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line V genes (reviewed by Baltimore, 1981; Gearhart, 1982). However, the work of Bothwell et al. (1981)on the nucleotide sequences ofVH genes neighbouring ...
The EMBO Joumal Vol. 1 No.5 pp.635-640, 1982

Immunoglobulin V region variants in hybridoma cells. Recombination between V genes Renate Dildrop*, Marianne Bruggemann, Andreas Radbruch, Klaus Rajewsky, and Konrad Beyreuther Institute of Genetics, University of Cologne, Weyertal 121, D-5000 Cologne 41, FRG Communicated by K. Beyreuther Received on I June 1982

The mouse hybridoma line B1-8.61 secretes a monoclonal IgD, Xl anti44-hydroxy-3-nitrophenyl)acetyl (NP) antibody with defined idiotypic determinants. Two spontaneous V-region variants (B1-8.V1/V2) with altered idiotope pattern were selected and the structural variation was located to the variable region of the heavy chain. The amino acid sequences of the B1-8.61 and variant heavy chain V regions were determined. The variant VH regions are identical. Wild-type and variant VH regions differ in 10 positions. Single amino acid exchanges are found in the first and second framework at positions 20 and 43. The majority of replacements (eight substitutions) is clustered in the second complementary-determining region (CDR 2). There are no differences in CDR 1 and CRD 3 and the JH region. The variant, which at first glance appears to have undergone a series of point mutations, arose by recombination, possibly gene conversion, between the rearranged VDJ gene of the wild-type (B1-8.61) and a neighbouring germ line VH gene encoding all of the substitutions. Key words: V-region variant/protein sequence/recombination between VH genes/somatic diversification

Introduction Immunoglobulin variable regions are composed of individual genetic elements belonging to different multigene families. The random somatic combination of these germ line segments produces much of the diversity exhibited by immunoglobulins at the protein level (Brack et al., 1978; Sakano et al., 1979, 1980; Max et al., 1979; Early et al., 1980). Superimposed on this recombinatorial joining process generating variation particularly in the third hypervariable region are point mutations which amplify antibody diversity at the somatic level (Weigert and Riblet, 1976; Bernard et al., 1978; Pech et al., 1981; Bothwell et al., 1981; Crews et al., 1981). The analysis of somatic mutation(s) occurring after V gene rearrangement is the subject of the present study. Spontaneous somatic variants of the hybridoma line Bi -8.61 which have lost an antigenic V-region determinant (idiotope) were isolated by cell sorting using monoclonal anti-idiotope antibodies. Two representative variants, B1-8.V1 and B1-8.V2 were chosen for further investigation. They carry a mutation in the variable region of the heavy chain and are indistinguishable from each other in terms of isoelectric focussing pattern and by serological analysis (Bruggemann et al., 1982). The wild-type line B1-8.61 (IgD/AI, anti-NP) was derived as a spontaneous class switch variant from the hybridoma B1-8 /4 (IgMAl, anti-NP) (Neuberger and Rajewsky, 1981). *To whom reprints requests should be sent.

© IRL Press Limited, Oxford, England. 0261-4189/82/0105-0635$2.00/0.

II.

The sequence of the B1-8A VH region (Bothwell et al., 1981) is used as reference sequence for the present studies in which we determine the amino acid sequences of wild-type and variant heavy chain V regions. We present evidence that recombination occurred in vitro between the rearranged VH gene expressed in B1-8.61 and another VH gene in the same VH gene cluster. Results Isolation of cyanogen bromidefragments IgD antibodies secreted by the B1-8.61 cell line and the two V-region variants are dimers composed of two heavy and light chains linked by single interchain disulphide bridges. The apparent mol. wt. determined by SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions is 22 500 for the light chain and 60 000 2000 for the glycosylated heavy chain. In the schematic CNBr peptide pattern of the Bl-8-IgD molecule in Figure 1 the intra- and interchain disulphide bridges are indicated. Most light chain fragments and heavy chain constant region fragments are linked to each other by disulphide bridges. The three CNBr peptides corresponding to the variable region of the heavy chain differ from the former in size and can be easily purified by gel chromatography. The CNBr peptides derived from the parental and the two variant proteins gave similar elution profiles when separated on Sephadex G-50. Fractions containing variable region fragments were pooled as indicated in Figure 2, desalted, and lyophilized. CNBr peptides HI and H3 were rechromatographed on the same column after complete reduction and carboxyamidomethylation. Amino acid composition of CNBr fragments Aliquots of the three isolated CNBr fragments were hydrolyzed and applied to a Beckman 121 M amino acid analyzer. The amino acid compositions of parental and variant peptides were compared to those of the B1-8y progenitor of the B1-8.61 parent (Table I). The analysis of fragment HI (1-34) shows a single Leu-Val exchange in the two variants. Fragment H2 (35- 81) carries several substitutions. Fragment H3 ~sm

rS

s

r-

l

oI9-s-

Q

1 H2

HI

H3

o laaI S

H4

I -5

5

V-Region

1

s

HlO

S

I

---s

----nP) LS

LI

Ls

HS8H9

H6

H5

I

a

I_

Cgl

L

S

s

L2 L

aI

-5J

r

C-Region

Fig. 1. CNBr peptide pattern of B1-8.61 and variant antibodies. The alignment of the heavy chain CNBr fragments and the positions of carbohydrate attachment sites (open arrow heads) were previously described by

Dildrop and Beyreuther (1981).

635

R. Dildrop et al.

protein fragments. This is suggested by the presence of isoleucine and histidine in these preparations. Sequence and peptide analysis The HI fragments were completely blocked by N-terminal pyroglutamic acid and partial blockage was encountered in the case of the H3 fragments. Therefore, only fragment H2, which was not blocked, was sequenced directly by automated Edman degradation. Tryptic, thermolytic, and tryptic-thermolytic double digestions were performed with all CNBr fragments and peptides were separated two-dimensionally on cellulose thin-layer plates. The isolated peptides were identified according to their electrophoretic mobilities at different pH values and further characterized by amino acid analysis and manual Edman degradation. The result of the sequence and peptide analysis is illustrated in Figure 3 where residues identified by sequencing and peptides analyzed in amino acid composition are indicated. The B1-8.61 VH region shows no differences to the B1-8y reference sequence. Sequence comparison of the two variants B1-8.V1 and B1-8.V2 indicates that they are identical and presumably represent the progeny of the same mutated cell. The sequences of the B1-8.61 parent and the V-region variant are compared in Figure 4. The variant carries 10 substitutions distributed over the V region between positions 20 and 66. Most of the exchanges are located in the second hypervariable region. There are no differences in the D and J region. Comparison ofprotein sequences with germ line VH sequen-

(82- 128) seems to be unaltered in amino acid composition at least for the B1-8.V2 derived peptide. The differences in the B1-8.61 and B1-8.V1 derived H3 fragments are most likely due to contamination of the peptide preparations with other

1.0

H8-HIO 100

80

60

120

140 Fraction number

ces

From the DNA sequences of the NP-VH gene cluster (Bothwell et al., 1981) we know that the B1-8.61 parent expresses a germ line-encoded VH gene segment (V186.2). Comparing the variant sequence with those of the other members

Fig. 2. Elution profile of CNBr-cleaved B1-8.61 and variant proteins. Gel filtration of 100 mg CNBr-cleaved protein was performecd on Sephadex G-50 (2 x 200 cm column) in 6 M urea, 0.1 M formic acid. Fractions of 3 ml were collected and variable region peptides of the heavy chains were pooled as indicated. -

Table L. Amino acid compositions of purified CNBr fragments

HI B1-8 61

VI

is 1 2 4

0.9 2.0 3.8

0.8 2.0 3.8

6 2 3 3 3

5.7 2.1 3.5 3.2 2.9 0.1 3.0 1.9 1.0 0.2 nd 2.6 0.1 nd

5.5 2.1 3.3 3.1 3.7

B1-8 Asx CMC Thr Ser Glx HS Pro

Gly Ala Val

Ile

-

Leu Tyr Phe His HSL

3 2 I

Lys Arg Trp

-

+ 3 -

1

H3

H2 B1-8 61

VI

A

0.7 1.9 3.7

4 4 4

3.8 4.1 4.2

4.7 4.3 4.0

5.5 1.8 3.1 3.3 3.9

4 3 5 2 2 2 2 2 I 1 + 6 3 2

4.1 2.7 4.8 2.5 1.9 1.5 2.4 2.7

4.8 3.1 3.6 2.7 2.0 1.6 2.5 2.2 1.1 1.8 + 4.6 1.7 +

V2

-

-

2.0 2.0 1.1 0.1 + 2.7

2.1 2.0 1.0 -

+ 2.6

-

-

nd

+

B1-8

i.2 0.9 nd 5.5 2.5 +

B1-8 61

VI

V2

A

5.0 -4.1 3.9

6 4 8

4.9 3.8 6.8

5.3 4.6 7.2

5.3 4.0 7.6

4.6 3.2 4.02.2 2.0 1.6 2.2 2.3 1.1

5 1 5 2 2

4.8 1.5 5.2 3.0 2.3 0.5 3.2 5.4 1.4 0.4 + 1.7 1.1 nd

4.8 1.2 5.2 2.7 2.2 0.3 3.6 5.9 1.2 0.3 + 1.1 0.9 +

4.8 1.0 5.2 2.2 2.0

V2

1.77 +

5.0-4.1.94.+

B1-8

-

3 7 1 -

+ 1 1 1

-

3.3 6.8 1.1 -

+ 1.1 1.0 +

CMC; carboxyamidomethyl-cysteine; HS, homo-serine derivative of methionine; HSL, homo-serine-lactone derivative of methionine; +, qualitatively determined; nd, not detected. The B1-8i compositions were calculated according to the cDNA sequence of Bothwell et al. (1981). Composition of fragment H3: residues (82-120), Bothwell et al., 1981; residues (121-128), &-constant region, this work.

636

B1-8,t

.-

Recombination between VH gnC

(1) QVQLQOPGAELVKPGASVKLSCK(23)

B1-861

Spe

1) OVOLOOPGAELVKPGASVKVSCK (23)

_.

--4o

_ _~~~~~~~--

124) ASGYTFTSYWMHWVKORPGRGLEWIGR (50) -

,-

. 4-----

--

FT---

_V --7 -V-7

-4-4--*

_HS7 _

151) IOPNSGGTKYNEKFKSKATLTVDKPSSTAYM 181) -_ --. _-

(I1)

-(50

_S

-4

N-- -7__ KK-L _ _ 7

(82)

_-

*

_

_

lHPSDSDYNYNQKFKGKATLTVDKPSSTrAYM (8)

-7 -7

(82)OLSSLTSEDSAVYYCARYDYYGSSY (106) _

-

4~~~~~9

-.

4,-,

-7

7-T-

124) ASGYTFTSYWMHWVKORPGQGLEWIGRR

-

--7-7

-

OLSSLTSEDSAVYYCARYDYYGSSY (106)

_

-7 -7

471-4 7-

7

-

7-

Wt7)

FDYWGQGTTLTVSSGNEKGPDM (128)

(107)

FDYWGOGTTLTVSSGNEKGPDM (128) -7 7-

-

*

-

7--- 7

77-7 7

_

> -_ C

V REGION

-REGION

Fig 3. Sequence and peptide analysis of B1-8.61 and two variant VH regions. Peptides whose amino acid composition was detrmined are indicated by thick arrows

(pep) and each residue identified by automated or manual Edman degradation by thin arrows (seq). 2n

10

BI-8

Q V Q L

Variant

- - - - - - - - - - - - - - - - - - - V

Q P GA E L V K P

30

S V K L S C K

C. A

-

A

S

G Y

CDRI

T F T SY W

40 HiW V

K

Q

R

V 186.2

V 102

- - - - - - - - - - - - - - - - - - -

0

Variant S0

COR2

P G R G L I W1 I G^ R I D P 11 S G G T K r NI E K F K S K A T L T V D It P S S T A Y - -

Q

-

- - - - - - -

H

-

S D SD

-

N

100

90

Q L S S L T S E D S

- -

A

V

Y V

CAR

Y

D

V

Q

- - -

G

OIJO 0

80

70

60

0

1

20

40

60

80

98

- - - - - - - - - - - - - -

D Y G s sY F D Y

110

xG Q G

J

120

T T L T V S S

Fig. 4. Amino acid sequence comparison of the BI-8.61 parent and the variant VH region.

of the VH gene family (Figure 5) we discovered that the substituted amino acids are all encoded by the VH gene V102. This gene is distinguished from the V186.2 gene by two additional non-silent substitutions (codons 75 and 98). At these positions the variant sequence is identical to the parentally expressed V186.2 gene (in codon 1 of the published V102 gene sequence the third base turned out to be G instead of T; A. Bothwell, personal communication). We conclude that the V-region variant expresses a modified V186.2 gene whose 5' end, at least up to codon 66, has been substituted by the correspnding part of the V102 gene. Discussion

Primary structure and variant phenotype The present data establish that the VDJ regions of the two

Fig. 5. Comparison of the variant V region protein sequence with germ line encoded VH gene sequences. Sequence differences between the V186.2 and V102 VH genes are indicated with black and white boxes. The V-region variant expresses all except two of the different amino acids encoded by the V102 gene, in these positions the corresponding amino acids of the parentally encoded V186.2 gene were found.

somatic immunoglobulin variants BI-8.VI and -V2 are identical in amino acid sequence and differ from the VDJ region of the B1-8.61 wild-type by 10 amino acid substitutions. The results obtained by amino acid sequencing are in line with the following observations. First, the amino acid compositions of the three CNBr fragments that comprise the VDJ regions of the three proteins agree within experimental error with the sequence analysis. Second, identical sequences are found for the two variants; this agrees with the demonstration that the variants are indistinguishable serologically and in isoelectric focussing pattern (Bruggemann et al., 1982). Third, the amino acid sequence of the VDJ region of the Bl-8.61 antibody agrees in all positions with the nucleotide sequence of the VDJ segment expressed in the original Bl-8y cell line (Bothwell et al., 1981). Because of their sequence identity we consider it likely that the two variants Bl-8.V1 and -V2 derive from the same mutant cell in the BI-8.61 population. The antibody secreted by 637

R. Dildrop et al.

this cell is mutated, in that 10 amino acid substitutions are found within the N-terminal 66 residues of the heavy chain. Which of these substitutions could be responsible for the variant phenotype which is characterized by the loss of an idiotypic determinant (idiotope Ac 146, Bruggemann et al., 1982). The first substitution is in the first framework at position 20 altering a buried residue of the antiparallel strand 2 of the four-stranded ,B sheet (assignment according to Amzel and Poljak, 1979). There is no reason to think that the conservative replacement at this position of a leucine by a valine residue influences the idiotypic specificity of the variant protein. The second framework of the variant heavy chain has acquired a mutation at codon 43 resulting in the exchange of arginine by a glutamine residue. In this case, a "surface" residue possibly located at the end of the fourth "back loop" (bl 4 according to Amzel and Poljak, 1979) connecting strands 3 and 4 of the five-stranded :-pleated sheet (3(1-3 35) is affected. The loss of a positive charge at this position is very close to positions 101 and 102 of the light chain. The majority of the amino acid substitutions in the variant, eight of the 10 amino acid replacements, are clustered in the second hypervariable region (positions 52- 66). They are found at positions 52 (aspartic acid to histidine), 54 (asparagine to serine), 55 (serine to aspartic acid), 56 (glycine to serine), 57 (glycine to aspartic acid), 59 (tyrosine to asparagine), 62 (glutamic acid to glutamine), and 66 (serine to glycine). Only half of the alterations in CDR 2 represent isopolar replacements of amino acid residues. Clearly the substitutions in CDR 2 are most likely to be responsible for the loss of idiotope Ac146, in the variant. However, as discussed in the preceding paper (Bruggemann et al., 1982), this does not imply that in the wild-type, sequences of CDR 2 are selectively or even directly involved in the construction of idiotope Ac146. Idiotopes appear to be complex structures and the drastic changes in CDR 2 in the variant may change the conformation of other parts of the variable region. Together with idiotope Ac146, the variant has largely lost its NP-binding property (Bruggemann et al., 1982); again one would like to attribute this to the alteration in CDR 2. In this case there is a suggestion that the aspartic acid in position 52, changed to histidine in the variant, may be directly involved in hapten binding (Reth et al., 1981). V gene recombination instead of point mutation A minimum of 11 base substitutions is required to convert the B1-8.61 VDJ gene into that expressed in the variant. The two amino acid substitutions in the framework and seven of the eight replacements in CDR 2 could be achieved by single base substitutions. Only the replacement of serine 55 by aspartic acid requires two base substitutions. Thus, at first glance, the variant appears to be derived from the wild-type by a series of point mutations and to resemble, in this respect, previously described somatic immunoglobulin variants which were identified by comparing variable regions expressed in myeloma and hybridoma cells with those encoded by germ line V genes (reviewed by Baltimore, 1981; Gearhart, 1982). However, the work of Bothwell et al. (1981) on the nucleotide sequences of VH genes neighbouring the VH gene expressed in the Bl-8,u cell line allows us to interpret the mutation in our variant cells in an entirely different way, namely as a recombination between the rearranged VH gene (V186.2) and a neighbouring VH gene (V102). The multitude of the sequence differences between wild-type and variant and their perfect fit with the V102 sequence make it clear beyond doubt that this

638

interpretation must be correct. Recombination between the two genes must have occurred in the third framework, between codon 66 (encoding the last V102-specific amino acid in the variant heavy chain) and codon 75 (which encodes a V186.2-specific proline residue) (Figures 4 and 5). In this region the two genes exhibit almost complete homology. Sequence homology was also encountered in other cases of somatic recombination between antibody structural genes. It has been made responsible for a recombination resulting in a recombinant heavy chain constant region (Birshtein et al., 1980) and seems to play a role in immunoglobulin switch recombination (Honjo et al., 1981) and in V-J joining (Brack et al., 1978). Sequence homology is also a prerequisite for the process of gene conversion which occurs at high frequency in yeast and involves two recombination events (reviewed by Radding, 1978). The variant described in this paper could well be the result of gene conversion since the V186.2 and V102 genes exhibit striking sequence homology not only 3' but also 5' of CDR 2 and this homology extends into the 5'-flanking region (Bothwell et al., 1981). Conversion between the two genes would result in part of the rearranged "acceptor" V186.2 gene becoming identical in sequence to the "donor" gene V102, the latter remaining unchanged. We initially favoured gene conversion in the interpretation of the B1-8.61 variant since the V102 gene product was thought to have histidine at the N-terminus, in contrast to the product of V186.2 (Bothwell et al., 1981). We now know that both genes encode glutamine at this position (A. Bothwell, personal communication). However, restriction analysis of the cloned rearranged VH gene of the variant again indicates that the mutation involved a double crossover event (U. Krawinkel, personal communication). Clearly, analysis of the B1-8.61 variant at the level of the DNA is needed to clarify this matter, and is presently underway. The V186.2 gene and its neighbours are of C57BL/6 origin (Reth et al., 1978) and B1-8.61 cells carry only a single C57BL/6 Igh locus (Sablitzky et al., 1982). A recombination between V186.2 and V102 would therefore have to occur between sister chromatids or between the two genes on the same chromatid. It cannot be excluded, however, that the B1-8.61 hybridoma carries a V102-like gene on a BALB/c-derived chromosome and that the variant cells underwent transchromosomal recombination. V gene recombination and antibody diversity The data presented in this paper again raise the old question of whether recombination between V genes contribute to antibody diversity (Edelman and Gally, 1967; Smithies, 1967). Gene conversion is particularly attractive as a mechanism for generating and limiting diversity in multigene families (Seidman et al., 1978; Slightom et al., 1980; Baltimore, 1981) and recent data suggest that it may indeed have played a role in the evolution of antibody V and C genes (Clarke et al., 1982; Schreier et al., 1981). In the case of the variant described in this paper, recombination takes place in a somatic cell, between a V gene which is presumably in the germ line context and a rearranged, expressed V gene. Does this mechanism contribute to the somatic generation of the antibody repertoire in the immune system? The somatic mutations so far identified in antibody V genes have been interpreted as point mutations generated by a special mechanism which operates at some step of B cell differentiation (Bothwell et al., 1981; Gearhart et al., 1981; Crews et al., 1981). This interpretation is supported by two pieces of evidence, namely that V

Recombination between VH genes

genes carrying the corresponding mutations were not found in the germ line and that, in some instances, mutations also occurred in the flanking sequences, including those downstream of the rearranged J segment. The latter finding strongly argues against V gene recombination being involved in the generation of the corresponding mutants. The former represents negative evidence and is thus not definitive. In addition, somatic mutation has so far mostly been studied in families of antibodies which had similar antigen binding specificities. Mutations of the type encountered in the present variant which expresses changed antigen binding specificity may therefore have gone unnoticed. The question of whether recombination between V genes is a frequent event in differentiating B cells can thus only be resolved by direct experiments. Recombination between V genes (including gene conversion) would be an efficient mechanism by which antibody diversity could be expanded somatically. It allows, for example, as exemplified by the present variant, to re-assort CDRs within similar frameworks. Kabat and his coworkers (Kabat et al., 1978, 1979) have summarized the evidence for such reassortment as derived from V region amino acid sequences. It also assigns a biological role to "pseudo" V genes which appear to represent a large fraction of the V genes carried in the germ line (Givol et al., 1981; Bothwell et al., 1981). The recombinatorial process which has occurred in the B1-8.61 variant may operate most efficiently within V gene families exhibiting strong sequence homology such as the NP-VH family. Since apparently the products encoded by at least some of the latter genes (example given V102 and V186.2) share idiotypic properties but differ in antigen-binding specificity, recombination between the genes would result in the production of a highly heterogeneous collection of VH regions which would be idiotypically related but associated with different antigen binding specificities. It is attractive to think that the idiotypic network (Jerne, 1974) might be involved in the selection of somatic variants of this type.

Materials and methods CNBr cleavage Affinity-purified IgD antibodies (Bruggemann et al., 1982) (62A2) were cleaved in 70Gb (v/v) formic acid with a 5-fold excess of CNBr over protein (w/w) for 24 h at 8°C and dried under a stream of nitrogen. The fragments were separated on Sephadex G-50 superfine (2 x 200 cm column) in 6 M deionized urea, 0.1 M formic acid. Pooled fractions were desalted on Sephadex G-10 (3 x 40 cm column) in 0.5 M formic acid and lyophilized. Peptides linked to each other by disulphide bridges were rechromatographed after complete reduction and carboxyamidomethylation. Tryptic and thermolytic digestions Isolated CNBr fragments were digested with trypsin and/or thermolysin using a ratio of enzyme to protein of 1:50 (w/w) in 50 mM ammonium hydrogen carbonate, 6 mM calcium chloride, pH 8.0-8.5 for 12-15 h at 37°C. After lyophilisation the resulting peptides were separated two-dimensionally by a conventional fingerprint technique on cellulose thin-layer plates (20 x 20 cm, Scheicher and Schull) (Beyreuther et al., 1977). First dimension: electrophoresis at pH 2.1 in acetic acid/formic acid/water (4/1/50, v/v/v) or at pH 6.5 in pyridine/acetic acid/water (100/4/900, v/v/v) at 20 V/cm for 75-90 min. Second dimension: ascending chromatography in pyridine/butan-lol/water (35/35=30, v/v/v) at 220C for 16 h. The plates were sprayed with 0.05% (w/v) fluorescamine, 0.507o (v/v) pyridine in ethanol and viewed under u.v. light. The electrophoretic mobilities of the peptides at various pH values were determined according to Offord (1966). The peptides were eluted either directly with a 6 N HCI, 0.1 7% (v/v) phenol, hydrolyzed, and analyzed on a Beckman 121 M amino acid analyzer or with 5007o (v/v) acetic acid for N-terminal manual Edman degradation

(Chang et al., 1978).

Sequence determination Automated Edman degradations of CNBr fragments were performed using an updated Beckman 890 B sequencer. Degradations were performed in the presence of the non-protein carrier Polybren (Tarr et al., 1978) using a 0.2 M Quadrol program (Beyreuther et al., 1977). Phenylthiohydantoin derivatives obtained after conversion using the Sequemat P6 autoconverter were identified by h.p.l.c. (Johnson et al., 1979).

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft through SFB 74 and by the Minister fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen.

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