Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

Abstract

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wildtype cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5-to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.Glycoprotein components of mammalian cell surfaces are thought to play vital roles in such processes as regulation of cell growth, intercellular recognition, and cellular adhesion (8,31,32). A powerful approach for investigating relationships between glycoprotein structure and functions and for elucidating the biosynthesis of the oligosaccharide moieties of these molecules is to examine the phenotypes of somatic cell mutants with altered glycoprotein synthesis (5,10,14,16,18,20,22). One notably successful tactic for the isolation of such glycoprotein mutants has been the selection of cells resistant to cytotoxic lectins (4, 6, 9, 11, 23-25, 30, 33). Such resistance presumably reflects alterations in cell surface glycoproteins, particularly those which are part of or adjacent to the lectin receptors. Recent studies have suggested that more than one sugar may be involved in the surface recognition system for some lectins (2).We have initiated studies based on an alternative method for selection of glycoprotein mutants with altered oligosaccharide moieties. Our

Membrane mutants of animal cells: rapid identification of those with a primary defect in glycosylation.

“…The first three groups of mutants were selected for lectin resistance in single-step protocols, B4-2-1 survived a selection for a mannose-6-P receptor deficiency, and the remaining mutants were survivors of 3H-labeled sugar suicide protocols. All of the mutants had been previously tested for their lectin resistance or lectin binding properties or both with one or more lectins and, in many cases, exhibited phenotypes similar to those of mutants from our repertoire: 15B cells (isolated about the same time as Lecl CHO cells; 29) possessed a typical Lecl phenotype (1, 9); clone 13 (a novel phenotype when isolated; 2) and the abrin-resistant CHO-Ki mutants (18) appeared similar to each other and to Lec8 cells; the Jl LecR mutants isolated recently (14) exhibited phenotypes typical of Lecl (17B), Lec2 or Lec3 (19A), and Lec8 (21B) CHO cells; 1B4-2-1 was initially shown to be ricin resistant (21) and, subsequently, to be deficient in dolichol-P-mannose synthetase, resulting in the synthesis of altered N-linked carbohydrates (33); and the LecR properties of mutants 687 and 699 were consistent (for five different lectins) with those of a Lecl rtutant (699) and a related phenotype (10). The Cl mutant is a recent isolate which has not been extensively characterized.…”

Addition of truncated oligosaccharides to influenza virus hemagglutinin results in its temperature-conditional cell-surface expression.

“…The majority of such mutant cell lines were obtained using cytotoxic plant lectins to select from populations of mutagenized cells those bearing altered carbohydrate moieties on cell-surface molecules. Glycosylation mutants have also been found among survivors of suicide protocols using [3H]sugars (Hirschberg et al, 1981(Hirschberg et al, , 1982 and in cells resulting from selections aimed at individual glycoprotein membrane receptors such as Thy-1 antigen Hyman, 1975, 1979;Trowbridge et al, 1978), the mannose-6-phosphate receptor (Robbins et al, 1981;Stoll et al, 1982), and the low density lipoprotein receptor (Krieger et al, 1981;Kingsley et al, 1986). We have developed a protocol for the isolation of secretory pathway mutants of animal cells that uses a cell sorter and a cell line that constitutively expresses large amounts of a wellcharacterized membrane protein, the HA of influenza virus (Hearing et al, 1989, accompanying paper).…”

Control of carbohydrate processing: the lec1A CHO mutation results in partial loss of N-acetylglucosaminyltransferase I activity.

“…The LeclA mutants previously described in the literature (10,20) were both obtained by protocols that did not preclude the isolation of a rare mutant carrying more than one mutation. If, however, the leclA lesion is due to a single genetic event, it might be expected to arise at a frequency close to 10-5 or 10-6, as observed for the lecl mutation (24).…”