The overall objective of our studies is to identify and define structure/function relationships involving cell surface carbohydrates. We have isolated a family of lectin-resistant (Lec?R?) glycosylation mutants of Chinese hamster ovary (CHO) cells. All of the mutants express altered carbohydrate structures at the cell surface. Experiments have shown that some of the mutants are altered in their abilities to form tumors in nude mice. In-depth characterization of some mutants is our major aim. The experimental approach is to define the structural carbohydrate change corresponding to each mutant type and on the basis of this knowledge to search for the gene product responsible for each glycosylation defect. Animal viruses such as vesicular stomatitis virus and Sindbis virus will be used where possible to localize the carbohydrate defect expressed by each mutant type. Structural studies of radio-labeled carbohydrate moieties will be performed using lectin-affinity and gel filtration columns with the aid of specific glycosidase enzymes. Direct structural analyses of mg quantities of altered carbohydrates will be performed using 'H-NMR spectroscopy in conjunction with gas liquid chromatography/mass spectrometry. Once the structural lesion is defined for a particular mutant, assays will be devised to search for the enzymic basis of the defect. Preliminary studies indicate that all the mutants to be studied with highest priority result from mutations affecting specific glycosyltransferase enzymes. The defects in the three dominant mutants, LEC10, LEC11, and LEC12, have been characterized in studies of the carbohydrates synthesized by the mutants and their revertants. LEC10 CHO cells synthesize carbohydrates containing the """"""""bisecting"""""""" N-acetylglucosamine (GlcNAc) residue and also possess the appropriate glycosyltransferase activity (GlcNAc-TIII). Parental CHO cells do not possess detectable GlcNAc-TIII enzyme, and their carbohydrates do not contain the """"""""bisecting"""""""" GlcNAc. Both the LEC11 and LEC12 mutants synthesize carbohydrates containing alpha-(1,3)-linked fucose residues and also possess alpha-(1,3)-fucosyltransferase activities. Parental CHO cells do not possess detectable alpha-(1,3)-linked fucose residues. The conclusion is that the mutations which result in the LEC10, LEC11, and LEC12 phenotypes do so by inducing the expression of glycosyltransferase enzymes not expressed by parental cells. (A)
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