Conformational and dynamic characterization of blood group-related carbohydrate tumor-associated antigens. Relations to antibody binding specificity
Abstract
The monoclonal antibody CO 19-9 was raised in mice against human colon carcinoma tissue. CO 19-9 is specific for the tumor-associated 19-9 tetrasaccharide, NeuAc$\alpha$1$\to$3Gal$\beta$1$\to$3$\beta$GlcNAc(4$\gets$1$\alpha$Fuc) and does not cross-react with the 19-9 antigen's Le$\sp{\rm a}$ blood group component, Gal$\beta$1$\to$3$\beta$GlcNAc(4$\gets$1$\alpha$Fuc). In order to gain further information about the binding of CO 19-9 to the tetrasaccharide, a conformation comparison of the two carbohydrate antigens was carried out on synthetic versions of the antigens using $\sp1$H NMR spectroscopy and hard-sphere energy calculations. Significant chemical shift differences between the two molecules were detected only for protons at or near the linkage site of NeuAc to the Le$\sp{\rm a}$ trisaccharide core. Coupling constants were not significantly affected by NeuAc linkage, and thus the average conformation of Le$\sp{\rm a}$ appears unchanged by NeuAc attachment. However, in both Le$\sp{\rm a}$ and 19-9, a significant number of NOESY crosspeak integral values differed from those predicted by hard sphere energy calculations assuming a rigid structure model. The data indicate that the terminal NeuAc linkage is more flexible than the inter-residue bonds of the core trisaccharide whose dynamics appear more restricted upon NeuAc attachment. This analysis provides evidence in favor of NeuAc as an epitope-creating unit involved directly at the antibody binding site. However, these results do not preclude crucial roles in antibody recognition for regions on the 19-9 antigen that are distanced from NeuAc. Experiments were also conducted on a Y blood group-related antigen, Fuc$\alpha$1$\to$2Gal$\beta$1$\to$4GlcNAc(3$\gets$1$\alpha$Fuc), whose aberrant expression is associated with various malignancies. Those Y tetrasaccharide NOESY crosspeak integrals involving the methyl groups on the Fucose residues indicate increased mobility of both methyls relative to the overall tumbling rate. Both Fucose residues have been implicated in the binding of the Y antigen to BR55.2, an antibody raised in mice against human breast carcinoma cells. This result is in contrast to the Fucose methyl group on the 19-9 antigen whose local rate of motion appears to be equivalent to the overall tumbling rate of the molecule. The local motions at areas of these carbohydrate antigens such as the NeuAc/Gal glycosidic bond and the methyl groups may be features that are essential to antibody recognition. These data, therefore, point to the need for the incorporation of variable dynamics into the epitope concept.
Subject Area
Biophysics|Molecular biology
Recommended Citation
Bechtel, Bethany Suzanne, "Conformational and dynamic characterization of blood group-related carbohydrate tumor-associated antigens. Relations to antibody binding specificity" (1990). Dissertations available from ProQuest. AAI9026519.
https://repository.upenn.edu/dissertations/AAI9026519