Molecular characterization of antibodies bearing Id-460. II. Molecular basis for Id-460 expression.

Abstract

We have previously described an experimental system for studying the cellular and molecular basis of idiotype expression (1-5). BALB/c mice produce antidinitrophenyl (DNP) ~ antibody expressing a recurrent idiotypic determinant, Id-460. Two genes controlling the expression of this determinant map to the heavy and kappa chain variable regions (VH and V,j). Thus, the determinant is likely to be a combinatorial determinant involving both VH and V,l domains. 1d-460 is also found in normal serum, where much of it binds Pasteurella pneumotropica (P. pneumotropica), and none to DNP. Furthermore, P. pneumotropica immunization induces production of non-DNP-binding, Id-460 + antibodies. To understand the structural basis for this shared idiotypy, we have undertaken a molecular analysis of monoclonal Id-460 + antibodies. We have previously described the development of molecular probes for and the structure of the genes encoding the MOPC-460 (M460) protein, the prototypic DNP-binding antibody in the Id-460 system. We have also reported the structure of two Id-460 + hybridomas, LB8, which binds P. pneumotropica, and D35, which is DNP specific. These three proteins are encoded by closely related VH and V, gene segments, but differ in other gene segments (5). In the present experiments we use these probes to establish the molecular and genetic relatedness of other Id-460 + monoclonal antibodies in an attempt to determine the molecular basis of shared idiotypy by antibodies of differing antigen binding specificity.We have produced a panel of monoclonal antibodies selected for Id-460 expression. We obtained these Id-460 + monoclonal antibodies in two ways: by anti-Id-460 immunization or by DNP immunization. In this report, we demonstrate that all of the Id-460 ÷ hybridomas tested share immunoglobulin (lg) gene rearrangements and DNA sequence homologies for both Vu and VL with M460.

Structural characterization of the second major cross‐reactive idiotype group of human rheumatoid factors. Association with the VH4 Gene Family

“…Monoclonal proteins. The hybridoma product, C6B2, was the generous gift of Dr. S. Hoch (1 l), and the MOPC460 protein was contributed by Dr. J. F. Kearney (12). Dr. T. Logtenberg contributed the V, 5 IgM protein, A224, and the V, 6 anti-DNA antibody, L16 (13).…”

“…The latter was used as a positive control for the anti-VH4-HVZa reagent. MAb M460 from the 36-60 murine V, family shares the VH4-HV2c sequence, and served as a positive control for this antiserum in blotting experiments (12). The M460, VH6-L16, and VH4-C6B2 H chains also share an identical VH6-FRI sequence, and they served as positive controls for the anti-VH6-FR1 antibody.…”

Organization of the murine immunoglobulin VH complex in the inbred strains.

“…A 1.3-kb BamHI-EcoRI fragment contains the JH3 and J segments. VH family-specific probes were isolated as a 1.3-kb BamHI-EcoRI fragment from VH 3609P (Riblet et al, 1986), a 270-bp PstI fragment from the cDNA 93G7 (Sims et al, 1982) that hybridizes to the J558 VH gene family, a 600-bp EcoRI-XbaI fragment from M460 that is specific for the 3660 VH family (Dzierzak et al, 1985), a 500-bp XholI fragment from VGAM3-8 that hybridizes to the VGAM3-8 VH family (Winter, et al, 1985), a 450-bp PstI fragment from S107 (Crews et al, 1981), a 600-bp fragment from pBV14 that detects the J606 family, a 500-bp EcoRI-PstI fragment from VH 39 that hybridizes to the X24 family (Hartman and Rudikoff, 1984), a 320-bp EcoRI-HindEll fragment from VHQ52 , and a 280-bp PstI-EcoRI fragment from VH 81X that recognizes the 7183 VH gene family (Yancopoulos et al, 1984). Southern filter hybridization Fifteen micrograms of liver or B cell DNA was digested with the indicated restriction endonuclease (Boehringer Mannheim), applied to an 0.8% agarose gel and electrophoresed in the presence of 0.094 M Tris, pH 8.0, 0.02 M sodium citrate, 0.0175 M NaCI and 0.2 mM ethylenediaminetetraacetic acid at 85 mAmps for 16-18 h. The size-fractionated DNA was then transferred to nitrocellulose (Schleicher and Schuell) and pre-hybridized in the presence of 50% formamide (BRL) as described (Sims et al, 1982).…”

Amino acids at the site of V kappa-J kappa recombination not encoded by germline sequences.

“…The antigalactan light chains appear to be an exception . We have previously reported that 16 17 antigalactan light chains express Ile at position 96, the point of V,-J, recombination (Ile is not found at this position in any other murine VK region) . Since this amino acid could not readily be generated by sequences usually found at the 3' end of VK and the 5' ends of JK genes, we have cloned the rearranged and germline genes used in this response to determine the origin of the Ile codon at position 96 .…”

“…The actual site of recombination is thus involved in the determination of the last amino acid in the third CDR . Interestingly, in the mouse the length of the third CDR is highly conserved, with very few variants having been reported (14)(15)(16)(17)(18), although, from the three-dimensional structure of the murine K chain (19), there is no obvious constraint that would predict such a conservation . In fact, considerable size variation is observed in this region of rabbit is chains (5).…”