Production of IgM hexamers by normal and autoimmune B cells: implications for the physiologic role of hexameric IgM.

Secreted IgM is predominantly found as pentameric molecules, but IgM can also be secreted as hexamers by B cell lines. Murine hexamers activate the complement cascade more efficiently than pentamers, but the physiologic significance of hexameric IgM remains unknown. Here, we report that IgM hexamers and pentamers are cleared from the circulation with similar kinetics, suggesting that the predominance of pentameric IgM in vivo reflects the regulation of polymer assembly and secretion in responding B cells. Normal IgM-secreting B cells, particularly those from the peritoneal cavity, are capable of secreting abundant hexameric IgM in vitro. The disparity between the ability of B cells to secrete IgM hexamers in vitro and the paucity of this polymer in vivo suggest that IgM hexamers might be deleterious. In support of this, we demonstrate that the autoantibodies from a number of patients with cold agglutinin (CA) disease include both IgM hexamers and pentamers. The CA IgM hexamers lyse human erythrocytes in the presence of human complement more efficiently than CA IgM pentamers, suggesting a potential role for hexameric IgM in the pathogenesis of this autoimmune syndrome.

Functional effects of a naturally occurring amino acid substitution in human thymidylate synthase.

A major mechanism underlying the cytotoxicity of fluoropyrimidine analogs such as 5-fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd) occurs via the formation of 5-fluoro-2'-deoxyuridylate (FdUMP), a tight-binding inhibitor of thymidylate synthase (TS). Genetic variation in the structure of the TS molecule is an important determinant of response to fluoropyrimidines, because such variation may affect the binding of FdUMP to the enzyme. Previous studies have shown that the colonic tumor cell line HCT116 expresses two structurally distinct TS polypeptides that differ by the presence of tyrosine or histidine at residue 33. Compared with the Tyr-33 form, the His-33 form confers a 3-4-fold level of FdUrd resistance to cells; this was postulated to be derived from the reduced affinity of the enzyme for FdUMP and N5,N10-methylenetetrahydrofolate, ligands required for the formation of a stable inhibitory complex. In the present study, the Tyr-33 and His-33 forms have been purified to homogeneity, and their properties have been compared in detail. The Km values for dUMP and N5,N10-methylenetetrahydrofolate in the TS reaction were not significantly different between the two enzymes. In contrast, the catalytic efficiency (kcat) was 8-fold lower for the His-33 form. Kinetic and equilibrium binding measurements demonstrated that the dissociation constant for FdUMP binding into the ternary complex was 3-4-fold higher for the His-33 form; this was shown to be due to both a decrease in the rate of FdUMP association with the enzyme and an increase in the rate of FdUMP dissociation from the ternary complex. A TS form containing phenylalanine at residue 33 was created by site-directed mutagenesis and was shown to be very similar to the Tyr-33 enzyme with regard to kcat, pH/activity profile, and effect on FdUrd response. Thus, it is the presence of histidine at residue 33, rather than the absence of tyrosine, that is responsible for the alterations in catalytic and ligand-binding functions exhibited by the His-33 form. Possible mechanisms by which the histidine residue perturbs the structure of the TS active site are discussed.

Genetic variation in thymidylate synthase confers resistance to 5-fluorodeoxyuridine.

The human colorectal tumor cell line HCT 116 was resident to the cytotoxic effects of 5-fluorodeoxyuridine (FdUrd). The response to FdUrd was increased only slightly by the presence of 10 microM folinic acid (CF). HCT 116 formed FdUMP and CH2H4PteGlu polyglutamates after exposure to FdUrd and CF. The sensitivity to FdUrd correlated well with the extent of TS inhibition. The role of TS in the resistance of the cells to FdUrd was examined. HCT 116 expresses two TS enzymes, which differ in pI. The more basic TS has been detected in only HCT 116 cells. The other TS is identical in pI to the enzymes detected in other human cells. The variant TS differs from the common by His replacement of Tyr at residue 33. The variant TS exhibited a 3-fold lower affinity for FdUMP than the common TS. The enzymes co-expressed in HCT 116 exhibited an FdUMP binding constant similar to that of the variant TS. TS-deficient cells were transfected with cDNAs encoding the two TS polypeptides. Transfectants expressing the variant TS were more resistant to FdUrd cytotoxicity than cells expressing the common TS. Thus, the structural variation in TS reduced enzyme affinity for FdUMP and conferred resistance to FdUrd.