DUARTE ALLELE IMPAIRS BIOSTABILITY OF GALACTOSE-1-PHOSPHATE URIDYLTRANSFERASE IN HUMAN LYMPHOBLASTS

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.