The mannose 6-phosphate receptor of Chinese Hamster ovary cells. Isolation of mutants with altered receptors.

A method has been developed for the isolation of mutant Chinese hamster ovary cells with altered mannose 6-phosphate receptors.Mutants were selected for resistance to a conjugate composed of w-(6-phospho)pentamannose oligosaccharides covalently linked to ricin (Man 6P-ricin).Cells were incubated with this hybrid reagent in the presence of 0.1 M lactose, in order to block the normal binding of the ricin moiety to carbohydrates on the cell surface.Under conditions where binding of Man 6P-ricin occurred solely through the mannose 6-phosphate receptor, viability of the par- ent cells was reduced to 0.1%.To isolate Man 6P-ricinresistant mutants, mutagenized cells were treated with the conjugate, survivors were replicated, and the replicate colonies were screened for incorporation of [3sS] methionine into protein after a second incubation with Man 6P-ricin.Eleven of 20 mutants isolated according to this procedure were found to exhibit an increased resistance (2-to 9-fold) to Man 6P-ricin.Six of the resistant mutants showed decreased uptake of bovine testicular &galactosidase, which enters Chinese hamster ovary cells via the mannose 6-phosphate receptor.Studies of enzyme binding were conducted with two mutants that displayed 10% of the parental level of uptake: decreased binding of P-galactosidase to mutant A10-2-4 was found to reflect, at least in part, an increased rate of dissociation of enyzme from the receptor; mutant B4-2-1 exhibited reduced binding at low concentrations of P-galactosidase and greater than parental binding at elevated levels of enzyme.Unexpectedly, all mutants resistant to Man 6P-ricin also showed increased resistance (1.3-to 4-fold) to ricin, although binding of ricin to the mutants was equal to or greater than binding to the parent.Uptake of exogenous acid hydrolases into fibroblasts has been shown to depend upon the presence of a mannose 6phosphate recognition marker on those enzymes (1-6).