Expression, Localization, and Biochemical Characterization of Nicotinamide Mononucleotide Adenylyltransferase 2

Abstract

Nicotinamide mononucleotide (NMN) adenylyltransferase 2 (Nmnat2) catalyzes the synthesis of NAD from NMN and ATP. The Nmnat2 transcript is expressed predominately in the brain; we report here that Nmnat2 is a low abundance protein expressed in neurons. Previous studies indicate that Nmnat2 localizes to Golgi. As Nmnat2 is not predicted to contain a signal sequence, lipid-binding domain, or transmembrane domain, we investigated the nature of this interaction. These experiments reveal that Nmnat2 is palmitoylated in vitro, and this modification is required for membrane association. Surprisingly, exogenous Nmnat2 is toxic to neurons, indicating that protein levels must be tightly regulated. To analyze Nmnat2 localization in neurons (previous experiments relied on exogenous expression in HeLa cells), mouse brains were fractionated, showing that Nmnat2 is enriched in numerous membrane compartments including synaptic terminals. In HeLa cells, in addition to Golgi, Nmnat2 localizes to Rab7-containing late endosomes. These studies show that Nmnat2 is a neuronal protein peripherally attached to membranes via palmitoylation and suggest that Nmnat2 is transported to synaptic terminals via an endosomal pathway. Nicotinamide mononucleotide (NMN)2 adenylyltransferases (Nmnat) catalyze the synthesis of NAD from NMN and ATP (1-3). Humans and mice express three isoforms, each from a separate gene: Nmnat1, -2, and -3; whereas invertebrates such as Drosophila melanogaster have only one (4). Nmnat2 is unique in that its transcript is expressed predominately in the brain (5-7). In contrast, Nmnat1 and -3 transcripts are widely expressed, although they do not necessarily overlap (2). Furthermore, Nmnat2 localizes to Golgi (8), whereas Nmnat1 is nuclear (8, 9), and Nmnat3 associates with mitochondria (8, 10). This suggests that 1) Nmnat1, -2, and -3 have evolved specialized roles in vertebrate NAD metabolism, and 2) the subcellular location of NAD synthesis is cell type-specific.In addition to its role as a cofactor, NAD is also a substrate for numerous enzymes including sirtuins, poly(ADP-ribose) polymerases, and ADP-ribosyl cyclases (e.g. CD38) (11). These enzymes regulate diverse cellular processes including transcription, apoptosis, and calcium signaling (12)(13)(14). In eukaryotes, NAD is synthesized either de novo from tryptophan or recycled from nicotinic acid, nicotinamide, or nicotinamide riboside (1, 11). Because neurons require an enormous amount of energy to propagate action potentials, it is not surprising that insufficient dietary intake of NAD precursors results in severe neurological dysfunction (15,16).It is unclear what specific role Nmnat2 has in the brain that cannot be met by Nmnat1 or -3. To address this question, we have first sought to understand how Nmnat2 interacts with the Golgi, its cellular and developmental protein expression, and its localization in brain cells. We report here that Nmnat2 is a developmentally regulated, low abundance neuronal protein that localizes not only to Golgi but also to vesicles ...

Decreased SMG7 expression associates with lupus-risk variants and elevated antinuclear antibody production

“…At 1q25, rs2022013 located in the first intron of nicotinamide mononucleotide adenylyltransferase 2 ( NMNAT2 ) was associated with SLE in GWAS (p=1.1×10 −7 , OR=0.85) and in a large-scale replication study (p=1.5×10 −3 , OR=0.92) using European-derived subjects,2 3 thus suggesting NMNAT2 as a SLE risk locus. NMNAT2, mainly expressed in the brain, is a central enzyme in the nicotinamide adenine dinucleotide biosynthetic pathway that is known to delay axon degeneration 4 5. Given no apparent clues for its involvement in immune dysregulation, we fine-mapped NMNAT2 and its neighbouring genes in four major populations, confirmed NMNAT2 association with SLE in European American (EA) and Amerindian/Hispanic (HS) ancestries and identified independent association at SMG7 in EA only.…”

Rescue of Peripheral and CNS Axon Defects in Mice Lacking NMNAT2

“…Other mouse strains and animal work C57BL/6OlaHsd-Wld (Wld S ) mice were obtained from Harlan UK, FLPe transgenic mice (Rodríguez et al, 2000) were obtained from Martin Turner (Babraham Institute), and Nmnat2 gtBay mice, derived from gene trap ES cell clone RRF238 (BayGenomics), were described previously (Mayer et al, 2010).…”

“…Nmnat2 gtBAY/gtE compound heterozygotes, with further reduced NMNAT2 levels, are also outwardly healthy up to this age. Given that NMNAT2 is a low abundance protein (Mayer et al, 2010), this sets a surprisingly low threshold of NMNAT2 for normal development and animal survival, although enzyme activity will amplify its effects. The finding that conditional silencing of a single Nmnat2 allele in primary culture is sufficient to trigger distal neurite degeneration was therefore both unexpected and intriguing.…”

“…Samples were microfuged at 10,000 ϫ g to pellet cell debris, and the supernatant was mixed with 2ϫ Laemmli sample buffer. This step was included to facilitate detection of NMNAT2, which is a nonabundant protein (Mayer et al, 2010). There was no significant loss of NMNAT2 in the pellet.…”

“…We crossed Nmnat2 ϩ/gtE mice to mice heterozygous for an alternative gene-trapped Nmnat2 allele (termed Nmnat2 gtBay to distinguish it from the Nmnat2 gtE allele), which suppresses expression from the trapped allele by only ϳ50% (Mayer et al, 2010). This produced compound heterozygotes (Nmnat2 gtBay/gtE ) expressing Nmnat2 mRNA and NMNAT2 protein at ϳ25% of wild-type levels (Fig.…”