Eur. J. Immunol. 1991.21: 1837-1841

Frank Charles Hay, Andzriej Jan Soltys, Gordon Tribbick. and H. Mario Geysen. Division of Immunology, St George’s Hospital Medical School, London and Coselco Mimotopes Pty., Melbourne

RF framework sequences exhibiting aggregated IgG binding

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Framework peptides from xIIIb rheumatoid factor light chains with binding activity for aggregated IgG Most monoclonal human rheumatoid factors (RF) and some RF from rheumatoid patient’s synovia are restricted in their light chains, using predominantly the xIIIb subfamily.Very few sequence differences are found between these light chains. Light chains with similar variable region framework sequences are also found in some mouse monoclonal RF derived from mice stimulated with lipopolysaccharide or secondarily immunized with protein antigens. There are two likely explanations for this restriction in framework sequences between the two species: (a) the sequences are important for the immunoregulation of RF production or (b) the sequences are concerned with the antibody binding specificity of the RF. We have examined overlapping octapeptides from the xIIIb light chain variable region and show that some framework peptides have the ability to bind aggregated IgG. Replacement of amino acids within the peak binding peptide have indicated the critical amino acids necessary for binding.

1 Introduction Rheumatoid factors (RF), anti-globulin antibodies which bind to the Fc region of IgG, have provided a useful diagnostic aid for rheumatoid arthritis since their serendipitous discovery by Erik Waaler [l] and their later rediscovery by Rose et al. [2]. Evidence is now revealing that RF are coded by a limited number of germ-line genes and that these genes are expressed in germ-line form during fetal development [3]. Autoantibodies formed in rheumatoid arthritis and other non organ-specific diseases are intrinsically different from those found in the organ-specificforms.Whereas antibodies such as anti-thyroglobulin show the characteristic high specificity of normal antibodies, those such as RF show broad cross-reactivity, with low affinity for individual antigens.This phenomenom is seen with natural antibodies [4] and those derived from CD5+ B cells [5]. As well as being characteristic of rheumatoid arthritis, RF are also produced in non-rheumatoid diseases such as chronic lymphocytic leukemia. Prior to sequence analysis, studies with anti-idiotypic sera revealed that human RF could be classified into three groups [6].The main Wa group composed about 60% of RF, while the Po group accounted for about 20%. Examination of the L chains from a number of Wa group RF has revealed that they share closely similar L chain V regions of the same xIIIb subgroup,with almost identical sequences suggesting that this sequence may be important for RF specificity [7-91. Normal mice treated with polyclonal activators such as bacterial LPS or by hyperimmunization produce IgM RF.L [I 93091 Correspondence: Frank C. Hay, Division of Immunology, St George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, Great Britain Abbreviations: CDR Complementarity determining region FR: Framework region RF: Rheumatoid factor 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

chain diversity is also restricted in mouse RF but to a lesser degree than with human RF. In one study only 3 out of 28 known L chain familieswere found to contribute to RF [lo, 111.These RF differed in their complementarity-determining regions (CDR), those areas normally making contact with the antigen, while having very similar sequences in frameworks (FR) 2 and 3,whereas randomly chosen mouse L chains differed markedly in FR regions. This led to the suggestion that, unconventionally, it might be the FR sequences which were responsible for RF activity rather than the normal CDR. Comparison of human and mouse RFL chains revealed that the same conserved FR sequences were present in both species supporting the view that these FR regions are responsible for RF activity [121. A contrary view, that FR have nothing to do with RF activity, has been expressed [13] on the basis that homologous L chain sequenceswere found on both RF and non-RF antibodies. This has led to some controversy in the literature, over the role of FR sequences for RFactivity [14,15].To test this framework hypothesis, a series of overlapping octapeptides, homologous with human RF L chain SIE encompassingCDRl to CDR3 have been used to determine whether FR peptides might have the ability to bind aggregated and monomeric IgG.

2 Materials and methods 2.1 Synthesis of peptides Octamer peptides, overlapping by seven amino acids, spanning residues 29-94 of the x m b L chain SIE [16] were synthesized on the tips of polystyrene pins in the form of a 96-well microtiter plate [17]. Peptides from L chains GAR [8], GOT [8],WOL [16], PAY [8], NEU [S] were synthesized where the amino acid sequences differed from SIE (Fig. 1). Analogues of the peptide QRPGQAPR, from sequence PAY, one of the optimal binding peptides, were similarly synthesized on pins. Each amino acid in turn was substituted for by the other 19 common natural amino acid alternatives. 0014-2980/91/0808-1837$3.50+ .25/0

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F. C. Hay, A. J. Soltys, G. Tribbick and H. M. Geysen FRI

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Eur. J. Immunol. 1991.21: 1837-1841 FR3

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Figure 1. Sequences of human RF L chains. continuous lines indicate sequence identity with the top sequence. (data taken from

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2.2 Preparation of aggregated and monomeric IgG Human IgG was isolated from Cohn fraction I1 (Sigma, Poole, GB) by ion exchange chromatography on DEAEcellulose equilibrated with 20 mM phosphate buffer [18]. IgG was aggregated by heating a 27 mg/ml solution at 63 "C for 10 min, and then immediately cooled in ice. Monomeric IgG obtained by ion exchange chromatography was prepared by passing through a Sephacryl HR400 gel filtration column, or was an Intragam preparation. (Commonwealth Serum Laboratories, Parkville, Australia)

detected by reaction in the dark for 45 min at 25°C with 0.15 ml of freshly prepared substrate solution. This consisted of 50 mg 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Sigma), 0.03 ml H202 (30%. 100 vol; BDH) in 100 ml of 0.1 M phosphate 0.08 M citrate buffer, pH 4.0. Color development was monitored by eye and stopped (by pin removal) when the most intense color corresponded to an approximate absorbance value of 2.0. Plates were then read in an ELISA reader (Titertek Multiskan, Flow, Irvine, Scotland) at 405 nm against a reference wavelength of 492 nm.

2.3 Immunoassay for binding of IgG to L chain peptides

3 Results

ELISA reactions on the pins were performed in microtiter trays containing IgG and conjugate solutions. The tips of the pins holding bound peptides were immersed for 1 h at 25 "C in 0.2 ml of a buffer containing 2% BSA (Fraction V, Sigma) and 0.05% Tween 20 (BDH, Poole, GB) in PBS pH 7.2 to block nonspecific adsorption of antibodies. A 10 pg/ml solution of IgG was prepared in the same buffer and the pins were incubated in 0.175 ml overnight at 4°C with agitation. Pins were then washed four times in PBS/Tween (PBS containing 0.05% Tween 20) for 10 min to remove unbound IgG. Biotinylated goat anti-human IgG (Amersham Int., Amersham, GB) was made up in a diluting buffer containing 1% sheep serum, 0.1% casein and 0.05% Tween 20. The pins were immersed in 0.175 ml of this solution and incubated for 1h at 25 "C with agitation. The pins were again washed vigorously four times in P B S m e e n for 10 min. Bound anti-IgG was detected by reaction with streptavidin peroxidase (Amersham Int.) diluted 1/2000 in diluting buffer for 1 h at 25 "C in 0.175 ml with continuous agitation. Following a further four vigorous washes with PBSl'lkeen the presence of enzyme on the pins was

3.1 L chain peptides with RF like activity Synthesized octapeptides from the middle of CDRl to within CDR3 of L chain SIE bound aggregated human IgG. Maximal binding was seen with peptides from outside the CDR (Fig. 2 a), although some binding was seen in peptides overlapping CDR2 and FR 3. Low nonspecific binding of aggregated IgG to pin-bound peptides was inferred from the observation that many peptides gave low extinctions in the immunoassay used. This low binding was similar to that found with the peptides PLAQGGGG and GLAQGGGG synthesized as controls (data not shown). Monomeric IgG bound in a similar fashion to that of aggregated IgG but at a lower level (Fig. 2 b).

3.2 Binding of IgG to L chain peptides derived from alternative RF The lcIIIb RF L chains shown in Fig. 1differ by only a few amino acids from protein SIE. To see if these sequence

Figure 2. (a) Binding of aggregated IgG to octapeptides synthesized from the middle of CDRl to within CDR3 of light chain SIE.The sequence of each octapeptide tested is given in single-letter code below the bar indicating its binding to aggregated IgG. Reverse print indicates positions of CDR. (b) Binding of monomeric IgG to octapeptides synthesized from the middle of CDRl to within CDR3 of L chain SIE.The sequence of each octapeptide tested is given in single-lettercode below the bar indicating its binding to monomeric IgG. Reverse print indicates positions of CDR.

Eur. J. Immunol. 1991. 21: 1837-1841

RF framework sequences exhibiting aggregated IgG binding

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changes made a difference in the RF activity, we examined peptides from the variant L chains for binding to aggregated and monomer IgG. Whereas most changes made no difference to the binding of IgG (results not shown), the change of lysine at position 39 to arginine in protein PAY had a marked effect on binding IgG (Fig. 3).

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We compared the effect of this change from lysine to arginine, at position 39, by synthesising eight octapeptides, walking along the L chain sequence so that this variant amino acid was, in turn, at positions 8 t o 1in the peptide. This showed that substitution at position 7 or 8 in the octapeptides made no difference to the binding of aggregated IgG but higher binding was observed with arginine in positions 6 to 1.The effect was especially noticeable, for binding aggregated IgG, in peptides WYQQRJGQ, YQQRPGQA and QQRPGQAP where low or nonexistext binding was founxwith lysine but strong reactivity was seen with the arginine variants (Fig. 3 a). The change from lysine to arginine also increased the binding of monomeric IgG, particularly for peptides RPGQAPRL, - (Fig. 3 b). Q E G Q A P R and AWYRPG

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Figure3. (a) Binding of aggregated IgG to the eight variant octapeptides representing the region from the middle of CDRl to within FR2 of L chain SIE (closed bars) and L chain PAY (open bars) .The amino acid lysine (SIEsequence) is replaced by arghiine (PAY sequence) stepping through each possible peptide so that the replacement goes from position 8 to position 1 in turn. The sequence of each octapeptide tested is given in single-letter code below the bar indicating its binding to aggregated IgG. The lysine to arginine interchange is boxed. (b) Binding of monomeric IgG to octapeptides synthesized from the middle of CDRl to within FR2 of L chain SIE (closed bars) and L chain PAY (open bars). The sequence of each octapeptide tested is given in single-letter code below the bar indicating its binding to monomeric IgG. The lysine to arginine interchange is boxed.

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The peptide QRPGQAPR, from protein PAY, with maximum binding of monomeric IgG was selected for further study. Peptide analogues were synthesized so that the effect of substituting all naturally occurring amino acids in each position, on binding of aggregated IgG and monomeric IgG, could be analyzed. The binding of each analogue was expressed as a percentage of the mean binding obtained from all the copies of the parent peptide. With binding of aggregated IgG, varying degrees of replaceability were obtained with some positions such as the first glutamine and glycine at position 4 being replaceable with almost any amino acid (Fig. 4). Substitutions for either of the two arginines had the greatest effect on binding with many of these substitutions destroying binding activity completely. A similar replaceability pattern was obtained for each of the arginines, in that binding was retained for peptides in which the arginines were substituted for by the aromatic amino acids tryptophan, tyrosine and phenylalanine. Monomeric

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Eur. J. Immunol. 1991. 21: 1837-1841

F. C. Hay, A. J. Soltys, G. Tribbick and H. M. Geysen

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Figure 5. Binding of IgG to replacement net octapeptidesfrom the peptide QRPGQAPR (from L chain PAY). In each individual graph the bars represent peptides in which the position markedby an asterisk is replaced in turn by alternative amino acids as indicated below the bars. The mean binding obtained with the parent amino acid in the starred position is shown by the shaded bar. The sequence of each octapeptide tested is given in single-letter code below the bar indicating its binding to monomeric IgG.

IgG bound with a similar pattern although the parent either slight differences in the inherited gene or are the sequence was rather more critical than with aggregatedIgG result of somatic mutation. Most of the resultant amino (Fig. 5). Again the two arginines were most critical to acid changes made no difference to the binding of IgG but binding, and could be effectively replaced by aromatic the substitution of an arginine for a lysine at position 39 amino acids and to a lesser extent by the acidic aspartate made a dramatic change in the binding. Aggregated IgG and glutamate residues. Although the specificity for the was bound more strongly to the arginine variant, as did parent residue was greatest for arginines in position 2 and 8, monomeric IgG. The difference in binding observed in comparison with aggregated IgG, a greater specificity between arginine- and lysine-containing peptides was surwith monomeric IgG was also observed for Q1, P3, Q5 and prising as this is a relatively conservative substitution, both lysine and arginine being basic, positively charged amino P7. acids, which have a mean equivalence of about 50% in antigenic determinants [23]. It is appealing to speculate 4 Discussion that such changes might contribute to the conversion of a non-pathogenic low-affinity autoantibody, responsible for RF use a limited set of germ-line genes. Idiotypic and clearing immune complexes, into a higher affinity, possibly genetic studieshave revealed usage of the conservedHumV pathological, autoantibody of the type detected in synovial x325 germ-line gene in Wa idiotypic RF [19, 201. At the cells [24] but which is undetectable in serum as it is already same time, althoughVHIH chains are frequently associated complexed with monomeric IgG. That this binding is not with Wa group RF, the various RF have many heavy chain simply the result of a nonspecific charge effect is shown by amino acid sequence differences, particularly in the CDR the lower binding observed for analogues containing argin[21]. This together with the finding that mouse RF share ine or lysine substitutions at other residues in the binding conserved L chain FR regions which are virtually identical peptide QRPGQAPR. to human sequences has led to the proposition that FR sequences might be important for RF activity in contrast to Analysis of each amino acid in the key peptide the conventional CDR normally associated with antigen QRPGQAPR by replacing each amino acid in turn with all binding [lo, 111. 19 alternatives confirmed the importance of arginine in the binding.This is of interest as it parallels the work of Weigert Clearly our results show that FR regions 2 and 3, and the et al. [25] in which it was shown that somatic mutation overlap region with CDR2, have the ability to bind contributes to the development of autoimmunity in MRL aggregated IgG. That this is not simply the binding of mice and that the inclusion of arginine residues was an “sticky” aggregated IgG in a nonspecific manner to any important contribution to this autoimmune conversion. peptide is well shown by the many negative L chain peptides included in the assay. The binding of IgG to FR FR sequencesmay be necessary for RF binding but may not peptides from IgM SIE parallels that found with whole IgM themselves constitute the whole site. Reininger et al. [21] RF in that monomeric IgG is bound weakly by the peptides have shown that although one mouse mAb, with the when compared to the binding seen with aggregated IgG. conserved FR sequence, has RF activity, three others, with Presumably the binding of each individual peptide to a similar L chain but different H chain sequences, are devoid monomeric IgG is of relatively low affinity as shown for of RF activity.The H chain may influence the conformation single Fab regions of IgM RF [22], but the multivalent of the L chain and its RF activity. Indeed experiments presentation of peptides on the derivatized pins allows recombining Hand L chains from different antibodieshave high-affinity binding to multivalent aggregated IgG which shown that the x m B L chain may be necessary for RF sums to a higher overall avidity. activity but that the activity is only revealed when the d I I B L chain is combined with the correct H chain [26]. Hum Vx325 is a germ-line gene expressed in germ-line form in Wa group RE The few alterations from this In the mouse RF L chains the sequence of the octapeptide sequence seen in the outbred human population reflect corresponding to the strongest binding peptide identified in

Eur. J. Immunol. 1991. 21: 1837-1841

RF framework sequences exhibiting aggregated IgG binding

our study is QKPGQSPK not QRPGQSPR. Although the peptide in w h i a both ariinines were substituted by lysines at the same time was not tested in this study, replacement of either arginine alone by lysine resulted in less than 50% of the binding being retained. Substitution of both arginines by lysines in the octapeptides may result in weaker binding and so be on the borderline for detection as RF. It is of interest that mouse RF binding is far weaker than for human antibodies. Usually sensitive ELISA systems are needed to reveal mouse RF activity whereas human RF are able to cause strong agglutination. Intriguingly, of two mouse monoclonal RF [13] one had this conserved V, sequence while the other, which lacked this conserved sequence, had a potential glycosylation sequence Asn-Asn-Ser in the CDW of theVH region. This complements the pattern that we have noticed before in human RF,where RF related to the Wa idiotypic group tend to have conserved VlcIII sequences, while members of the Po idiotypic group have H chains which have potential V region glycosylation sites. Such glycosylation is rarely seen outside the VHIII subgroup to which Po group W belong [27]. This leads us to propose that there are two basic types of Wactivity, one based on theVxIII amino acid sequence, the other working through Fab sugar residues perhaps linked to the Fc glycosylation changes seen in rheumatoid arthritis [28-311. Received February 5, 1991; in revised form March 25, 1991.

5 References 1 Waaler, E., Acta Pathol. Microbwl. Scand. 1940. 17: 172. 2 Rose, H. M., Regan, C., Pearce, E. and Lipman, M. O., Proc. SOC. Exp. Biol. Med. 1946. 66: 1. 3 Kipps,T. J., Robbins, B. A. and Carson, D. A., J. Exp. Med. 1990.171: 189. 4 Avrameas, S., Matsiota-Beranard, €!, Guilbert, B., Ternynck, T., in Clot, J., Sany, J., Brochier, J., (Eds.), Septi2me Symposium International D’lmmuno-Rhurnatologie, Editions de 1’Interligne, Paris 1989, p. 127. 5 Nakamura, M., Burastero, S. E., Notkins, A. L. and Casali, I?, J. Immunol. 1988. 140: 4180. 6 Kunkel, H. G., Agnello,V., Joslin, F. G.,Winchester, R. J. and Capra, J. D., J. Exp. Med. 1973. 137: 331. 7 Andrews, D. W. and Capra, J. D., Proc. Natl. Acad. Sci. USA 1981. 76: 3799. 8 Ledford, D. K., Goni, F., Pizzolato, M., Franklin, E. C., Solomon, A. and Frangione, B., J. Zmmunol. 1983. 131: 1322.

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9 Goni, F., Chen, F! P., Pons-Estel, B., Carson, D. A. and Frangione, B., J. Immunol. 1985. 135: 4073. 10 Shlomchik, M. J., Nemazee, D. A., Sato,V L., van Snick, J., Carson, D. A. and Weigert, M. G., J. Exp. Med. 1986. 164: 407. 11 Shlomchik, M., Nemazee, D. ,Van Snick, J. and Weigert, M.,J. Exp. Med. 1987. 165: 970. 12 Hay, E C., Br. J. Rheumatol. 1988. 27 (suppl IZ): 157. 13 Reininger, L., Spertini, E ,Takanori, S.,Jaton, J. C. and Izui, S., Eur. J. Zmm. 1989. 19: 2123. 14 Shlomchick, M. and Weigert, M., Eur. J. Immunol. 1990.20: 2529. 15 Reininger, L., Jaton, J.-C. and Izui, S., Eur. J. Immunol. 1990. 20: 2530. 16 Andrews, D. W. and Capra, J. D., Biochemistry 1981. 20: 5816. 17 Geysen, H. M., Rodda, S. J., Mason,T. J.,Tribbick, G. and I . Immunol. Methods 1987. 102: 259. Schoofs, I?, . 18 Hudson, L. and Hay, F. C., Practical Immunology, Blackwell Scientific Publications, Oxford 1989. 19 Radoux,V., Chen, I? I?, Sorge, J. A. and Carson, D. A., J. Exp. Med. 1986.164: 2119. 20 Chen, F! F!, Goni, F., Fong, S., Jirik, E, Vaughan, J. H., Frangione, B. and Carson, D. A., J. Immunol. 1985. 134: 3281. 21 Andrews, D. W. and Capra, J. D., Biochemistry 1981. 20: 5822. 22 Dissanayake, S., Hay, F. C. and Roitt, I. M., Immunology 1977. 32: 309. 23 Geysen, H. M., Mason,T. J. and Rodda, S. J., J. Mol. Recog. 1988.1: 32. 24 Robbins, D. L., Benisek,W. F., Benjamin, E. and Wistar, R. Jr., Arthritis Rheum. 1987.30: 469. 25 Shlomchik, M. J., Marshak-Rothstein, A., Wolfowicz, C. B., Rothstein,T. L. and Weigert, M. G., Nature 1987. 328: 805. 26 Newkirk, M. M., Gram, H., Heinrich, G. F., Oestberg, L., Capra, J. D. and Wasserman, R. L., J. Clin. Invest. 1988. 81: 1511. 27 Kabat, E. A., lki,T. W., Reid-Miller, M., Perry, H. M. and Gottesman, K. S., (Eds.) Sequences of proteins of immunological interest, 4th-Edn., U.S. Department of Health and Human Services, Bethesda 1987. 28 Parekh, R. B., Dwek, R. A., Sutton, B. J., Fernandez, D. L., Leung, A., Stanworth, D., Rademacher, T. W., Mizuochi, T., Tmniguchi,T., Matsuta, K.,Takeuchi, E, Nagano,Y., Miyamoto, T. and Kobata, A., Nature 1985. 316: 452. 29 Roitt, I. M., Dwek, R. A., Parekh, R. B., Rademacher,T.W., Alavi, A., Axford, J., Bodman, K. B., Bond, A., Cooke, A., Hay, F. C., Isenberg, D. I., Lydyard, F! M., Mackenzie, L., Rook, G., Smith, M. andSumar, N., Rec. Prog. Med. 1988.79: 314. 30 Sumar, N., Isenberg, D. A., Bodman, K. B., Soltys, A. J., Young, A., Leak, A. M., Round, J., Hay, F. C. and Roitt ,I. M., Ann. Rheum. Dis. 1991, in press. 31 Bond, A., Cooke, A. and Hay, F. C., Eur. J. Imrnunol. 1990.20: 2229.

Framework peptides from kappa IIIb rheumatoid factor light chains with binding activity for aggregated IgG.

Most monoclonal human rheumatoid factors (RF) and some RF from rheumatoid patient's synovia are restricted in their light chains, using predominantly ...
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