K. Lai et al., DUARTE ALLELE IMPAIRS BIOSTABILITY OF GALACTOSE-1-PHOSPHATE URIDYLTRANSFERASE IN HUMAN LYMPHOBLASTS, Human mutation, 11(1), 1998, pp. 28-38
The Duarte allele (D) is a missense mutation (N314D) that produces a c
haracteristic isoform and partial impairment of galactose-1-phosphate
uridyltransferase (GALT) inhuman erythrocytes, fibroblasts, and transf
ormed lymyhoblasts. The position of this amino acid is distant, howeve
r, from presumptive catalytic site(s) as deduced from a three-dimensio
nal model of crystallized Escherichia coli galT protein, To evaluate t
he mechanism(s) involved in the partial impairment of enzymatic activi
ty, we compared the activity abundance, biological stability, and mRNA
of GALT in human lymphoblastoid cell lines cultured from individuals
homozygous for wild-type (WT/WT) and Duarte alleles (N314D/N314D). No
other nucleotide differences were present in their GALT genes, The app
arent V-max was reduced in N314D/N314D) cells to 31 +/- 3.6 compared t
o WT/WT of 54 +/- 6.5 nmole UDP galactose formed/g cell protein/hour,
Both genotypes had similar apparent K(M)s for UDP glucose of 0.142 +/-
0.057 mM and 0.133 +/- 0.056 mM. This reduced V-max was associated fi
fth a reduced abundance of the 86kD GALT dimer as determined by Wester
n blots acid densitometry Using RNase protection assays, this reduced
GALT protein in the N314D/N314D cell lines was not associated with red
uced abundance of GALT mRNA, Using cycloheximide ethyl-2-oxocyclohexyl
)-2-hydroxyethyl]glutarimide) inhibition of de novo protein synthesis,
GALT enzyme activity and its dimeric protein had a biological T-1/2 o
f similar to 24 hours in N314D/N314D cell lines as compared to 50 hour
s for WT/WT lymphoblasts. Upon exposure to 50 degrees C for 15 minutes
, N314D/ N314D lymphoblasts retained 45% of GALT activity, whereas con
trols retained 77% activity, Reduced activity and thermal sensitivity
caused by the N314D mutation reverted to control values when a lysine
was substituted for a glutamic acid at amino acid 203 in cis (E203K).
In summary, N314D/N314D lymphoblasts have reduced GALT enzyme capacity
, dimeric protein abundance, biological, and thermal stability, We con
clude that the substitution of aspartate for asparagine at amino acid
314 in the human GALT protein reduces the biostability of the active e
nzyme in human lymphoblasts. (C) 1998 Wiley-Liss, Inc.