Human X-linked phosphoribosylpyrophosphate synthetase superactivity is associated with distinct point mutations in the PRPS1 gene.

B J Roessler,J M Nosal,P R Smith,S A Heidler,T D Palella,R L Switzer,M A Becker

doi:10.1016/s0021-9258(19)74339-7

Abstract

Superactivity of phosphoribosylpyrophosphate synthetase (PRS) is an X chromosome-linked disorder of purine metabolism, characterized by gout with uric acid overproduction and, in some families, neurodevelopmental impairment. Two highly homologous isoforms of PRS (PRS1 and PRS2), each encoded by a distinct X chromosome-linked locus, have been identified, and PRS1 and 2 cDNAs have been cloned. The entire 954-base pair translated regions of PRS1 and 2 cDNAs derived from cultured lymphoblasts and fibroblasts from two patients in whom purine nucleotide feedback resistance of PRS is associated with enzyme superactivity and neurodevelopmental defects were examined by direct sequencing after polymerase chain reaction amplification of PRS transcripts. Nucleotide sequences of PRS2 cDNAs from the patients and normal individuals were identical. In contrast, PRS1 cDNAs from the patients differ from normal PRS1 cDNA, each by a single base substitution. PRS1 cDNA from patient N. B. showed an A to G transition at nucleotide 341, corresponding to an asparagine to serine change at amino acid residue 113 of mature PRS1. A G to C transversion at nucleotide 547, indicating an aspartic acid to histidine change at amino acid 182, was found for PRS1 cDNA from patient S. M. Point mutations at the sites identified in the PRS1 cDNAs of the two patients were confirmed by the results of RNase mapping analysis. Normal, N. B., and S. M. PRS1 cDNAs were introduced into Escherichia coli BL21 (DE3)/pLyS, and recombinant N. B. and S. M. PRS1s showed the purine nucleotide feedback resistance phenotypes characteristic of PRS from patients' cells.

Highlights

Superactivity of phosphoribosylpyrophosphatesyn- regulatory substrate in the synthesis of purine, pyrimidine, thetase (PRS) is an X chromosome-linked disorder of and pyridine nucleotides (Becker et al, 1979)
The entire 954-base pair translated regiofnsP R S l and 2 cDNAs derived from culturedlymphoblasts and fibroblasts from two patients in whom purine nucleotide inorganic phosphate (Pi)and Mg2+ as activators (Kornberget dl., 1955; Fox and Kelley, 1971)
Superactivity of PRS is an X chromosome-linked inherited disorder (Yen et al, 1978) in which excessive enzyme activity is associated with uric acid overproduction and gout (Becker et al, 1979).Uric acid overproduction in individuals with PRS superactivity results from feedback resistanceof PRS is associated with enzyme increased production of PRPP and consequent acceleration superactivityand neurodevelopmentaldefects were of purine nucleotide synthesis de novo (Zoref et al, 1975; examined bydirect sequencing after polymerase chain Becker et al, 1987)

Summary

EXPERIMENTAL PROCEDURES

Cell Lines-Fibroblasts strains were initiated (Rosenbloom et al, 1968) from skin biopsies obtained from N. EcoRI restriction fragment containingthe complete translated region of normal human PRSl cDNAwasexcised from the previously described expression plasmid, pBCllO (Nosal etal., 1993)and cloned in appropriate orientationinto theEcoRI site of the multiple cloning region of the plasmid, pSPRBS. The latter vector consisted of the plasmid pSP72 (Promega) into which a ribosomal binding sequence (Shine and Dalgarno, 1974) had been cloned between the BglII and EcoRI sites of the plasmid. Inthe resulting plasmid, designated pSPRSlN, the Shine-Dalgarno sequence was 11bp upstream of the ATG translation initiation codon of PRSl cDNA and downstream of the T7phage RNA polymerase promoter (Studier etal., 1990).

PRPSl MutationHs uinmPaRnPP

RESULTS

DISCUSSION

Findings

PRPSl Mutations in Human PRPP Synthetase Superactivity