364 C. Silvain, zyxwvutsrq F! Aucouturier, I. Leduc et al. Eur. J. Immunol. 1993. 23: 364-368 z Christine Sivain, Pierre Aucouturier, Isabelle Leduc, Edith Mihaescoo+, Jean-Louis Preud’homme and Michel Cognd Laboratory of Immunology and Immunopathology, CNRS URA 1172, Immunology and Molecular Interactions, University Hospital and Faculty of Sciences, Poitiers and Laboratory of Immunochemistry and Immunopathology (INSERM U zyxwvutsr 108), Hospital Saint Louiso, Paris 1 Introduction A human myeloma IgA with a hybrid heavy chain resulting from putative somatic gene conversion A human monoclonal IgAl-IgA2h hybrid molecule was detected in a myeloma patient homozygous for the A2m(l) allotype during a systematic study of monoclonal IgA with subclass-specific monoclonal antibodies (mAb) and the lectin jacalin. This monoclonal immunoglobulin (GAU) reacted with both, although not with all, anti-a1 and anti-a2 mAb. Its heavy (H) chain contained an a1 hinge region as shown by jacalin reactivity, the presence of disulfide-linked H and light chains in spite of its belonging to the IgA2m(l) allotype and amino acid composition of the isolated hinge region. The complete sequence of the H chain was deduced from that of complementary DNA clones from bone marrow cells. The CH1 domain, hinge region and beginning of the CH2 domain and the membrane peptide were encoded by the a1 gene, with an insertion of an a2m(l) gene sequence accounting for the end of the CH2 and part or all of the CH3 domain (sequence identity between the two normal genes precludes a precise definition of breakpoints). The region of the 5‘ recombination site included a repeat of a six base pair sequence which might play a role in the genetic exchange. The GAU hybrid zyxwv a gene was undetectable in the patient’s genomic DNA from polymorphonuclear cells. The genetic event which occurred at the level of the proliferating plasma cell clone is most likely to be a gene conversion. Human IgA includes two subclasses, IgAl and IgA2, and two IgA2 allotypes, A2m( 1) and A2m(2). The A2m( 1) a2 gene likely emerged as a result of a conversion between the a1 and A2m(2) a 2 genes, in which most of the CH3 exon of the a2 gene was replaced by that of the a1 gene [l, 21. In hominoids carrying a duplication of the Cy-Cy-Ceca cluster, comparison of Ca gene sequences also demon- strated a concerted evolution of these genes through gene conversion [3]. A few human hybrid Ig H chains containing parts of two different C regions have been documented at the protein level. Hybrid IgG molecules were first reported. A yl-y3 (“lepore” type) hybrid H chain found in a normal serum [4] carried a y3 Fab fragment extending up to or beyond the hinge region and a yl Fc fragment [5]. A normal counterpart of a y2-y4 hybrid chain characterized in a Black myeloma patient [6] was found in certain sera in the Black population, suggesting that such peculiar Ig C regions may exist at the germinal level. In the single reported instance of a hybrid myeloma IgA molecule, protein ROU, a partial amino acid sequence of tryptic and chymotryptic fragments showed that the CH1 domain was related to the a1 gene, whereas the hinge and CH2 domain displayed an a 2 sequence of the A2m(l) allotype [7]. [I 109051 zyxwvu + This paper is dedicated to our lamented friend and colleague Edith Mihaesco. Correspondence: Christine Silvain, CNRS URA 1172, CHU, BP 577, F-86021 Poitiers Cedex, France Abbreviations: cDNA: Complementary DNA bp: Base pair aCH1-3 probe: a1 gene fragment encompassing the CH1, CH2 and CH3 exons amb probe: a1 gene fragment containing the membrane exon Key words: Myeloma zyxwvutsrqp I IgA I Gene conversion However, whether this recombination had occurred at the germinal or somatic level remains unknown. We report on the primary structure of a new human myeloma IgAl-IgA2h hybrid molecule (GAU). This pro- tein was detected during a study of myeloma IgA proteins with the lectin jacalin (which is IgA1- and IgD-specific by precipitation assays [8]) and subclass-specific mAb, in which a single protein out of 231 displayed features of both IgA subclasses [9]. The sequence of the corresponding complementary DNA (cDNA) and study of germinal DNA showed that protein GAU resulted from a somatic recom- bination event which was likely a conversion between the a genes. 2 Material and methods 2.1 Immunochemical studies IgA subclasses were determined with the mAb M4D8, HP6116, MH141-11, 1-7262, 69-11.4 and 69-7.1 (anti-al) and HP6109 and 512-H5.1 (anti-a2), by a competitive ELISA and with the lectin jacalin by immunoelectrophor- esis, as previously described [9]. IgA2 allotypes were assessed by hemagglutination inhibition with polyclonal Ab (kindly performed by Dr. G. de Lange, Amsterdam) and by ELISA with the anti-A2m(2) mAb 194-3.1. The mAb were provided by Dr. J. Mestecky (the UAB, Birmingham, AL), except for M4D8 which was purchased from the Binding Site Ltd. (Birmingham, UK). The presence of disulfide bridges between H and L chains was assessed by 10% SDS-PAGE followed by Western blotting [lo]. The protein GAU was purified from the patient’s serum by Sepharose 6B (Pharmacia, Uppsala, Sweden) and jacalin- Sepharose affinity chromatography, as previously described 00 14-298Ol9310202-0364$3.50 + zyxwvutsr .2510 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1993