Cloning and expression of the lifespan-associated protein Sir2 from Daphnia pulex

The activity of NAD⁺-dependent deacetylase Sir2, originally discovered in yeast, is known to be essential for effective longevity. The relationship between Sir2 and lifespan in Daphnia pulex was investigated by cloning and analysis the full-length 1901 bp cDNA. The Sir2 protein includes several zinc finger active sites and two readily hydrolysable low-complexity SD-rich regions. The three-domain structure of Sir2 has a distinct crevice that plays an important regulatory role in the binding of NAD⁺. D. pulex Sir2 shares 90% amino acid sequence identity with Sir2 from D. pulicaria, 89% with D. magna Sir2, 40% with Mus musculus Sir2, and 39% with the Homo sapiens protein. Expression of Sir2 mRNA was measured at 1, 10, 15, 20,25, 30 and 35 days by real-time PCR (p <.05), and was lowest at 1 day and highest at 20 days (p <.05), after which expression decreased with age. In situ hybridisation showed that Sir2 mRNA was expressed in the chest, the first and second antennae, and the gonadal gland in D. pulex. A Sir2 gene fragment was amplified by PCR, ligated into the pEASY-Blunt vector, and recombinant Sir2 was expressed in Escherichia coli and purified by Ni affinity chromatography. The recombinant protein was used as antigen to immunise rabbits, and antiserum was successfully purified using the protein A method, yielding a Sir2 polyclonal antibody. Western blotting showed that Sir2 is expressed at a 69 kDa protein in D. pulex, in accordance with the predicted value. Sir2 protein abundance increased gradually with age, but remained unchanged after 25 days.