Genetic defect in N-acetylglucosaminyltransferase I gene of a ricin-resistant baby hamster kidney mutant

The analysis of mutations associated with glycosylation-defective cell lines has the potential for identifying critical residues associated with the activities of enzymes involved in the biosynthesis of glycoconjugates. A ricin-resistant (Ric(R)) baby hamster kidney (BHK) cell mutant, clone Ric(R)14, has a deficiency in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and as a consequence is unable to synthesize complex and hybrid N-glycans, Here we show that Ric(R)14 cells transfected with wildtype GlcNAc-TI regained the ability to synthesize complex N-glycans, demonstrating that the glycosylation defect of RicR14 cells is due solely to the lack of GlcNAc-TI activity. With the use of specific antibodies to GlcNAc-TI, Ric(R) 14 cells were shown to synthesize an inactive GlcNAc-TI protein that is correctly localized to the Golgi apparatus. We have cloned and sequenced the open reading frame of GlcNAc-TI from parental BHK and Ric(R) 14 cells. A comparison of several Ric(R) 14 cDNA clones with the parental BHK GlcNAc-TI sequence indicated the presence of two different Ric(R)14 cDNA species. One contained a premature stop codon at position +81, whereas the second contained a point mutation in the catalytic domain of GlcNAc-TI resulting in the amino acid substitution Gly320 --> Asp The introduction of a Gly320,Asp mutation into wild-type rabbit GlcNAc-TI resulted in a complete loss of activity; the GlcNAc-TI mutant was correctly localized to the Golgi, indicating that the inactive GlcNAc-TI protein was transport-competent. Gly320 is conserved in GlcNAc-TI from all species so far examined. Overall these results demonstrate that Gly(320) is a critical residue for GlcNAc-TI activity.