Quantitative determination of tumor cell-surface sialic acids and their effects on MHC class I antigen-antibody binding

We selected a nontumorigenic leukemia murine T cell line, WB6, from its parental tumorigenic leukemia cell line called EL4 using wheat germ agglutinin (WGA).; To characterize the cell-surface sialic acids, new anion exchange chromatographic methods were developed. The new methods could separate sialic acids released from bovine submandibular mucin (BSM) using 60% methanol in 10 mM NH{dollar}sb4{dollar}OAc aqueous solution as mobile phase. Neu5Ac, Neu5, 9Ac{dollar}sb2{dollar} and Neu5Gc were completely separated from each other. The new anion exchange chromatography has great potential to be coupled with mass spectrometry to quantify and characterize sialic acids from cell surfaces and mucins. The new methods may be used for preparative purposes of sialic acids since the NH{dollar}sb4{dollar}OAc mobile phase is lyophilizable.; Reverse phase HPLC with fluorometric detection was used to quantify the sialic acids released from the cell-surface by Vibrio cholerae neuraminidase (VCN). The tumorigenic EL4 cell line has 1.7 times more total VCN-releasable cell-surface sialic acid than the nontumorigenic WB6 cell line. The majority of VCN-releasable cell-surface sialic acids on the nontumorigenic WB6 is Neu5Gc (69%), while all the VCN-releasable cell-surface sialic acid on the tumorigenic EL4 is Neu5Ac.; By using radioimmunoassay (RIA), the binding affinity between MHC class I antigen and its monoclonal antibody was determined to be 1.5 times higher on WB6 cell surface than on EL4 cell surface. In addition, removing sialic acids from both WB6 and EL4 cell surfaces by neuraminidase pretreatment increased the binding affinity by 1.7 fold.; Assuming the binding of antigen-antibody resembles the antigen-TCR (T cell receptor) binding, the results support the idea that tumor growth may stimulate neuraminidase activity to remove sialic acids from the tumor cell surface to increase the binding strength of TCR to the antigen. This may be one line of the immune defense to get rid of tumor cells.