Cloning, Expression and Characterization of Mouse (Mus musculus) Nicotinamide 5-Mononucleotide Adenylyltransferase-2

David      W. Raches; Suhong      Xiao; Praveen      Kusumanchi; Joel      Yalowitz; Paresh      Sanghani; Eric      C. Long; Asok      C. Antony; Hiremagalur      N. Jayaram

Abstract

Nicotinamide adenine dinucleotide (NAD+) is synthesized by the action of nicotinamide mononucleotide adenylyltransferase (NMNAT) from NMN and ATP. The mouse homolog of NMNAT-2 (mmNMNAT-2) was cloned, expressed, and subsequently identified using MALDI-TOF in conjunction with the ProFound database. Circular dichroism analyses of recombinant mmNMNAT-2 showed α helical and β sheet secondary structures, consistent with the known structure of the human isoform. Competition experiments using mouse pancreatic tissue lysates with recombinant mmNMNAT-2 demonstrated that the activity of the expressed protein was similar to the human isoform. Immunohistochemistry of mouse embryonic tissues with hNMNAT-2 also showed a tissue- and cellular-specific expression of this isoform. Therefore, our studies demonstrate for the first time the clear biological evidence for the existence of a mouse isoform of hNMNAT-2. These studies may help in future investigations aimed at understanding the regulation of this gene and its pathway, and in turn, will spur the development of novel therapies for diseases such as cancer and diabetes since mice are the most frequently used experimental system for in vivo studies.

Keywords: Circular dichroism, MALDI-TOF, mouse NMNAT-2, tissue expression, NAD, tiazofurin, Nicotinamide adenine dinucleotide, nicotinamide mononucleotide, adenylyltransferase, analyses, human isoform