As. Opat et al., Genetic defect in N-acetylglucosaminyltransferase I gene of a ricin-resistant baby hamster kidney mutant, BIOCHEM J, 336, 1998, pp. 593-598
The analysis of mutations associated with glycosylation-defective cell line
s has the potential for identifying critical residues associated with the a
ctivities of enzymes involved in the biosynthesis of glycoconjugates. A ric
in-resistant (Ric(R)) baby hamster kidney (BHK) cell mutant, clone Ric(R)14
, has a deficiency in N-acetylglucosaminyltransferase I (GlcNAc-TI) activit
y 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 rega
ined the ability to synthesize complex N-glycans, demonstrating that the gl
ycosylation defect of RicR14 cells is due solely to the lack of GlcNAc-TI a
ctivity. 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 lo
calized to the Golgi apparatus. We have cloned and sequenced the open readi
ng frame of GlcNAc-TI from parental BHK and Ric(R) 14 cells. A comparison o
f several Ric(R) 14 cDNA clones with the parental BHK GlcNAc-TI sequence in
dicated the presence of two different Ric(R)14 cDNA species. One contained
a premature stop codon at position +81, whereas the second contained a poin
t 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 Gl
cNAc-TI mutant was correctly localized to the Golgi, indicating that the in
active GlcNAc-TI protein was transport-competent. Gly320 is conserved in Gl
cNAc-TI from all species so far examined. Overall these results demonstrate
that Gly(320) is a critical residue for GlcNAc-TI activity.