MOLECULAR BASIS OF PYRIMIDINE 5'-NUCLEOTIDASE (P5N) DEFICIENCY AND FUNCTIONAL ANALYSIS OF P5N

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In this study, we screened 73 subjects with non-spherocytic hemolytic anemia of unknown pathogenesis, and diagnosed 5 of pyruvate kinase, 10 of glucose-6-phosphate dehydrogenase, 1 of adenylate kinase and 1 of pyrimidine 5'-nucleotidase (P5N-I) deficient cases.We identified five novel mutations in nine families with P5N-I deficiency : two missense mutations (425C, 721C), one splicing mutation (339C), one 1-bp insertion (251-insA-252) and one 9-bp deletion (del 192-200). All patients are homozygous for each mutation.We expressed mutant P5N-I with 425C and 721C in Cos-7 cells, and examined their intracellular stability. The L124P (425C) showed approximately 20% residual activity and immunoreactive protein of a wild-type control, suggesting that the L124P was an unstable variant. The G241R(721C) had been demonstrated as a kinetic variant with lower affinity for cytidine monophosphate. This mutant was as stable as a control in Cos-7 cells, being compatible with previous results. We could c … More onclude that Gly241 is important for the substrate binding. Haplotype analysis showed that the 721C, which had been identified in five unrelated families, was a founder mutation. Since proteasome inhibitors restored the stability of the L142P, the L142P increases the susceptibility to the degradation by ubiquitin-proteasome pathway.To examine physiological significance of P5N-I in non-erythroid cells, we established a line of transgenic mouse, which ubiquitously overexpressed human P5N-I. Transgene expression was detected in both liver and kidney. However, expression levels are within 2 times of normal controls. Effects of transgene-derived P5N-I on liver and kidney are being analyzed.The P5N-I gene can be induced by alpha-interferon, and the expression is augmented in lymphocytes of patients with HIV infection or autoimmune diseases such as SLE. From these observations, the P5N-I has been suggested to have any roles on immune response. We examined effects of the P5N-I on acute immunity of influenza infection ; however, overexpression of the P5N-I did not modify immune responses of infected cells. Further studies will require gaining insights on immunological roles of P5N-I. We thank Takako Hamada and Shin-ichi Okada for their excellent technical supports. Less