Immunochemical specificity of the combining site of murine myeloma protein CAL20 TEPC1035 reactive with dextrans.

Abstract

The immunochemical specificity of the combining sites of murine myeloma protein CAL20 TEPC1035 was studied by quantitative precipitin and precipitin inhibition assays. Myeloma protein CAL20 TEPC1035 precipitated with only three dextrans, B1355S4, B1498S, and B1501S, with high proportions of alpha(1 leads to 3) linkages, but not with any other dextrans, glycogen, and pullulan. Inhibition tests with various sugars show that the combining site of myeloma protein CAL20 TEPC1035 is most complementary to panose, a trisaccharide DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 4)DGlc. Panose was 3.3 times more potent than a tetrasaccharide DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 4)DGlc alpha(1 leads to 4)DGlc and 8, 23, 42, > 42 times more active than maltose, nigerose, isomaltose, and kojibiose, respectively. These findings were paralleled by their binding properties as determined by affinity electrophoresis. The association constants (Ka) of these three dextrans to myeloma protein CAL20 TEPC1035 ranged from 3.8 X 10(3) ml/g to 5.02 X 10(3) ml/g. The association constant of inhibitor (Kia) of panose was 8.19 X 10(3) M-1. Myeloma protein CAL20 TEPC1035 is an antidextran with specificity different from those of other murine myeloma antidextrans and from human antidextrans reported previously and its combining site size is at least as large as a trisaccharide. The binding constant of methyl alpha-D-glucoside (7.2 X 10(2)) was 73% of that of panose and comparable to that of myeloma protein W3129 (9.4 X 10(2)) with a cavity-type site and 600 times lower (1.6 X 10(0)) for QUPC52 with a groove type site, indicating that the terminal nonreducing residue is held in a cavity. Inhibition data with various alpha(1 leads to 4)-linked oligosaccharides also indicate that the internal portions of these inhibitors may react directly with a portion of the combining site. These findings suggest that myeloma antidextran CAL20 TEPC1035 has a partial cavity-type combining site in which the terminal nonreducing dGlc alpha(1 leads to 6) moiety is held in a cavity with the other two sugars forming a groove. However, oligosaccharides with one or more alternating [leads to 3DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 3)DGlcl leads to] units with and without terminal nonreducing DGlc alpha(1 leads to 6) or DGLc alpha(1 leads to 3) side chains remain to be tested to determine whether structures known to be present in the three dextrans which precipitate CAL20 TEPC1035 may not prove to be more active than panose.

Induction of a cross-reactive idiotype dextran-positive antibody response in two IgH-Cb mouse strains treated with anti-J558 cross-reactive idiotype antibodies.

“…This is clearly evident from the calculations of their relative binding avidities. Table I also shows that the binding specificity of the J558 myeloma protein measured in the present assay is very similar to that of its reported specificity in a precipitin assay (28). In contrast, purified )~l-positive IdX-negative conventional C57B1/6 antibodies have a very different binding pattern; they do bind to B1355, B1498S, and B1501S, but they do not bind at all to the remaining five Dex.…”

“…The binding pattern obtained with the purified antibodies was typical of the results obtained with the serum samples, and is shown in Table I. Also shown is the binding pattern of purified antia(l-3) Dex antibodies from hyperimmune BALB/c mice and from conventionally hyperimmunized C57B1/6 mice, as well as the binding of the reference J558 and MI04E myeloma antibodies (28,29).…”

Combining site specificities of mouse hybridoma antibodies to dextran B1355S.

“…When the antibodies are arranged in order of increasing cross reactivity, five groups are seen. Previously characterized mouse myeloma proteins CAL20 TEPC 1035, J558, and MOPC104E fit into groups 1, 3, and 5, respectively (10)(11)(12). From Fig.…”

“…The three members of group 3, Hdex 6, 9, and J558, also react with B742S. In addition, proteins in this group cross-react somewhat with three or four other dextrans: Hdex 6 and 9 with B1299S, B1355L, and B1255, and J558 (10,11) with B1299S, B1255, B1375, and B742L.…”

A comparative study of CE‐SDS, SDS‐PAGE, and Simple Western—Precision, repeatability, and apparent molecular mass shifts by glycosylation

“…To stay with the example of dextran: interactions between dextran and some proteins were described as early as in 1960 [51]. In addition, some (murine) myeloma proteins appear to show affinities to dextran [52, 53]. An interaction would reduce the migration speed of the proteins concerned and would also explain why the effects can be observed to different extents when comparing the methods.…”