THEJOURNALBIOLOGICAL OF CHEMISTRY Vol. 264, No. 8, Ienue of March 15, pp. 45134522,1989 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in C.S. J A. Variable Region Primary Structuresof a High Affinity Anti- fluorescein Immunoglobulin M Cryoglobulin Exhibiting Oxazolone Cross-reactivity* (Received for publication, September 2, 1988) Mary Ann Dombrink-Kurtzman$, Leslie S. Johnsong, Gary S. Riordang, William D. BedzykS, and Edward W. Voss, Jr.$ll From the tDeDartment of Microbwloev. Uniuersitv of Illinois, Urbana, Illinois 61801 and the SGenex Corporation, ” “ I I . GaithersburgsMaryhnd 20877 Previous studies of murine IgM hybridoma protein show abnormally high spontaneous polyclonal B cell activa- 18-2-3, derived from an (NZB/NZW)Fl mousefollow- tion (Izui et al., 1978). ing hyperimmunization with fluorescein (Fl)-conju- A high affinity (& = 2.9 X 10” M”) murine monoclonal gated keyhole limpet hemocyanin, demonstrated a high anti-fluorescein IgM antibody 18-2-3 displaying low temper- affinity for F1 (K.= 2.9 X 10” M-’) and cryoprecipi- tation that was abrogated upon F1 binding to the anti- ature insolubility in the absence of bound ligand has served body-combining site. V region sequences of 18-2-3 as a model to study Type I cryoprecipitation. Antigen binding were determined by Edman degradation and nucleo- site involvement was indicated since the presence of fluores- tide sequence analysis. The VH region of 18-2-3 was cein prevented cryoprecipitation (Ballard et al., 1983). Anti- encoded by a gene VHI(B)of the 6 5 2 VH family with body 18-2-3 was originally derived from an (NZB/NZW)FI 96%homology to anti-oxazolone antibody NQ7.5.3 but mouse, a strain showing a high incidence of autoimmunity. utilized a larger D region (De62 plus Nregion). The V, Studies by Ballard et al. (1985) suggested that 18-2-3 was region of 18-2-3 was encoded by a gene VJV with an amino acid sequence 97% homologous to that of anti- derived from a relatively rare B cell progenitor since exami- oxazolone antibody NQ 11.1.18. Although monoclonal nation of 37 IgM and IgG monoclonal antibodies of similar anti-F1 antibodies 18-2-3 and 4-4-20 possessed similar origin and specificity &d not reveal low temperature insolu- binding affinities and quenched bound fluorescein to bility or high binding affinity for fluorescein. Previous results the same extent (Q- > 96%),they utilized different indicated that cryoprecipitation occurred via electrostatic in- VH, D, V., and J. genes, but the same JH gene segment teractions involving 18-2-3 antibody-combining sites with (JH~). Solid-phase analyses showed that 18-2-3 was interactivesites in the Fc region of the homologousIgM not idiotypically related to 4-4-20 and 9-40, prototypic anti-F1 antibodies. Fine specificity binding patterns of (Dombrink-Kurtzman and Voss, 1988). F1 analogues by 18-2-3 IgM and IgM. were distinct In the present study, variable region sequences of heavy from other anti-F1 antibodies. Monoclonal antibody 18- and light chains derived from 18-2-3 have been determined 2-3 bound phenyloxazolonebovine serum albumin with through cloned cDNA synthesized from mRNA templates. a lower affinity than F1-bovine serum albumin. The Three DNA segments (VH,’ DH, and J H ) encode the VH region, for first hypervariable region of the 18-2-3 light chain while two DNA segments (V, and J.) encode the V, region showed homology to human cryoglobulins. This is the (Seidman et al., 1978; Sakano et al., 1979; Schilling et al., first variable region sequence of a murine IgM which self-aggregates at low temperature. 1980). VH and V. polypeptides both contribute to antigenic binding specificity of antibodies. The V, of 18-2-3 was nearly identical to thatof BALB/c anti-oxazolone antibodies (Berek et al., 1985),whereas the VH of 18-2-3 was highly homologous Cryoglobulins reversibly precipitate at temperatures below to other BALB/c anti-oxazolone antibodies (Griffiths et al., 37 “C and have been classified into three types based on the 1984).Although anti-fluorescein antibodies 18-2-3 and 4-4-20 molecular composition of the aggregate (Brouet et al., 1974). had similar high binding affinity and fluorescence quenching Murine IgM 18-2-3 is a Type I cryoglobulin consisting solely of the monoclonal 18-2-3component. Cryoglobulinshave been of bound fluorescein, they differed in VH and V, gene usage. observed in normal BALB/cmice, but occur at increased idiotypic and metatypic relatedness, and fine specificity. levels in autoimmune-prone strains (NZB, NZB/NZW, MRL/ CDRl of the 18-2-3 V, gene segment closely resembled the 1) correlated with age and disease severity (Andrews et al., human V, sub-subgroup IIIb, which has been preferentially 1978). Data suggest that autoreactive B cell precursors are in used by a group of human monoclonal IgM-RF cryoglobulins a proliferative state in these autoimmune strains since they (Kunkel et al., 1973). * This work wassupported in part by National Institutes of Health The abbreviations used are: VH, variable region of heavy chain; Grant AI 20960 (to E.W. V.). The costs of publication of this article amp, ampicillin; BSA, bovine serum albumin; CDR, complementarity were defrayed in part by the payment of page charges. This article determining region; DTT, dithiothreitol; FITC, fluorescein isothio- must therefore be hereby marked “advertisement” in accordance with cyanate; F1, fluorescein; H, heavy; IgM., monomeric subunit of IgM; 18 U.S.C. Section 1734 solely to indicate this fact. L, light; mAb, monoclonal antibody; PAGE, polyacrylamide gel elec- The nucleotide sequence(s) reported in thispaper has been submitted trophoresis; phOx, phenyloxazolone or oxazolone; RF, rheumatoid to the GenBankTM/EMBL Data Bank withaccession number(s) 504609 factor; SDS, sodium dodecyl sulfate; ssDNA, single-stranded DNA; and 504610. V,, variable region of light chain; HPLC, high performance liquid ll To whom correspondence should be sent. chromatography. 4513 4514 Variable Region Sequences of an Anti-F1 Cryoantibody EXPERIMENTAL PROCEDURES~ Anti-Pluorescein Hybridor Pmtain 18-2-3 Heavy Chain Vari8ble Re8ion Squenoa RESULTS Determination of NH2-terminal Amino Acid Sequences- -19 e K t Ala Val Leu Val Leu Phe Lau Cys Leu Val Ala Phe Pro Ser Cys The NHz-terminal amino acid sequence (43 residues) of 18- A C G G CT T A A AA G GCT GI7 CTC On: CTC TK: Crc Icc CTC OTr C C A ?CCA AGC TGT TI 2-3 light chain was determined by repetitive Edman degra- dation. After deblocking the amino-terminal residue of the 1 10 heavy chain of 18-2-3 with pyroglutamate aminopeptidase, Val Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Val L a Val Ala Pro Ser Gln OrC CTC TCC CAC G10 CAC CTC AAG GAG T C A GGA CCT C E CTC On: OCC CCC TCA CAG the NHz-terminal sequence (30 residues) was identified. Monoclonal antibody 18-2-3 utilized a VH gene of the Q52 family and a V, gene from the V,5 subgroup of Potter et al. 20 3o ID h Ser Leu Sur Ile Thr Cys T r Val Ser Gly pha Ser L u Thr Asn Tyr Gly Val His e (1982) or V,IV subgroup of Kabat et al. (1987). Southern blot ACC CTC T CTA T CA CC Icc A T OrC ‘ E T OGO ‘HT T C A T T A ACC AAC TAT CCT C T A CAC C hybridization was utilized to identify J, usage. Restriction fragments obtained from separate digests of 18-2-3 DNA with CfoI and EcoRII indicated J,5 was being used (datanot 40 Trp Val Arg Gln Pro Pro Gly LYE Gly Leu ’50 G1u Trp Leu Gly Val 110 Trp Ala Gly TCC CTC CGC CAG C C A T CC COA LAG CCT CTC GAG TCC CTC GGA C AT TA A TCC GCT CCT shown). Heavy C h i n Sequences-Six oligonucleotideswere synthe- CDR2 sized as primers in sequencing the heavy and light chain 70 60 I variable regions.Fig. 1 describes the oligonucleotides and Gly A Thr Aan Tyr Aan Ser Ala Lau Mt Ser Arg Leu Ser 110 Ser Lys Asp m Asn GGA LAC ACI AAT AAT TAT X A GCT CIC ATC TCC U i A CTC AGC A X AGC AAA C CA AA T locations to which they hybridized. The nucleotide and amino acid sequences of the VH segment of 18-2-3 are presented in 80 B Z a b c Figs. 2 and 3, respectively. The amino acid sequence was Ser Lys Ser Gln Vel Phe Leu LysMet Asn Ser Leu Cln Ile Asp Asp Thr Ala Ile TCC AAG AGC C A I G C “‘2 TTA AAA T ATG LAC ACT CTC C I A ATT GAT T CAC ACA GCC A A deduced by dideoxy sequencing of cDNA synthesized from mRNA. Amino acid residues comprising positions 2 through 90 -E3a b c d I €mRI VH Hindm Tyr Tyr Cys Ala LysArgLeu Glu Arg I l e me TyrTyr Ala Met Asp Tyr Trp Gly TIC TAC K T GCC AAA C C A CTC C I A CCA A C T m TT? TIC TAT CCT A GAC TAT TGC GGT I I ?!M 110 Cln Gly Thr Ser Val Thr Val Ser Ser CAA GGA ACC T C A GTC A C G N TCC C TCA c- MI3 FIG. 2. Nucleotide and deduced amino acid sequences the of 18-2-3 Vg gene segment, including leader peptide and 5‘- untranslated region. Numbering of the amino acids and comple- mentarity determining regions is according to Kabat et al. (1987). 31 determined by amino acid sequencing corresponded to those deduced from the nucleotide sequence. The VH region of 18-2-3 was encoded by a gene segment from subgroup VHI(B) (Kabat et al., 1987), belonging to the relatively com- - plex 652 VH family that contains approximately 15 genes (Brodeur and Riblet, 1984). Members of this family also encode the VH region of antibodies specific for oxazolone. I798 Anti-F1 antibody 18-2-3 possessed a D segment closely resem- 31 11 bling the DqS2 genesegment (Sakano et al., 1981; Kurosawa 7 > 1648 and Tonegawa, 1982), as shown in Fig. 4, but did not express MI3 the germ line sequence Gln-Leu-Gly since it differed at two * bases, resulting in a sequence of Arg-Leu-Glu. Additionally 18-2-3 exhibited variation inthe length of the CDR3 segment. Eight noncoded bases (N region; Alt and Baltimore, 1982) appeared to be present between D and J H ~ , resulting in a D region of six amino acids (Fig. 4). The precise boundaries FIG. 1. DNA sequencing strategy. l’he synthetic oligonucleo- tide primers used to sequence the clones of the heavy and light chain between V H and D gene segments and D and J gene segments variable regions of 18-2-3 are described. Primers numbered 1311, will only be known when germ line D and J gene segments 1648, 1798, and 2542 are oligonucleotides designed on the basis of are cloned. sequencing data obtained using primers 2507 and 2515. Specific A comparison of heavy chain variable region amino acid location within the genes where the oligonucleotides bound is shown sequences is also shown in Fig. 3. Interestingly, the 18-2-3 together with the direction and extent of the sequencing information sequence had much greater homology with antibodies of an- obtained. Key to the oligonucleotides: 1311, 5”dCTGCAGGTCAT GGTGACC-3’; 1648, 5‘-dTCTTACTCTCTCACAATC-3‘; 5‘- other specificity, anti-oxazolone (VH-Oxl and NQ7.5.3) than 1798, dATGACCTGCAGGGCCAGC-3‘; 2507, 5”dTGGATGGTGGGAA with anti-F1 antibodies (4-4-20 and 3-13). Monoclonal anti- GATG-3‘; 2515, 5’-dCAGGAGACGAGGGGGAA-3‘; 2542,B”dAAC body 4-4-20 had an affinity comparable to thatof 18-2-3 and TATGGTGTACACTGG-3’. The IT-rner, M13 sequencing primer quenched bound fluorescein to the same degree (Q- > 96%). (-20) was also used. The only similarity in various gene segments between the three anti-F1 antibodies was that they contained J H gene ~ Portions of this paper (including “Experimental Procedures,” segments (Sakano et al., 1980). The VH genes used by 18-2-3, Figs. 7-9, and Tables I and 11) are presented in miniprint at theend of this paper. Miniprint is easily read with the aid of a standard 4-4-20, and 3-13 belonged to VHI(B), VHIII(C), and VHII(A), magnifying glass. Full size photocopies are included in the microfilm respectively. The D region of 4-4-20 was truncated (Bedzyk edition of the Journal that is available from Waverly Press. et al., 1989), while that of 3-13 was unusually long, suggesting 45 15 -COR11 I 30 40 50 52 a b 18-2-3 S L T N Y G V H W V R Q P P G K G L E W L G V I W . . VH'Oxl "-s"""""""""-" . . NQ7.5.3 """""_""""""" . . 4-4-20 T F S D - W W N - - - - S - E - " " V A Q - R N K 3-13 T F S S - V L Y - - K - K - W A - - - - I - F - F P . "I..& I C 60 70 18-2-3 . A G G N T N Y N S A L M S R L S I S K D N S K S Q V VH'Oxl * - " s - " " " " " " " " " " - NQ7.5.3 " " -"""-"- " " " " - 4-4-20 P Y N Y E - Y - S D S V K G - F T - - R - O - - - s - 3-13 . Y N D G - K - - E K F K R - G T L T S - K - S - T A 80 82a b c 18-2-3 F L K M N S L Q I D D T A I Y Y C A K R L E R I F Y V VH-Oxl - - - - " - - T - " - M - - - - R D R G . . . . . NQ7.5.3 - - " - " - T " - - " - - - R D H G . . . . . 4-4-20 Y L Q - - N - R V E - M G - - - - T G S . . . . . - - 3-13 Y M E L S - - T S E - S - V - - - - R T G A D S S G - I c d e f 110 18-2-3 A M . . D Y W G Q G T S V T V S S v"-oxl s a . . A " - - - - L " - - A NQ7.5.3 . . . . - - -"""- 4-4-20 G - . . - - - " - " " " - 3-13 V R A " " " - " " " - FIG. 3. Comparison of heavy chain immunoglobulin variable region amino acid sequences. 18-2-3,4- 4-20, and 3-13 are anti-fluorescein antibodies. VH-0x1 and NQ7.5.3 are anti-oxazolone antibodies. Gapsare indicated by dots. Blanks indicate that the amino acid cannot be identified since one or two bases have not been determined. Amino acid sequences are given in a one-letter code (IUPAC-IUB Joint Commission on Biochemical Nomenclature, 1985). plus Dsp2.5) that it may be derived from D-D joining ( D F L ~ ~ . ~ 5 and 6, respectively. Dideoxy sequencing of cDNA, which (Liu et al.,1987). had been synthesized from mRNA, was used to determine the Light Chain Sequences-The nucleotide and amino acid amino acid sequence. Amino acid residues 1-43 determined sequences of the VL segment of 18-2-3 are presented in Figs. by amino acid sequencing of pure light chains were identical 4516 V a r ~ lReg~on e q ~of ~ An~i-F~ e ~ an e s C ~ ~ n ~ ~ ~ d ~ Germ-line C A A C T C G G A C DQ52 FIG. 4. Nucleotide sequences of D regions of immunoglobulin heavy chains using D ~ a r . Sequences are 18-2-3 aligned for maximal homology with the DwPgerm line gene segment. 18-2-3, 4- s43 4-20, and 3-13 are anti-fluo~scein anti- bodies. S43 and 22.8 are anti-&hydroxy- 28 2. 3-nitrophenyl and anti-poly (GI@- AlaS-Tyr*') (GAT) antibodies, respec- 4525 tively (Bothwell et al., 1981; Roth et al., 1985). Q5W is a myeloma clone (Sakano 04-01 et al., 1981), 04-01 is an anti-ssDNA autoantibody (Smith et al., 1988), and v"-oxl VH-Oxlis an anti-oxazolone antibody. 4-4-20 3-13 Anti-Fluorescein Hybridar Protein 18-2-3 Light C h i n idiotypic antisera were produced against anti-fluorescein an- Variable Region Sequence tibodies 4-4-20, 9-40, and 5-27. Each idiotype-anti-idiotype interaction was ligand-inhibitable: indicating that the idi- -22 otypic reagents may be interacting specifically with active site H i t Asp Phe Leu Val Gln Ile Phe S r Phe Leu u Leu IIe determinants or that tertiary structure was altered upon an- MATTCAMTACACA A l S G T T 7 CTC GTG CAC A'fT T C ACC A I I l l G CTA A T C tigen binding. Idiotype interactions were also inhibitable by 1 10 nonradioi~ated homologous proteins. Fig. 7, in the Mini- Sar A l a %r Val Ala Met %r Arg Gly Glu Aan Vel Leu Thr Gln Sur Fro Ala Ile print, shows inhibition of the idiotype-anti-idiotype interac- A ACT GCC K A ClT W A 1 0 TlX ACA GGA C M AAT GK CTC ACC CAG TCT CCA OCA ATC tions with increasing concentrations of unlabeled 4-4-20, 9- 20 D1 C R- - 1 21 a 40, and 5-27.One-half nanogram of unlabeled 4-4-20inhibited the 4-4-20-anti-4-4-20 interaction 50%while 1 ng of 9-40 Hat %r Ala S.r Pro Gly Glu Lys Thr Nt Thr Cy8 Are Ala %r %r %r Vel Val e A 1 0 TCT CCA TX CCA OOO GAL AAG CTC ACC A V JA C C TCC A f f i OCC Mic TCA A C T GTA i inhibited the 9-40-anti-9-40 interaction and 10 ng of 5-27 inhibited the 5-27-anti-5-27 interaction to thesame extent. 1 Inhibition of the idiotype-anti-idiotype interactions by in- 30 40 %r Sar Ser Tyr Leu His Trp Tyr Gln Gln lye %r Gly Ala %r Pro Lye Lnu Trp creasing concentrations of 18-2-3 IgM protein is shown in M;r1CCACTTACnr,CM:rrx;TM:CffiCffiAAGKAocrGCCnxcccAAAcn:Icc Fig. 7. In general, 18-2-3did not appreciably inhibit homolo- gous4-4-20 or the 5-27 idiotype-anti-idiotype interactions ' 50 D" C I -) Val Tyr Gly Thr %r A Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Gly Gly concentration tested (10 pg)in the 9-40-anti-9-40 interaction m U 60 over a wide protein concentration range. Only at the highest ~ T A T ~ A C A : C M C ~ ~ T C T ~ A C T C ~ G C T ~ ~ A l l ? ~ ~ O O O did 18-2-3 show any significant inhibition (40%). This rep- resented a much higher concentration (>10,000 times) of 18- IO Bo Sur Gly Thr %r Tyr Scr Lnu T r Ile Ser Scr Val Glu Ala Glu Asp Ala Ale T r h h 2-3 than required using homologous 9-40 protein. Thus, the TCT CCC ACC ZCT TAC TCT CTC ACA : A ACC All? CTC GAG CCT GAL GAT GCT WC ACT effect seen regarding 9-40 appeared to be nonspecific. Fine Specificity-Fine specificity binding patterns for 18- r p o " a n 3 7 100 2-3 IgMand IgMa weredetermined using five analogues of F1 Tyr Tyr Cys Gln Gln Tyr %r Gly Tyr Pro Lsu Thr Phe Gly AlaGly Thr Lys Leu covalently conjugated to BSA at similar epitope densities TAT TAC nC Cffi CAG TAC ACT GCT TAC CCA CTC A TIC GCT CCT CCC ACC A M C N ; W (erythrosinlo-BSA, eosinll-BSA, tetramethylrhodamine~ G1u Leu Lys BSA, rhodamines-BSA, substituted rhodaminels-BSA), plus GAG CTC A M dinitrophenol (dinitrophenobBSA). Table I, in the Mini- FIG. 5. Nucleotide and deduced amino acid sequences of the print, lists analogue concentrations required to inhibitbinding 18-2-3V. gene segment, including leader peptide and 5'- 50% to solid phase Fh,-BSA. Inhibition patterns for 18-2-3 untranslated region. Numbering of the amino acids and comple- were distinct from the other anti-F1 antibodies, indicating mentarity determiningregions is according to Kabat et at (1987). that monoclonal antibody 18-2-3possessed a nonidentical antigen-combining site. The only analogue showing 50% in- to those obtained from the nucleotide sequence. The V, region hibition was tetramethylrhodaminelc-BSAwhich inhibited was encoded by a member of the V,IV gene family (Kabat et the binding of 18-2-3 IgM and IgM. at concentrations of 440 al., 1987)which also encodes the V, regions of antibodies and 600 p ~ respectively. The highest concentration ofF1 , specific for oxazolone, dinitrophenol, arsonate, and a partic- analogues tested was 1 mM, while the highest concentration ular anti-idiotype. The J region of 18-2-3was encodedby J,5. of dinitrophenol tested was 10 pM. A comparison of light chain variable region amino acid Binding of 18-2-3 to Fl-BSA and phOx-BSA-A direct sequences is shown in Fig. 6. Again the 18-2-3sequence shared binding assay was used in studying the binding of 18-2-3 to much greater homology with anti-oxazolone antibodies (Vs- phOx-BSA because in preliminary inhibition assays it was 0x1 and NQ11.1.18) than with anti-F1 antibodies (4-4-20 and not possible to inhibit the high affinity binding of 18-2-3 to 9-40). Anti-Fl antibodies 4-4-20 and 9-40 and anti-single solid-phase FI-BSA with fluid-phase phOx-BSA. Binding of stranded DNA autoantibody 04-01 (Smith et aL, 1988) had 18-2-3 to phOx-BSA (Fig. 8,in the Miniprint) appeared to be similar light chain sequences (VJI) which were distinct from low affinity. Although the heavy and light chain variable 18-2-3 (V,IV). Reactivity of 18-2-3 with Anti-idiotype Antibodies-Anti- R. M. Bates and E. W. Voss, Jr., unpublished data. Variable Region Sequences of an Anti-F1 Cryoantibody 4517 region sequences utilized by 18-2-3 havebeen observed in Antibody 18-2-3 appeared to be using VH and D gene and J H ~ . different anti-oxazolone antibodies, no single anti-phOx an- segments from families residing most proximal to D and J, tibody used both variable sequences found in 18-2-3. Struc- respectively. Preferential utilization of D-proximal VH gene turally, the epitopes differ. The xanthenone portion of fluo- families has been observed in murine pre-B-cell lines (Yan- rescein is planar (Voss et al., 1976), whereas phenyoxazolone copoulos et al., 1984) and in hybridomas derived from non- has two nonrigid aromatic rings. Chemical structures ofF1- immunized 6-day-old BALB/c mice (Holmberg, 1987). BSA and phOx-BSA are shown in Fig. 9 (in the Miniprint). A comparison of heavy chain variable region sequences indicated that 18-2-3 and anti-phenyloxazolone antibodies DISCUSSION used homologous VH genes. The highest degree of homology Data reported here represent primary structural determi- (96% at the nucleotide and amino acid levels) with known nations of gene segments encoding the variable domains of sequences was with anti-phOx antibody NQ7.5.3 which had murine IgM 18-2-3, an antibody that self-aggregates in the been obtained 14 days following primary immunization. Meek absence of its cognate antigen F1. Antibodies 18-2-3 and4-4- et al. (1987) suggested that novel mechanisms were involved 20 had similar high intrinsic binding affinities (KO= 2-3 X in generation of D segments in autoantibodies. Although 18- ~ ~ 10" M ' and quenched bound fluorescein to thesame extent 2-3 utilized a gene segment resembling D Qthere were eight -) (Qm.. > 96%), yet they utilized different VH, D, V., and J, additional noncoded nucleotides between the D and J Hseg- ~ gene segments. The only similarity in gene segment usage ments. The GAATCTTT sequence was probably not attrib- J Gene utable to imprecise joining since it did not represent flanking between the two antibodies was that they contained H ~ . segments utilized by the 18-2-3 heavy chain variable region regions nor was D-D joining indicated. The D segment was appeared to be VHI(B) (a member of the v ~ Q 5 family), Dqsz, interesting in that the N segment was A,T-rich rather than 2 4518 Cryoantibody Variable Region Sequences an Anti-F1 of G,C-rich. Extra nucleotides may be a product of the activity subgroup IIIb, which is utilized by cryoimmunoglobulin RFs of terminal deoxynucleotidyltransferase(Alt and Baltimore, having anti-IgG activity. Ten of twelve amino acid residues 1982),although this enzyme, which polymerizes random deox- were the same. Differences were at positions 27 (Ser and Gln) ynucleotides at 3‘ ends show a preference for dG residues. and 34 (Hisand Ala) for 18-2-3 and V,IIIb, respectively. Alternatively, the possibility exists that the N segment was Additionally, molecular modeling of the antigen binding site germ line-encoded since germ line D genes have not yet been of 18-2-3 has indicated the presence of aromatic residues isolated. (tyrosines and tryptophan)! Tyrosine residues have been ~ The J Hsegment of 18-2-3 was identical to the J Hgerm ~ shown to be involved in thecombining site of RFs (Nardella line gene segment of the BALB/c strain (Sakano et al., 1980)) et al., 1985). The sequence VH listed as a murine V,IV gene in except for one silent substitution at the thirdbase of codon Kabat et al. (1987) appears to have been a mistaken classifi- 102 (T for C). Since 18-2-3 was derived from a NZB/NZW cation as a murine gene because VH had been reported by mouse, this difference may be an allelic form of the J Hgene. ~ Pech and Zachau (1984) to be related to human V. sub- ~ The entire J Hsegment was used by 18-2-3. Because anti-F1 subgroup IIIb. Thus, inaccord with the proposed evolution of antibodies 18-2-3, 4-4-20, and 3-13 all showed a high degree human V, genes and murine V, genes (Barker et al., 1972), of fluorescence quenching of bound fluorescein (Table 11, in human V. sub-subgroups IIIBand murine V,IV couldbe the Miniprint), this property may be related to utilization of considered related phylogenetically. Moynihan et al. (1985) ~ the J Hgene segment. observed restricted association of the VJIIB light chain sub- The sequence of 18-2-3 light chain indicated that the VL subgroup with &-heavy chain in normal human serum and V,5 segment was encoded by a gene belonging to the subgroup suggested that the KIIIb-p combination could represent a (Potter et al., 1982). The deduced amino acid sequence of the signal to prevent class switching. This may represent a way 18-2-3 VL segment was almost identical to the sequence of of generating high affinity IgM antibodies, as seen with 18-2- light chains in antibodies against 2-phenyloxazolone from 3. BALB/c mice. The light chain of anti-phOx antibody X-ray crystallographic analyses of F(ab’L fragments NQ11.18.1 utilized a V, gene that was 98% homologousat the (Amzel and Poljak, 1979) have indicated that the tertiary and nucleotide level and 97% at the protein level to the VL gene quaternarystructures of the antigen binding sitecan be used by 18-2-3. Hybridoma NQ11.18.1 had been obtained significantly influenced by the chemical nature of the amino following a secondary immunization 8 weeks after the primary acid at position 96 of the light chain. This residue occurs at with phenyloxazolone-conjugated chicken serum albumin. the V-J junction in CDR3 and is encoded by V and J genes. Both gene segments could have been derived from two germ A conserved leucine was located at position 96 in both anti- line genes in the same family or they could be related by oxazolone antibodies and 18-2-3. somatic mutation of the same gene. Somatic mutations have Genes utilized by autoantibodies appear to be present in been observed in IgM, but are more restricted than in IgG or normal individuals as well as in autoimmune patients. It has IgA (Chua et al., 1987). Alternatively, slight differences in been suggested that differences in the complex regulatory homology may indicate that different allelic genes were being pathways of the immune system are responsible for the ex- utilized since 18-2-3 was derived from an (NZB/NZW)Fl pansion in autoimmune patients of clones that wouldbe mouse and the anti-phOx antibody NQ11.18.1 was from a down-regulated in normal individuals (Sanz and Capra, 1988). BALB/c mouse. Findings indicate that autoantibody production in NZB mice The J, gene segment utilized by 18-2-3was J,5, a relatively results because NZB marrow-derived immature B cells ab- uncommon occurrence, since 80% of splenic B cell V-J re- normally resist tolerance induction due to defective clonal arrangements utilized either JJ or J,2 (Wood and Coleclough, inactivation (Cowdery et al., 1987). During the secondary 1984; Nishi et al., 1985). Anti-oxazolone antibodies preferen- immune response in normal humans and animals, IgM RFs tially utilized J,5 (Griffiths et al., 1984). It is possiblethat the are regularly synthesized. Rheumatoid factors may have been V. segment utilized by 18-2-3 and anti-phenyloxazolone an- maintained during evolution because they have the ability to tibodies preferentially rearranged to J,5. remove opsonized bacterial and parasites (Clarkson and Mel- The ability of monoclonal antibody 18-2-3 to bind to phOx low, 1981). may correlate with self-aggregation at low temperatures Studies investigating the genetic origin of murine autoanti- (Dombrink-Kurtzman and Voss, 1988).The structure of phOx bodies have indicated that autoimmune mice do not possess may simulate dipeptidyl conformational or sequential epi- unique IgVH genes (Kofler et al.,1985b).The genetic elements topes (e.g. Phe-His) in the Fc region of 18-2-3 to which the (V, D, J segments) used to encode autoantibodies and anti- antigen binding region of 18-2-3 can bind. Chemical modifi- bodies against foreign antigens are not obviously different cation studies have indicated that histidines are involved in (Kofler et al., 1985a; Manheimer-Lory et al., 1986). Although the Fc region of human IgG and tyrosines on both antigenic somatic mutations canbe a contributory factor(Diamond and and antibody sides of the interactions of two IgG-rheumatoid Scharff, 1984), germ line genes can encode autoantibodies factors (RFs) (Nardella et al., 1985). (Naparstek et al., 1986).Recent findings indicate that unmod- Although RFs are typically IgM and form immune com- plexes by binding Fc determinants on IgG molecules, self- ified or scarcely modified human VH germ line genes encode systemic lupus erythematosus-derived anti-DNAautoanti- association of IgM (Tsai et al., 1977), and IgG antibodies bodies (Dersimonian et al., 1987). Studies based on idiotypic (Pope et al., 1974; Nardella et al., 1981) has been observed. In such cases each molecule serves as an antibody as well as an of and structural characteristics human monoclonal cryoglob- antigen, as with 18-2-3. Antibody 18-2-3 did not appear to ulins with RF activity have indicated that different VH genes have RF activity since it did not bind to IgG molecules, but are utilized, but only a limited set of VL genes are present bound to both human and murine IgM molecules.4 (Kunkel et dl., 1973). An inherent restriction in the immune Interestingly, there was a high degreeof homology between response to self-antigens was suggested by the preferential the CDRl region of 18-2-3 V, and that of human V, sub- association of KIIIb light chains with monoclonal human IgM. RF autoantibodies (Ledfordet al., 1983).The high degree M. A. Dombrink-Kurtzman, M. J. Lacy, and E. W. VOSS, Jr., of primary structure homology and cross-reacting idiotypes manuscript in preparation. indicated that the majority of human IgM RF light chains Variable Region Sequences of an Anti-Fl Cryoantibody 4519 were derived from a single germ line V, gene or a family of Glisin, V., Crkvenjakov, R., and Byus, C. (1974) Biochemistry 1 3 , closely related VJII germ line genes (Goiii et aL, 1985). 2633-2637 For murine RFs, no clear consensus exists. Part of the Go%, F., Chen, P. P., Pons-Estel, B., Carson, D. A., and Frangione, B. (1985) J. Zmmunol. 135,4073-4079 divergence is due to thevariety of strains andconditions (e.g. Griffiths, G.M., Berek, C., Kaartinen, M., and Milstein, C. (1984) unmanipulated, polyclonally activated, antigen-injected) used Nature 312,271-275 in the different studies. An additional consideration is the Gubler, U., and Hoffman, B. J. (1983) Gene (Amst.) 25,263-269 actual number of genes that comprise a family. Originally the Hanahan, D. (1983) J. Mol. Biol. 166,557-580 5558 VH gene family was thought to have -60% of the Holmberg, D. (1987) Eur. J. Immunol. 17,399-403 IUPAC-IUB Joint Commission on Biochemical Nomenclature (1985) H approximately 100 germ line V genes (Brodeur and Riblet, J.Bwl. Chem. 260,14-42 1984). Recent evidence indicated that 500-1000 genes exist in Izui, S., McConahey, P. J., and Dixon, F. J. (1978) J. Zmmunol. 1 2 1 , the 5558 family (Livant et al., 1986). 2213-2219 Since the anti-fluorescein response is diverse, it was not Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M., and Gottes- surprising that antibodies 18-2-3and 4-4-20used different VH man, K.S. (1987) Sequences of Proteins of Immunological Interest, and V, genes, had unrelated idiotypic and metatypic structures United States Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD (Voss et al., 1988) and demonstrated different fine specificities Kofler, R., Noonan, D. J., Levy, D. E., Wilson, M. C., Mdler, N. P. regarding structural analogues. Yet similarity exists between H., Dixon F. J., and Theofilopoulos, A. N. (1985a) J. Exp. Med. these two antibodies since they both exhibit high instrinsic 161,805-815 affinity for fluorescein and >96% quenching of bound ligand. M., Noonan, D. J., Dixon, F. J., and Kofler, R., Perlmutter, R. Although differing in primary structure, the three-dimen- Theofilopoulos, A. N. (1985b) J. Exp. Med. 162,346-351 sional structure of their respective antigen binding sites may Konigsberg, W. H., and Henderson, L. (1983) Methods Enzymol. 91, be similar. X-ray crystallographic studies are in progress to 254-259 Kranz, D. M., and Voss, E. W., Jr. (1981) Mol. Immunol. 1 8 , 889- determine such correlations. 898 Kunkel, H. G., Agnello, V., Joslin, F. G., Winchester, R. J., and Acknowledgments-We thank Maria A. Kyroudis for technical Capra, J. D. (1973) J. Exp. Med. 137,331-342 assistance in theanti-idiotype and fine specificity studies and Angela Kurosawa, Y., and Tonegawa, S. (1982) J. Exp. Med. 155,201-218 Cox for typing the manuscript. Ledford, D. K., Go%, F., Pizzolato, M., Franklin, E. C., Solomon, A., and Frangione, B. (1983) J.Immunol. 131,1322-1325 REFERENCES Levy, S., Mendel, E., and Kon, S. (1987) Gene (Amst.) 54,167-173 . Liu, Z., Wood, C., and Wu, T T. (1987) Nucleic Acids Res. 15,6296 Alt, F. W., and Baltimore, D. (1982) Proc. Natl. Acad. Sci. U.S. A. Livant, D., Blatt, C., and Hood, L. (1986) Cell 47,461-470 79,4118-4122 Makela, O., Kaartinen, M., Pelkonen, J. L. T., and Kajalainen, K. Amzel, L. M., and Poljak, R. J. (1979) Annu. Reu. Bwchem. 48,961- (1978) J. Exp. Med. 148,1644-1660 997 Manheimer-Lory, A. J., Monestier, M., Bellon, B., Alt, F. W., and Andrews, B. S., Eisenberg, R. A., Theofilopoulos, A.N., Izui, S., Bona, C. A. (1986) Proc. Natl. Acad. Sci. U.S. A. 83,8293-8297 Wilson, C. B., McConahey, P. J., Murphy, E. D., Roths, J. B., and Maniatis, T.,Fritsch, E., and Sambrook, J. (1982) Molecular Cloning: Dixon, F. J. (1978) J. Exp. Med. 148,1198-1215 A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Ballard, D. W., Kranz, D. M., and Voss, E. W., Jr. (1983) Proc. Natl. Harbor, NY Acad. Sci. U.S. A. 80,5071-5074 Margolies, M. N., Marshak-Rothstein, A., and Gefter, M. L. (1981) Ballard, D. W., Bates, R. M., and Voss, E. W., Jr. (1985) J.Zmmunol. Mol. Immunol. 18,1065-1077 135,433-439 Meek, K., Jeske, D., Alkan, S., Urbain, J., and Capra, J. D. (1987) Barker, W. C., McLaughlin, P. J., and Dayhoff, M. 0. (1972) in Atlas Monogr. Allergy 22,109-119 of Protein Sequence and Structure (Dayhoff, M. O., ed) pp. 31-38, Moynihan, J. A., Looney, R. J., and Abraham, G. N. (1985) Zmmu- National Biomedical Research Foundation, Washington, D.C. nology 54,207-213 Bates, R.M., Ballard, D.W., and Voss, E. W., Jr. (1985) Mol. Naparstek, Y., Andrk-Schwartz, J., Manser, T., Wysocki, L. J., Breit- Zmmunol. 22,871-877 man, L., Stollar, B. D., Gefter, M., and Schwartz, R. S. (1986) J. Bedzyk, W. D., Johnson, L. S., Riordan, G. S., and Voss, E. W., Jr. Exp. Med. 164,614-626 (1989) J.Biol. Chem. 264,1565-1569 Nardella, F. A., Teller, D. C., and Mannik, M. (1981) J. Exp. Med. Berek, C., Griffiths, G. M., and Milstein, C. (1985) Nature 316,412- 154, i12-125 418 Nardella. F. A.. Teller. D. C.. Barber. C. V.. and Mannik., M. (1985) . . Birnboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7,1513-1523 J. Exp: Med.'162, i811-1824 Bothwell, A.L.M., Paskind, M., Reth, M., Imanishi-Kari, T., Ra- Nishi, M., Kataoka, T., and Honjo, T. (1985) Proc. Natl. Acad. Sci. jewsky, K., and Baltimore, D. (1981) Cell 24,625-637 U.S. A. 82,6399-6403 Brodeur, P. H., and Riblet, R. (1984) Eur. J.Zmmunol. 14,922-930 Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2 , 161-170 Brouet, J.-C., Clauvel, J.-P., Danon, F., Klein, M., and Seligmann, Pech, M., and Zachau, H. G. (1984) Nucleic Acids Res. 12,9229-9236 M. (1974) Am. J. Med. 67,775-788 Podell, D. N., and Abraham, G. N. (1978) Biochem. Biophys. Res. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. Commun. 81,176-185 J. (1979) Biochemistry 18,5294-5299 Pope, R. M., Teller, D. C., and Mannik, M. (1974) Proc. Natl. Acad. Chua, M.-M.,Goodgal, S. H., and Karush, F. (1987) J. Zmmunol. Sci. U.S. A. 71,517-521 138,1281-1288 Potter, M., Newell, J. B., Rudikoff, S., and Haber, E. (1982) Mol. Clarkson, A. B., and Mellow, G. H. (1981) Science 214,186-188 Zmmunol. 19,1619-1630 Cowdery, J. S., Jacobi, S. M., Pitts, A. K., and Tyler, T. L. (1987) J. Reinitz, D. M., and Voss, E. W., Jr. (1984) Mol. Zmmunol. 2 1 , 775- Zmmunol. 138,760-764 784 Dersimonian, H., Schwartz, R. S., Barrett, K. J., and Stollar, B. D. Reinitz, D.M., Strich, R., Scott, J. F., and Voss, E. W., Jr. (1988) (1987) J. Zmmunol. 139,2496-2501 Mol. Zmmunol. 25,621-630 Diamond, B., and Scharff, M. D. (1984) Proc. Natl. Acad. Sci. Roth, C., Rocca-Serra, J., SommB, G., Fougereau, M., and Thkze, J. U.S. A. 81,5841-5844 (1985) Proc. Natl. Acad. Sci. U.S. A. 82,4788-4792 Dombrink-Kurtzman, M.A., and Voss, E. W., Jr. (1988) Mol. Zm- Rusche, J. R., and Howard-Flanders, P. (1985) Nucleic Acids Res. 1 3 , munol. 25,1309-1320 1997-2008 Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W., and Howard, J. Sakano, H., Huppi, K., Heinrich, G., and Tonegawa, S. (1979) Nature C. (1977) Nature 266,550-552 280,288-294 Gell, P. G. H., Harington, C. R., and Rivers, R. P. (1946) Br. J. Exp. Sakano, H., Maki, R., Kurosawa, Y., Roeder, W., and Tonegawa, S. Pathol. 27,267-286 (1980) Nature 286,676-683 Gerpen. J. P.. Stern. R. H.. and Wensink. P. C. 11979) Nucleic Acids . . Sakano, H.. Kurosawa, Y., Weigert, M.,. and Tonegawa, S. (1981) - . R ~ S7.2115-2136 . Nature 290,562-565 . 4520 Variable Region Sequences of an Anti-F1 Cryoantibody Sanger, F., Nicklen, s.,and Coulson, A. R. (1977) Proc. Natl. Acad. (1977) Proc. Natl. Acad. Sci. U. S. A . 74,4591-4594 Sci. U. S. A. 74,5463-5467 Vieira, J., and Messing, J. (1982) Gene (Amst.) 1 9 , 259-268 Sanz, I., and Capra, J. D. (1988) J. Immunol. 140,3283-3285 Voss, E. W., Jr., Eschenfeldt, W., and Root, R. T.(1976) Immuno- Schilling, J., Clevinger, B., Davie, J. M., and Hood, L. (1980) Nature ChmktQ' 139 447-453 283,35-40 Voss, E. W., Jr., Miklasz, S. D., Petrossian, A., and Dombrink- Seidman, J. G., Leder, A., Nau, M., Norman, B., and Leder, P. (1978) Kurtzmmy M, A. (lgm) 'mmunoL*'' 751-759 Science 2 0 2 , l l - 1 7 Wallace, R. B., Johnson, M. J., Hirose, T., Miyake, T., Kawashima, Smith, R. G-, Ballard, D.w9 B k p. R.,Pace, p. E., Bothwell, A. E. H., and Itakura, K. (1981) Nucleic Acids Res. 9,879-894 Wood, D. L., and Coleclough, c. (1984) proc. N&. A C ~ sei, . . L. M., Herron, J. N., Edmundson, A. B., andVoss,E. W., Jr. (1988) u. s. A. 8 1 , 4756-4760 J. Indinn Inst. Sci., in press Yancopoulos, G . D., Desiderio, S. V., Paskind, M., Kearney, J. F., Tsai, C.-M., Zopf, D. A., Yu, R. K., Wistar, R., and Ginsburg, V. Baltimore, D., and Alt, F. W. (1984)Nature 311,727-733 EXPERIMENTAL PROCEDURES Variable Region Sequences of an Anti-F1 Cryoantibody 4522 Variable Region Sequences of an Anti-Fl Cryoantibody :L, 9-40/anl1-9-40 018-2-3 - - 5-27/nmt-5-27 1 I I I 0 , 1 - 2 - 1 0 1 2 3 4 - 2 - 1 0 1 2 3 4 log [nu Inhlbilor/Well] 8- ? 4 c 7- 6- P 4 3 2 1 0 Log Antibody Concentration (ng) Figure 8. Direct blndlng of 18-2-3 to nolld phase FI-BSb and phox-BSL. Fl-BSL Or PhOX-BSA ULa adSOPbed onto well$ and incubated ulth 50 yl afflnlty pupifled antl-fIUOrF4Cein antlbody 18-2-3. Bound antibody wa3 detected wILh '%]-anti- IgH (Y chaln Ppeclficl (- 5 I lo4 epm). HN-C-qH-(CH2)4-PROTEIN PROTEIN S Fluorescein-5-ES& 2-Phenyloaozolone-BSA Figure 9. StrYQtUre 01 lluarereein compared to 2-phenyloi~zolone Covalently coupled to bovine 4epum albumin.
Pages to are hidden for
"J. Biol. Chem.-1989-Dombrink-Kurtzman-4513-22"Please download to view full document