xequence of the H10 Fragment Fragment HlO is composed of 40 residues (Tables 4 and 5). Peptides released after chymotrypsin digestion or obtained by acid hydrolysis were separated by ion-exchange chromatography, respectively on Dowex 50x2 and resin P (Fig.6). Many fractions obtained from the acidic hydrolyzate contain free amino acids. Tryptic peptides were directly isolated by high-voltage electrophoresis on paper at ph 6.5, and further purified by a second run at ph 3.5. Tryptic peptides lot1 to 10T9 account for the entire H10 fragment. Sequence data are presented on Fig.7. Direct sequencing of fragment Hi0 automatically determined on a Socosi sequencer makes it possible to order peptides lot1 to 10T5. Peptides 10C3 and 10A2 make it possible to order tryptic peptides 10T5, 10T6 and 10T7. Peptide loc6 imposes the order lot8-10t9, the latter peptide containing the glycyl COOH-terminus of the heavy chain. The overlap 10T7-10T8 is contained in chymotryptic peptide loc4. It can be seen (Fig.6) that peptides loa3 to 10A6 must be located within peptide 10T7. All peptides of this region were consistently obtained in very low yield, and an ambiguity remained for the

8 430 Sequence of a Mouse Fc Fragment 10 Pro-Glu-Asp-~le-Tyr-VaT-Glu-Trp-Tha:-Asn-Asn-Gly-Lys-T~-Clu-Leu-Asn-Tyr- ' I T 1 9T2 t 1 ; -I c 1 9C2,. 9c3 '-77r' -, '77 I 9S g 5 I 20 Lys-Asn-Thr-Gln-Pro-Val-Leu-Asp-Ser-Asp-Gly-Ser-Tyr-Phe-Rsr T----?.?-r' 9T I - L. - - I r -r 9c4 '9s ;L- 7-7 I I Fig. 5. Amino-acid sequence of fragment H9. The first residues were directly determined with an automatic sequencer. Sequence of peptides was done by the dausyl-edman (7) method and by the use of carboxypeptidases A and B (-). In the case of H9 fragment a kinetic was made (5 min-1 h at25 "C) (1) c u' 0 - E! E 0. = Effluent volume (ml) w io - m al c f 2 0.1c < 0.0S 0 0 1x) Effluent voiurne (ml) Fig. 6. Separation of peptides obtained by chymotryptic and acid cleavage. (I) Separation of chymotryptic peptides from 20mg of the H10 fragment on Dowex 50x2. Incubation proceeded at 37 "C for 25 min. Enzyme-substrate ratio: 2O/, (w/w). (11) Separation of peptides resulting from acidic cleavage of 20 mg of H10 with 6 N HCl, at 37 "C for 18 h, on resin P. Elution conditions as on Fig.1. Absorbance at 570 nm after alkaline hydrolysis and ninhydrin coloration

9 A. Bourgois, M. Fougereau, and J. Rocca-Serra 431 Table 4. Amino-acid composition of tryptic peptides from HI0 fragment Results me expressed in number of residues per mole of peptide. Yield is based on nanomoles of peptides isolated compared with nitnomoles of Hi0 fragment digested. Electrophoretic mobility and net charge at ph 6.5 are given Aminoacid ~~~~ ~ Amount in peptide Sum H10 l0tl lot2 10T21 10T22 10T3 10T4 10T5 10T6 10T7 10T8 10T9 Tl-T9 residues/mol peptide Carboxamidomethylcysteine Asparticacid asparagine Threonine Serine Glutamic acid or glutamine Proline Glycine Valine Leucine Tyrosine Phenyl alanine Histidine Lysine Arginine TrvDtophan 1& 1& 1.4b Total "0 Yield Nobility Charge ti I +I 0 a Determined by fluorescence. b Determined by spectrometry. assignment of two residues at position 30 and 31. Since peptide lot7 contains only one leucine, at position 3 (position 26 of the Ht0 fragment), positions of peptides ioa3 and loa4 are easily deduced. The histidyl residue (position 27 of H10) is contained in 10A5, which allows sequence determination up to residue 6 of lot7 (29 of H10). That sequence Thr- Lys of peptide loa6 must be at the COOK-terminus of 10T7 is confirmed by analysis of the chymotryptic peptide 10C4, which contains the overlap iot7 to 10T8. Thus, only two positions could not be determined in 10T7 and therefore in fragment H10: positions 30 and 31, Val-Ser or Ser-Val. Net charge determination of trvyptic peptides and of the chymotryptic peptide ioc2 allowed straight forward identification of all acidic and amide residues of the H10 fragment. DISCUSSION The amino-acid sequence of the entire Fc fragment of the MOPC 173 IgG2a immunoglobulin molecule is presented on Fig.8. Results described in detail in this paper extend from residue 235 (in Eu numbering) to the COOH-terminal residue 446, with the exception of the H5 data published separate- Em. J. Biochem. 43 (1974)

10 ~ ~~~~ 432 Sequence of a Mouse Fc Fragment Table 5. Amino-acid composition of acidic and chymotryptic peptides from fragment HI0 Results are expressed in number of residues per mole of peptide. Yield is based on nanomoles of peptides isolated compared with nanomoles of Hi0 digested. Electrophoretic mobility and net charge at ph6.5 are given Aminoacid Amount in peptide loal 10A2 10A3 10A4 10A5 10A6 loa7 loa8 loc1 10C2 loc3 10C4 1OC5 10C6 residues/mol peptidc Carboxamidomethylcysteine 0.9 Sspartic acid or asparagine 1.o 1.0 Threonine o 1.1 Serine Glutamic acid or glutamine Proline 1.o 0.9 Glycine 1.o o Valine Leucine Tyrosine Phenyl alanine Histidine Lysine o 1.0. I Arginine ~~ Total % Yield Mobility 0 Charge 0 _- ly [18]. Only one deletion had to be included for homology, at position 355 (Eu numbering). The adjacent sequence of the hinge region previously reported [3,4] is also included in Fig.8. Comparison of the MOPC 173 Fc sequence with homologous regions of a human yl chain [16,17] is in complete agreement with the order of the CNBr fragments which was proposed in the first paper of the series [2], and that was based upon the conservation in evolution of cysteyl residues and typical linear arrangement of intra-chain disulfide bridges [19,20]. By looking at homologies between the three species (Fig.8), it can be seen that a clear contrast is apparent whether one considers the inter-heavychain region or the Fc fragment itself. As already pointed out [19], the inter-heavy-chain region is high- ly isotype and species-specific, and therefore, no particular homology is expected at this level. Sequences immediately adjacent to the hinge region are clearly homologous between the three species compared, although the intraspecies sub-class diversification may slightly hinder a straight forward vertical comparison [21]. For instance one cannot exclude the possibility (although it does not seem very likely from the data published so far), that a given C gene product be under particular strong selective pressure that would insure maximal interspecies homology to a very restricted number of sub-classes. When rabbit, human and mouse Fc sequences are compared (Fig.8), it can be seen that extensive stretches appear highly conserved in evolution. On the proposals that the general evolution of immuno- Eur. J. Biochern. 43 (1974)

11 A. Bourgois, M. Fougercau, and J. Rocca-Serra 433 bo 20 Tyr-Ser-Lys-Leu-nrg--Val-Gl~-Lys-Lys-Asn-Trp-V~l-Glu-AP-g-Asn-Ser-Tyr-Ser-Cmc*-Ser- H10 t lotl 10T2 la 10T4 10T 5 1oT21,, 1m22, t 1x2-1 c-2% L loal, c Val-Val- iis-gln-gly-leu-his-asn-his(val,se~)thr-lys-~er-phe-ser-a~g-tlir-p~o-g~y I 10T6 10T t I C A4. 10C5 I kf=$ -7 10A2 7777' '7 a A3 I C6 '-7 7 i i A5,lOA6,, 10A7 I 10A8 Fig. 7. Amino-acid sequence of HlO. Apart from the beginning of the fragment, sequence was manually determined by the dansyl-edman method (-) I kbbit yg D D UP DE(V d Q Z)F I TSHF W Y VI IB N B V E EN. Q V R T Q E D P Q V K F N W Y V D G V Q V H N 420 Fig.8. Comparison of the sequences of the hinge region and of the Pc fragment of the mouse IgGZa heavy chain with the corresponding regions of the human yl chain [15] and that of the rabbit y chain [16,17]. Boxes indicate positions that are identical between the three species. A, alanine; G, glycine; E, glutamic acid; P, proline; T, threonine; I, isoleucine; S, serine; K, lysine; C, cysteine; D, aspartic acid; M, methionine; N, asparagine; L, leucine; V, valine; Y, tyrosine; W, tryptophan; I?, phenyl alanine; H, histidine; Q, glutamine; R, arginine

12 434 Sequence of a Mouse Fc Fragment Table 6. Interspecific identities for domains VHIII, C H and ~ CH3 This table was computed from sequences presented on Pig.8 and 9 Domains Number of residues Identical positions Additional identical positions between 2 species between the 3 species human/rabbit or human/mouse mouse/rabbit or human/guinea pig mouse/guinea pig CH2 107 (232 to 338) 63 ch3 108 (339 to 446) 46 VHIII 108 (1 to 108) T I S R N D S K N T L Y L N M N S L R P Z B T A I F l N u A v 1 Q [ S K V N i F l T I S R D D G K N T L Y L Q M N S L R E D T A Fig.9. Comparison of the sequence of the variable region of the mouse y2a heavy chain (MOPC 173 (51) with that of the human VH~II subgroup and that of a Guinea pig y2 chain (291. For basic sequence of VHIII residues 1 to 34 see [27 and 281 and sequence of protein Nie [27] for residues 35 to 108. For one-letter notation of amino acids see legend to Fig. 8 globulin chains emerged through successive duplications of an ancestor gene coding for about 110 amino-acid residues [17], Edelman proposed that each of these regions was folded within a compact domain which would have been selected for in evolution for a given function [22]. When homologous regions of the Fc fragment are compared (Fig.8), it can be observed (Table 6) that the ch2 domain (residues 232 to 338) seems to be more conserved than the ch3 domain (residues 339 to 446). Although the nature of the function and/or conformation requirements on which the selective pressure has been acting is not known, this observation suggests that the weight of selection may differ from one homology region to the other, in aggreement with the proposals that each domain evolved independently [22]. As the ch2 domain seems to be involved in complement fixation [23,24], it might be thought that stronger conservation of this region be related to maintaining this function. That such a simple linear relationship between the aminoacid sequence and a given function cannot be established is amply proven by the fact that the same degree of homology applies to the ch2 domain of the human y4 chain [25], which is known not to fix complement. It was previously shown that the VH region of MOPC 173 chain was strikingly homologous to human VHI~I subgroup [26]. Such a comparison is extensively presented on Fig. 9, together with that of t,he sequence of the guinea pig y2 chain [29]. The degree of conservation of this VH domain is of the same order of magnitude as that observed for the constant region (see Table 6): 58 residues (out of 108) are identical between the three species whereas 63 are identical for ch2 and 49 for ch3. Variable positions are grouped in hyperdiversity regions [30] in the VH domain as opposed to a relative widespread pattern in the constant regions. This implies that, if one excepts the

13 A. Bourgois, M. Fougereau, and J. Rocca-Serra 435 hypervariable region of the VB. domain, the conservation of large continuous stretches of sequence is more pronounced in the V region than in the C region. This observation leaves little space for speciesspecific residues [30] and strongly suggests that selective pressure is just as high (if not higher) on the V region and on the C region. This situation, even though it may not apply to all V regions (see for instance the complexity of the mouse Vx system [31]) should be kept in mind when discussing theories of variability [ As Hood and Talmage pointed out : "if multiple genes for the common region of the 1c chains existed in this ancestor (to human and mouse), then it is difficult to explain how in the face of the normal mutation rate all copies within each species maintain the same amino-acid sequence". The same reasoning (evidently applied in a broader sense of general conservation) should apply to the V region as well, and should be taken into consideration to account for the striking parallel conservation in evolution of both the V and the C domains. If one accepts this parallelism (and in the present case, it seems difficult to avoid the evidence) one is led to propose either that both V and C regions are encoded by a limited number of genes, the variability being gained by somatic processes or that, if variability is indeed germinal, it may be not so obvious that C regions cannot be encoded by multicistronic systems. In this provocative situation one then might be expected to find more isotypic variants of the constant regions such as the Oz [361 and the Kern systems [373 than those described to date. Skilful technical assistance of Miss Michele Milili is greatly acknowledged. We are also grateful to J. If. Kehoe for having performed partial sequence of the H6 fragment with a sequenator. This work was partially supported by a grant of the Eondation pour la Recherche Mkdicale Erangaise. REFERENCES 1. Potter, M., Appella, E. & Geisser, S. (1965) J. Mol. Biol. 14, Boureois. A. & Foueereau.. M. (1970)., Eur. J. Biochem. 12," " 3. de Preval, C., Pink, J. R. L. & Milstein, C. (1970) Nature (Lond.) 228, de Preval, C. & Fougereau, M. (1972) Eur. J. Biochem. 30, Bourgois, A., Fougereau, M. & de Preval, C. (1972) Eur. J. Biochem. 24, Gibbons, I. & Perham, R. N. (1970) Biochem. J. 116, Jolles, J., Jauregui-Adell, J., Bernier, I. & Jolles, P. (1963) Biochem. Biophys. Acta, 78, Milstein, C. (1966) Biochem. J. 101, Offord, R. E. (1966) Nature (Lond.) 211, Goodwin, T. W. & Morton, R. A. (1946) Biochem. J. 40, Gray, W. R. (1967) Methods Enzymol. 11, Capra, J. D. & Kunkel, H. G. (1970) Proc. Nut. Acad. Sci. U. S. A. 67, Edman, P. & Sjoquist, J. (1956) Acta chem. Scad. 10, Van Orden, H. 0. 8: Carpenter, F. H. (1964) Biochem. Biophys. Res. Commun. 14, Edelman, G. M., Cunningham, B. A., Gall, W. E., Gottlieb, P. P., Rutishauser, U. & Waxdal, M. J. (1969) Proc. Natl. Acad. Sci. U. S. A. 63, Fruchter, R. G., Jackson, S. A., Mole, L. E. & Porter, R. R. (1970) Biochem. J. 116, Hill. R. L.. Lebovitz. H. E.. Fellows. R. E.. Jr & Delaney, R: (1967) in Gamma Globulins, (J. J. Killander, ed.), pp , Almqvist and Wiksell, 18. Kehoe, J. M., Bourgois, A., Capra, J. D. & Fougereau, M., Biochemistry, in press. Frangione, G., Milstein, C., & Pink, J. R. L. (1969) Nature (Lond.) 221, Gall, W. E. & Edelman, G. M. (1970) Biochemistry, 9, Milstein, C. & Pink, J. R. L. (1970) Progr. Bwphys. Mol. Biol. 20, Edclman, G. M. (1970) Biochemistrv, 9, Icehoe, J; M., Fougereau, M. & Bouiiois, A. (1969) Nature (Lond.) 224, Ellerson,'J. R., Yasmeen, D., Painter, R. H. & Doming ton, K. J. (1972) PEES Lett. 24, Pink, J. R. L., Buttery, S. H., De Vries, G. M. & Milstein, C. (1970) Biochem. J. 117, Bourgois, A. & Fougereau, M. (1970) FEES Lett. 8, Ponstingl, H., Schwartz, J., Reichel, VV. & Hilschmann, N. (1970) Hoppe-Seyler's Z. Physiol. Chem. 351, Pink, J. R. L. & Milstein, C. (1969) in Gamma Globulins. Structure and Biosynthesis (F. Franck & D. Shugar, eds) Academic Press, London. 29. Cebra, J. J., Ray, A., Benjamin, D., Birshtein, B. (1971) in Progress in Immunology (B. Amos, ed.), pp , Wu,T.T. & Kabat,E.A. (1970) J. Exp. Med. 132, Hood, L., McKean, D. & Potter, M. (1970) Science (Wash. D. C.) 170, Hood, L. & Talmage, D. W. (1970) Science (Wash. D. C.) 168, Gallv, J. A. & Edelman, G. M. (1970)., Nature (Land.) 227, Jerne, N. K. (1971) Eur. J. Immunol. 1, Cohn,M. (1971) Ann. N. Y. Acad. Sci. 190, Ein, D. (1968) Proc. Natl. Acad. Sci., U. S. A. 60, Hess, M., Hilschmann, N., Rivat, L., Rivat, C. & Ropartz, c. (1971) Nature New Biol. 234, Weber, K. & Konigsberg, W. H. (1967) J. Biol. Chem. 242, A. Bourgois, M. Fougereau, and J. Rocca-Serra, Centre de Biochimie et de Biologie Molkculaire du C.N.R.S., 31 Chemin Joseph-Aiguier, F Marseille-Cedex-2, France