LEC32 IS A NEW MUTATION IN CHINESE-HAMSTER OVARY CELLS THAT ESSENTIALLY ABROGATES CMP-N-ACETYLNEURAMINIC ACID SYNTHETASE-ACTIVITY

LEC29.Lec32 is a glycosylation mutant that was isolated from a selecti on of mutagenized Chinese hamster ovary (CHO) cells for lectin resista nce. Compared with LEC29 CHO cells, the double mutant exhibited an unu sually high sensitivity to the toxic lectin, ricin, indicating increas ed exposure of galactose residues on cell surface carbohydrates. Struc tural analysis of LEC29.Lec32 cellular glycoproteins showed a nearly c omplete lack of sialic acid residues. Genetic analysis demonstrated th at the lec32 mutation is recessive and novel. Biochemical analysis sho wed that the mutant cells contained less than 5% of the cytidine 5'-mo nophosphate N-acetylneuraminic acid (CMP-NeuAc) present in parental CH O cells (1.6 nmol/mg of cell protein). A sensitive radiochemical assay used to measure CMP-NeuAc synthetase activity showed that the propert ies of this enzyme in parental CHO cells were essentially identical to those of CMP-NeuAc synthetase in various mammalian tissues. However, no CMP-NeuAc synthetase activity was detected in LEC29.Lec32 extracts. Mixing experiments provided no evidence for an inhibitor in the mutan t CHO cells, and two revertants, which expressed only the LEC29 phenot ype, had normal CMP-NeuAc synthetase levels. The combined evidence ind icates that the lec32 mutation resides in either the structural gene e ncoding CMP-NeuAc synthetase or in a gene that regulates the productio n of active enzyme.