Nucleotide sequence of the cDNAs encoding the variable region heavy and light chains of a myeloma protein specific for the terminal nonreducing end of alpha(1----6)dextran.

Abstract

The complete nucleotide sequences of the cDNAs encoding the heavy-and light-chain variable regions (VH and VL) of myeloma protein W3129, an anti-a(1--+6)-dextran with a unique combining site, have been determined. The VH region is encoded by a germ-line gene highly homologous to VH441, which also appears to be used in some anti-galactans and anti-levans. W3129 VH uses a diversity region that is not found in any of the galactan-or levan-specific antibodies and whose germ-line counterpart is unknown. The W3129 light chain differs from complete light-chain sequences thus far reported but is identical in the rwst 23 amino acids with NZB IgA myeloma PC118.Dextran B512 is a homopolymer of glucose consisting of 95% a(1-6)-linked glucose, with 5% a(1-*3)-linked glucose at branch points (1, 2), and dextran-specific antibodies have been described in humans (3, 4), rabbits (5), and mice (6-10). The simplicity of the antigenic determinant and the availability of oligosaccharides provided a molecular ruler for measuring the sizes of anti-dextran combining sites (3, 4). Isolation of mouse myelomas specific for a(1--6)dextran allowed detailed immunochemical mapping of the combining site sizes and shapes of homogeneous populations of antibodies. These monoclonal antibodies recognize either the internal linear portion of the molecule or the terminal nonreducing ends; the former were loosely described as having groove-shaped sites, and the latter as having cavityshaped sites. Two such myeloma proteins, QUPC52 and W3129, served as prototypes for groove-type and cavity-type combining sites, respectively. QUPC52 has a combining site size complementary to 6 a(1-*6)-linked glucose units, whereas the site size ofW3129 is 5 glucose units, with glucoses from the nonreducing end (11, 12), and possibly internal residues (13), contributing =95% of the binding energy. This distinction has been made by inhibition of precipitation of a(1-6)dextran with dextran-specific antibodies, using oligosaccharides ranging in size from 2 to 7 a(1-*6)-linked glucose units (11), by competition in equilibrium dialysis (12), and by fluorescence quenching experiments (12,14). Moreover, a synthetic linear dextran of 200 glucose units (15) behaves as a monovalent nonprecipitating ligand for W3129 since it has but one terminal nonreducing end (12), whereas it precipitates with QUPC52, since any 6 of the 200 a(1-36)-linked glucoses could constitute a determinant, and thus it would be multivalent toward groove-type sites. Precipitation with synthetic linear dextran thus provides a rapid method of distinguishing between these two kinds of sites.Numerous mouse hybridomas secreting antibodies specific for a(1-36)-linked dextran have been generated in this laboratory; all are reactive with the internal linear portion of the dextran molecule (6-8). Thus, although cavity-type antibodies have been described in the serum of rabbits (16), only two monoclonal antibodies, W3129 and W3434, have been reported (11) that recognize the terminal nonreducing end ofthe dextra...

The mouse antibody response to infection with Cryptococcus neoformans: VH and VL usage in polysaccharide binding antibodies.

“…They also suggest that priming either with a glycoconjugate (56,57), an antiidiotype, or other TD crossreactive antigens could increase the number ofresponder mice in infection. It is interesting to note that dextran, which elicits antibody responses in mice that use some of the same genetic elements that we have described above (58), has been used by AIDS patients as an antiviral agent (59). Given that the ability to mount an antibody response to CNPS in infection may depend on prior exposures to other antigens, it is conceivable that the administration of dextran to AIDS patients could influence their ability to make antibody to CNPS.…”

Molecular characterization of the humoral responses to Cryptococcus neoformans infection and glucuronoxylomannan-tetanus toxoid conjugate immunization.

“…Although compared with antiprotein responses antipolysaccharide responses are typically characterized as being "restricted" with regard both to isotype and clonotype (57)(58)(59)(60)(61)(62)(63)(64), restriction is a relative term. For example, although mAbs specific for c~-(1-*6)-dextran have been shown to use a limited subset of V. genes, there is considerable junctional diversity and variation in the combinations of V. and VL genes utilized (60,65,66). Likewise, the response to group A streptococcal polysaccharide has been shown to be characterized by a heterogeneous population of antibodies (59).…”

Model-building study of the combining sites of two antibodies to alpha (1----6)dextran.

“…The amino acid sequence of W3129 has been deduced from cDNA (5), but the sequence of QUPC52 has not been determined. However, many hybridoma proteins with groove-type sites have been sequenced (6).…”