J. Biol. Chem.-1989-Dombrink-Kurtzman-4513-22 by shuifanglj


              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
                                                                                                              (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,
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.

4514                               Variable Region Sequences of an Anti-F1 Cryoantibody
                EXPERIMENTAL PROCEDURES~                                               Anti-Pluorescein Hybridor Pmtain 18-2-3 Heavy Chain

                                                                                                     Vari8ble Re8ion Squenoa

   Determination of NH2-terminal Amino Acid Sequences-                         -19
                                                                               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
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
                                                                   Ser Leu Sur Ile Thr Cys T r Val Ser Gly pha Ser L u Thr Asn Tyr Gly Val His
(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
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
                                                                   Trp Val Arg Gln Pro Pro Gly LYE Gly Leu
                                                                                                          G1u Trp Leu Gly Val 110 Trp Ala Gly
                                                                   TCC CTC CGC CAG C C A
                                                                                    CC     COA LAG CCT CTC GAG TCC CTC GGA C AT
                                                                                                                            TA A  TCC GCT CCT
  Heavy C h i n Sequences-Six oligonucleotideswere synthe-
sized as primers in sequencing the heavy and light chain
                        70                                                              60

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
                                                                   Cln Gly Thr Ser Val Thr Val Ser Ser
                                                                   CAA GGA ACC T C A GTC A C G N TCC
                                                                                          C        TCA
                                                             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.
                                                                      Anti-F1 antibody 18-2-3 possessed a D segment closely resem-
                      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
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       .

                           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 -

                          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

  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
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.


               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-
                                    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-
                                                                                        tions with increasing concentrations of unlabeled 4-4-20, 9-

                                                          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
               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.
                                                                                           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
                     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
                                                                                        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
                                                                                        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
           ;                                        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
4518                                                                   Cryoantibody
                                    Variable Region Sequences an Anti-F1
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
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)
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
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4520                                      Variable Region Sequences of an Anti-F1 Cryoantibody
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                          EXPERIMENTAL PROCEDURES
Variable Region Sequences of an Anti-F1 Cryoantibody
4522   Variable Region Sequences of an Anti-Fl Cryoantibody


                                               -                                                         -             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]




                                  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

                              Fluorescein-5-ES&                                         2-Phenyloaozolone-BSA
          Figure 9.      StrYQtUre 01 lluarereein compared to 2-phenyloi~zolone Covalently
          coupled to bovine      4epum     albumin.

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