Immunochem~stry, 1975, Vol 12 pp 50~509

Pergamon Press

Printed m Great Bntam

THE NATURE OF 'SPECIES-SPECIFIC' AMINO ACID RESIDUES* AN-CHUAN WANGL H. HUGH FUDENBERG and HERVE BAZIN Department of Me&cme. Umverslty of Cahfornm School of Medicine, San Francisco. California, U S A and the Department of Experimental Medicine. University of Louvaln, BruxeUe, Belgium (Recewed 5 September 1974)

Abstract--Amino acid sequences for the first 40 N-terminal residues of three rat 1,-type Bence-Jones (B -J ) proteins and for the first 23 residues of pooled rat immunoglobuhn hght chains were determined Comparison of these sequences with those of human and mouse x chains indicates that it is difficult to find any amino acid residue which IS absolutely 'speoes-specLfic' or "phylogenetically-assocmted' Therefore, the 'speoes-speclfic" or 'phylogenetlcally-associated' residues cannot he used as a strong argument ,n support of either the somatic mutation or the somatic recombination hypothesis regarding the genetic control of antibody varmbdity

INTRODUCTION The immune system appears to be an Ideal model for investigating the genetic mechanisms governing the &fferentlatlon in higher organisms. Each immunoglobuhn (Ig) molecule is characterized by one (or more) basic umt(s) consisting of two identical heavy (H) and two identical light (L) polypeptlde chains (Gaily and Edelman, 1972). Each L-chain consists of two homologous repeating units designated as domains (Gaily and Edelman, 1972), and each heavy chain consists of three to five of such domains (Wang and Fudenberg, 1974a) Each domain is approximately 110 amino aods in length and has an lntracham disulphide bond connecting two cysteme residues which are approximately 60 amino acid residues apart It has generally been accepted that all existing Ig genes have evolved by repeated gene duphcation and fusion from a common precursor gene coding for a polypeptlde chain which is equivalent to a domain (Hdl et al, 1966; Singer and Doolittle, 1966). Amino acid sequence studies of homogeneous Igs indicated that two genes are revolved m the synthesis of each Ig polypeptlde chain, one gene coding for the constant (C) region and another coding for the varmble (V) region of each chain (Dreyer and Bennet, 1965; Wang et al., 1970) Serological, biochemical as well as pe&gree studies showed that mul-

tlple genes control the production of both the C- and the V-regions of both the H- and the L-chams. Most investigators agree that a small number of genes are revolved m the synthesis of the Ig C-regions However, the number of genes coding for V-regions has been the center of a controversy regarding the genetic control of antibody varmbihty (Cohn, 1971). Both the somatic mutation (Lederberg, 1959; Brenner and Milstein, 1966; Jerne, 1971, Franek and Novotny, 1972; Capra et al., 1973) and somatic recombination (Edelman and Gaily, 1967; Smithies, 1970) hypotheses propose that each V-region subgroup is coded for by a single gene, and that antibody varmbthty is generated by mutation or recombination in somatic cells. In contrast, the germ-hne hypothes~s (Dreyer and Bennet, 1965; Hood and Talmage, 1970) proposes that the V-regions of each antibody are coded for by a specific pair of VH and VL genes, and that each individual has 1000 or more of V-region genes m the genome In the past, most of the structural stu&es on lgs were done on monotypIc proteins derwed from plasma cell tumors m man and m mice (Potter. 1967. Putman, 1972). Amino acid sequence data on the~ Igs showed repeated amino acid substltuhons w~thm a gwen V-region group (Hood et al, 1973) and subgroup (Wang et al., 1971); thus, in favor of the germhne hypothesis. On the other hand, "specles-speclfic' or 'phylogenetlcally-assocmted' amino acid residues have been desenbed and interpreted as evidence m support of the somatic mutation hypothesis (Kabat, 1967, Capra et al., 1973). The recent discovery of a transplantable plasma cell tumor in rats (Bazm et al, 19'72) offered an excellent opportunlty to further investigate the genetics and evolution of lg V-region genes. A comparison of the V-region amino acid sequences between two closely related species such as rat and mouse and between those of remotely related species such as rat and man or mouse and man should produce meaningful data regarding the

* This work was supported m part by USPHS grants (AI-09813 and HD-05894), National Science Foundation grant (GB-38153X), American Cancer Society grant (IM16G), USPHS Career Development Award (AI-70738) to A.C.W and the Fonds Cancerologique de la CGER, Brussels, Belgmm ACW ~s the reclplent of an American Cancer Society Faculty Research Award (FRA-125) H B is a staff member of EURATOM Biological Dwlslon, Pubhcation No. 1073 t All correspondence to. Dr An-Chuan Wang, Dept of Medicine (475-HSW), University of Callforma, School of Medicine, San Francisco, California 94143, U S A 505

506

AN-CHUAN WANG, H HUGH FUDENBERG and HERVE BAZIN RESULTS

'phylogenetically-associated' or 'species-specific' amino acid residues and their implications on the genetic control of antibody variability. This communication provides such comparison.

AND DISCUSSION

B.-J. proteins were isolated from LOU/C/Wsl rats bearing three different immunocytomas with plasma cell tumors. The B.-J. proteins from one of the transplantable tumors (IR-52) had three homogeneous MATERIALS AND METHODS fractions when they were purified by liquid chromatography on a DE-32 ion exchange column, indicating B.-J proteins were prepared from urine, serum, or ascmc the existence of charge differences among the B-J. fired of inbred LOU/C/Wsl rats bearing lmmunocytoma. The proteins were isolated by preclpltaUon wRh 65~ proteins in these fractions. Light chains were prepared ammomum sulphate followed by ton exchange chromat- from pooled IgGs from inbred LOU/C/Wsl rats withography on a DEAE-cellulose (DE-32, Whatman) column, out disease. All purified preparations were subjected and subsequently by gel filtration on a Sephadex G-100 to N-terminal amino acid sequence analysis. column (Querinjean et al., 1972). IgG molecules were preThe results of such analyses are shown m Table pared from normal LOU/C/Wsl rats by precipitation with 1. Thus far, the amino acid sequences of all B.-J. pro185/o sodmm sulphate followed by mn exchange and gel teins have been deterdmned up to position 40. Two filtratmn liquid chromatography procedures (Wang et al, 1974) The purified IgGs were completely reduced and of. the three B.-J. proteins, namely, IR-52 and IR-248, alkylated, and the resultant L and H chums were separated are identical at 37 of the first 40 positions, differing by gel filtration on a G-100 column (Wang et al., 1973a) from each other only at positions 4, 28 and 32. In Alkylation with C 14 mdoacetic acid facilitated the identifi- contrast, both IR-52 and IR-248 differed markedly from IR-158; 1R-52 differs from IR-158 at 12 posication of cysteine residues Purified B.-J. proteins and L-chains were subjected to tions, namely, positions 4, 9, 10, 15, 17, 18, 21, 22, amino acid sequence analysis on an automatic Protein 24, 25, 32 and 40; and IR-248 dtffers from IR-158 Sequencer (Beckman Model 890) according to a previously at 11 positions, (positions 9, 10, 15, 17, 18, 21, 22, described procedure (Wang et al, 1971), modified from 24, 25, 28 and 40). Immunoglobulin ~c chain V-regions that of Edman and Begg (1967) DMAA (dlmethylallyarcane trifluoroacetic acid) was substituted for quadrol-tri- have been classified into subgroups according to fluoroacetic acid since a small fraction of the latter was degree of amino acid sequence homology m man often washed together with the cleaved thiazolinone den- (Smith et al., 1971; Gaily and Edelman, 1972), mouse vative into the tubes m the fraction collector About 10 mg (Hood et al., 1973) and rabbit (Thunberg et al., 1973; of polypeptide chain were dissolved m 0"5 ml of trlfluoro- Braun and Jaton, 1973). Our data indicate that rat acetic acid immediately prior to apphcatmn to the K chains can also be placed into subgroups. It is sequencer. After each cycle of degradation, one residue likely that B.-J. proteins IR-52 and IR-248 belong to was cleaved from the amino-terminal (N-terminal) end of one subgroup whereas IR-158 belongs to another subthe polypepUde chain in the form of an anflinothlazolinone group (Table 1). denvaUve. This derivative was converted into a phenylthloThe three fractions of B.J. protein IR-52 have differhydantoin by incubation m 1 N HC1 at 80°C for 10 mm Phenylthiohydantoin amino acids were Identified by gas ent peptide maps but similar mobility on SDS polychromatography using 10~ DC 560 resin (Pisano and acrylamide gels, and similar amino acid composition Bronzert, 1969). Phenylthiohydantom amino acids were following hydrolysis by 6 N HC1. The N-terminal 40 also hydrolyzed to their corresponding amino acid with residues of these fractions have similar amino acid 6 N HC1 for 20 hr at 150°C under reduced pressure. These sequences except at position 6 where Gin was found amino acids were then analyzed either on an atmno acid m IR-52-I and IR-52-II but Glu was found in IR-52analyzer, or by high voltage electrophoresis on Whatman III. This difference may reflect post synthetic deaml3 MM filter paper. The break down product of phenylthio- dation, since such deamldation has been observed hydantoin tryptophan by hydrolysis IS fluorescent under in wtro m many synthetic small peptldes (Robinson, ultrawolet hght. Peptide mapping was carried out by the procedure of 1974). However, additional work is needed in order Wang and Sutton (1965). Molecular sizes of the B.-J. pro- to document this point. The amino acid sequence teIns were estimated by electrophoresls on sodmm dodecyl data available to date on the N-terminal 23 positions sulfate (SDS) polyacrylamlde gels as described by Fmr- on Ig ~c chains of man, mouse and rat are shown banks et al. (1971) in Table 2, in which alternative amino acid residues Table 1. N-terminal amino acid sequences of Bence-Jones proteins from inbred LOU/C/Wsl rats. Solid lines indicate identity of amino acid sequences to that listed on the top hne Proteins 1

IR-158 Ir-248 Ir-52-I IR-52-II IR-52-III

21

IR-158 IR-248 IR-52-I IR-52-II IR-52-III

5

10

15

20

Asp-Ile-Gln-Met-Thr-Gln-ser-Pro-Ala-Ser-Leu-Ser-Ala-Ser-Leu-Gly-Glx-Thr-Val-Thr -Ser-Leu. .Val Asx-Arg.-Leu -Ser-Leu Val Asx-Arg-Leu .Ser-Leu. .Val Asx-Arg~ Leu Glu Ser-Leu. -Val Asx-Arg-25

30

35

40

Ile-Glx-Cys-Arg-Gly-Ser-Glx-Asx-Ile-Tyr-Asx-ser-Leu-Ala-Trp-Tyr-Glx-Glx? -Pro Leu-Ser Lys-Ala Ser Lys-Leu Leu-Ser Lys-Ala-Tyr Lys-Leu Leu-Ser Lys-Ala-Tyr Lys-Leu Leu-ser Lys-Ala Tyr Lys-Lcu

I 100

194 V6

B56 Z37 K4 T3

172 Vt8 T5 N5

D77 El8 N5

D 100

2

196 M2 V2

1

D67 E28 K3 B2

3

V52 Z48

Q 100

V70 Q20 T5 L5

QS0 V48 Z2

4

MM 1.,46

M67 L33

M59 L31 T5 V5

M62 L38

5

T 100

T 100

T95 15

T98 12

6

Z 100

Q 100

P 100

Q98 E2

7

S 100

S 100

$77 TI8 B5

$98 T2

8

P 100

P 100

P88 T5 E5 Q2

P 100

9

$61 A39

$67 A33

B5

A45 $23 LI8 T5 K5

$50 G21 LI2 AI2 B5

10

L52 S27 T8 F7 Y6

L67 $33

$61 FI4 TI I 19 L2 Y2

$58 T40 P2

11

L 100

L 100

LTI M20 D5 T5

L98 V2

12

$72 TI I A9 L4 V4

S |00

$55 A25 IX) F2 L2 T2 V2 Q2

$80 P13 A3 T2 V2

13

A60 V26 P$ L6

A 100

V59 A27 M7 T7

A43 L27 V23 T5 M2

14

$83 TI I P6

S 100

$75 T20 A5

$82 TI3 V3 P2

15

V65 L28 P7

V67 L33

L36 V23 I14 AI4 P7 $5 T2

V52 P43 L5

16

G92 Z8

G 100

G95 $5

G98 R2

17

B53 7.47

B67 Z33

E43 DI9 ZI4 QI2 K7 L2 B2

D54 E27 ZI6 B3

b Sequences of mouse L-chains were taken from Hood et al., 1973.

"Sequences of human L-chains were taken from Sn~th et a l , 1971; Pink et al., 1971; Wang et al., 1973b; Gergely et al., 1973

Rat (Pooled L--chains)

Rat

Motls¢ b

Man"

Spectes

P o s l t l o ~ "~

R57 TI6 SI2 G6 V5 L4

R67 T33

R44 K21 SI0 T5 E5 P5 Z5 L3 B3

R84 P14 L2

18

V73 A27

V 100

V73 A27

V54 A44 12

19

T85 SI I 14

T 100

T65 $35

T83 S15 A2

20

L51 149

L67 133

173 LI2 MI2 Y3

170 L30

21

$54 T28 BI0 Z8

$67 Z33

$58 T42

T48 $43 A3 B4 Z2

22

C 100

C 100

C 100

C 100

2~

Table 2. A summary of the N-terminal amano acid sequences of immunoglobuhn r chains of man, mouse and rat. The frequencies of alternative amino acid residues are represented by percentages as they occur in myeloma proteins or as yields as they occur in pooled L-chains. The anuno acid residues are numbered from the N-terminal end using the numbering system for the prototype amino acid sequences of human V r subgroups (Gaily and Edeiman, 1972). Residues representing "insertmns' or "deletions' are not included m the calculation of frequencies. The frequencies for rat Bence-Jones proteins should not be taken seriously, since only three chains were analyzed. The one letter atmno acid code is used: A, Ala; B, Asx; C, Cys, D, Asp, E, Glu; F, Phe; G, Gly; H, His, l, Ile, K, Lys; L, Leu; M, Met; N, Ash; P, Pro, Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; Y, Tyr; and Z, Glx.

,,,.i

f') gS" go

r.

go

508

AN-CHUAN WA G, H. HUGH FUDENBERG and HERVE BAZIN Table 3. Amino acid sequences between posmons 24 and 34 of rat Bence Jones proteins analyzed m this work and of several human myeloma L-chains Solid hnes indicate Identity of amino acid sequences to that of the top hne m each spemes Species Rat

Protein IR-52 IR-248 IR-158 VKII Tak Per TII Lea Pol

Ref 24 30 34 Lys-Ala-Ser-Glx-Asx-lle-Tyr-Asx-Tyr-leu-Ala Set S e r - Arg-Gl) -Ser-Arg-Ala-Ser-Gln-Ser-Val-Ser-Asn-Ser-Tyr-Leu-Ala Ser [GAP][GAP]Leu Arg[GAP]

-

1 1 1 2 3 2 4 4

References l = this paper, 2 = Pink et al, 1971, 3 = Gergely et al. 1973, 4 = Wang et al, 1974 a n d their relative frequencies at each position are summarized. A l t h o u g h the n u m b e r of rat K chains is too small for a quantitative analysis, the data, nevertheless show that no a m i n o acid residue is absolutely •specms-speclfiC' or "phylogenetically-assoclated' Therefore. these terms can be used only in a n operational sense to indicate that a certain a m i n o acid residue is p r e d o m i n a t e but not excluswely present at a certain p o s m o n o n the V-region of a certain kind of Ig L- or H-chain m a given species, whereas a different a m i n o acid residue is predominately, but not excluswely, found at the same p o s m o n of the corresponding L- or H-chain in a different species Consequently, the existence of 'species-specific' or "phylogenetlcally-associated" a m i n o acid residues o n Ig V-regions c a n n o t be taken as a strong evidence to support any of the various somatic hypotheses concerning the genetic control of a n t i b o d y diversity based o n small n u m b e r of V-region genes. To our surprise, the data obtained (Table 2) show that more concordance exists, m terms of the pred o m i n a n t a m i n o acid residue at a gwen position, between L-chains of m a n a n d rat t h a n between those of rat a n d mouse. F o r example, the p r e d o m i n a n t residue at p o s m o n 9 m Ig ~, chains in b o t h m a n a n d rat is serlne, but is alanme m mouse x chains. Similarly, at p o s m o n s 13 a n d 15, the p r e d o m i n a n t residues m h u m a n a n d rat Ig x chains are alanme a n d vallne, respectively, but most mouse ~, chains have vahne at p o s m o n 13 a n d leucme at position 15 These data are difficult to reconcde with the 'sudden gene expansion a n d contraction' hypothesis of H o o d et al (1970) for a n t i b o d y varlabfllt3., the data are compatible with the idea that different sets of Ig V-region genes may be expressed in different species (Wang a n d Fudenberg, 1974b; Wang, 1975) W u a n d K a b a t (19701 statistically analyzed the avadable a m i n o acid sequence data o n h u m a n ~:, h u m a n 2 a n d mouse h type chains a n d defined positions 24-34, 50-56 a n d 8%97 as "hypervariable' regions However, we observed no extra varlabdity between p o s m o n s 24 to 34 o n the three rat B.-J proteins we studied (Table 1) Similarly, L-chains of five unrelated h u m a n monotypac Igs also showed no hypervarlaNhty at this region (Table 3). Therefore, the validity of this first hypervarlable region in rat a n d h u m a n L-chains deserves re-evaluation This region may not be hypervarlable within a given subgroup

All five h u m a n L-chains o n Table 3 were classified as V K I I proteins in previous studies (Pink et al., 1971; W a n g et a l , 1973: W a n g et a l , 1973a). It is noteworthy, that three of them (TII, Lea a n d Pol) have a characteristic G A P when c o m p a r e d wxth other h u m a n V K I I L-chains. Perhaps these three L-chains may be members of a n undefined new subgroup. Acknowledgements--We thank Mmes S San Juan, Elaine Rames, J DeMets-Naze and Yee-mm Yang as well as Mr J P Kmts for their excellent techmcal assistance

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509

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