Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif

The human homologue of the Saccharomyces cerevisiae YSAl protein, YSAlH, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5'-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. K-m and k(cat) values with ADP-ribose were 60 mu M and 5.5 s(-1) respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg2+ or 100-250 mu M Mn2+ ions; fluoride was inhibitory, with an IC50 of 20 mu M. The YSAlH gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSAlH revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSAlH contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSAlH might be to remove free ADP-ribose arising from NAD(+) and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation.