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 (RicR) baby hamster kidney (BHK) cell mutant, clone RicR14, 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 RicR14 cells transfected with wild-type 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, RicR14 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 RicR14 cells. A comparison of several RicR14 cDNA clones with the parental BHK GlcNAc-TI sequence indicated the presence of two different RicR14 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 Gly320 is a critical residue for GlcNAc-TI activity.
Opat
A S
AS
Department of Pathology and Immunology, Monash University Medical School, Commercial Road, Prahran, Victoria 3181, Australia.
Puthalakath
H
H
Burke
J
J
Gleeson
P A
PA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D017124:Conserved Sequence; D006224:Cricetinae; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics*; D017354:Point Mutation; D011817:Rabbits; D051381:Rats; D012276:Ricin / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014162:Transfection
D012276:Ricin; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I