Human 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2) pharmacogenetics: gene resequencing, genetic polymorphisms and functional characterization of variant allozymes.
3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the sulfate donor cosubstrate for all sulfotransferase (SULT) enzymes. SULTs catalyze the sulfate conjugation of many endogenous and exogenous compounds, including drugs and other xenobiotics. In humans, PAPS is synthesized from adenosine 5'-triphosphate (ATP) and inorganic sulfate (SO2-4) by two isoforms, PAPSS1 and PAPSS2. Rare mutations that inactivate PAPSS2 are associated with human spondyloepimetaphyseal dysplasia and murine brachymorphism. To determine whether more common genetic polymorphisms that do not completely inactivate the enzyme might be one factor responsible for individual differences in sulfate conjugation, we previously cloned the human PAPSS2 gene. In the present studies, we 'resequenced' all twelve PAPSS2 exons and splice junctions, as well as approximately 500 bp of the 5'-flanking region, using 90 Polymorphism Discovery Resource (PDR) DNA samples from the Coriell Cell Repository. Twenty-two single nucleotide polymorphisms (SNPs) were observed, including four nonsynonymous coding region SNPs (cSNPs) that altered the following amino acids: Glu10Lys, Met281Leu,Val291Met and Arg432Lys. We also observed four insertions/deletions, including one sample that was homozygous for an 81-bp deletion in the 5'-flanking region 286 bp upstream from the site of transcription initiation. Transient expression studies showed that two of the nonsynonymous cSNPS, those that resulted in Glu10Lys and Val291Met alterations in encoded amino acids, showed significant decreases in levels of PAPSS activity. In the case of Glu10Lys, decreased activity was paralleled by a decrease in immunoreactive protein, while the Val291Met allozyme displayed a significant decrease in affinity for both ATP and Na2SO4 when compared to 'wild-type' enzyme, but without a significant alteration in level of immunoreactive protein. It will now be possible to test the hypothesis that these common, functionally significant PAPSS2 genetic polymorphisms might contribute to variations in sulfate conjugation in vivo.
Xu
Zhen-Hua
ZH
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Medical School-Mayo Clinic-Mayo Foundation, Rochester, MN 55905, USA.
Freimuth
Robert R
RR
Eckloff
Bruce
B
Wieben
Eric
E
Weinshilboum
Richard M
RM
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D015810:Linkage Disequilibrium; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D010597:Pharmacogenetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D013430:Sulfate Adenylyltransferase / Q000235:genetics*; D014162:Transfection
D017931:DNA Primers; D007527:Isoenzymes; D009097:Multienzyme Complexes; C022322:PAPS synthetase; D013430:Sulfate Adenylyltransferase