No abstract

We originally identified senescence marker protein 30 (SMP30) as a distinctive protein whose expression decreases in an androgenindependent manner with aging. Here, we report its sequence homology found in two kinds of bacterial gluconolactonases (GNLs) by using the BLAST search. Then, through a biochemical study, we identify SMP30 as the lactone-hydrolyzing enzyme GNL of animal species. SMP30 purified from the rat liver had lactonase activity toward various aldonolactones, such as D-and L-glucono-␦-lactone, D-and L-gulono-␥-lactone, and D-and L-galactono-␥-lactone, with a requirement for Zn 2؉ or Mn 2؉ as a cofactor. Furthermore, in SMP30 knockout mice, no GNL activity was detectable in the liver. Thus, we conclude that SMP30 is a unique GNL in the liver. The lactonase reaction with L-gulono-␥-lactone is the penultimate step in L-ascorbic acid (AA) biosynthesis, and the essential role of SMP30 in this synthetic process was verified here by a nutritional study using SMP30 knockout mice. These knockout mice (n ‫؍‬ 6), fed a vitamin C-deficient diet, did not thrive; i.e., they displayed symptoms of scurvy such as bone fracture and rachitic rosary and then died by 135 days after the start of receiving the deficient diet. The AA levels in their livers and kidneys at the time of death were <1.6% of those in WT control mice. In addition, by using the SMP30 knockout mouse, we demonstrate that the alternative pathway of AA synthesis involving D-glucurono-␥-lactone operates in vivo, although its flux is fairly small. aging ͉ osteogenic disorder ͉ vitamin C S enescence marker protein 30 (SMP30) is a 34-kDa protein whose tissue levels in the liver, kidney, and lung decrease with aging (1, 2). To examine the physiological function of SMP30, we established SMP30 knockout mice (3) and found that they were viable and fertile, although they were lower in body weight and shorter in life span than WT mice (4). Their livers were also far more susceptible to TNF-␣-and Fas-mediated apoptosis than those of WT mice, indicating that SMP30 may act to protect cells from apoptosis (3). The livers of SMP30 knockout mice showed abnormal accumulations of triglycerides, cholesterol, and phospholipids (4). In addition, the lungs of these knockout mice had enlarged alveolar airspaces during their first to sixth month of life (2). However, the molecular mechanism of SMP30 function has remained obscure.Recently, we reported that SMP30 acts as a hydrolase for diisopropyl phosphorofluoridate (5), a compound resembling chemical warfare nerve agents such as sarine, soman, and tabun. However, a physiological substrate for SMP30 must be present, because this compound is an artificial chemical. Our recent search for amino acid sequences resembling SMP30 was accomplished by using the BLAST program, which revealed that rat SMP30 is homologous with gluconolactonase (GNL) [EC 3.1.1.17], a lactone-hydrolyzing enzyme, of Nostoc punctiforme and Zymomonas mobilis (6). Therefore, we suspected that SMP30 is a GNL of animal species. In mammalian metabolism, GNL is...

No abstract

The inability of humans to synthesize L-ascorbic acid is known to be due to a lack of L-gulono-gamma-lactone oxidase, an enzyme that is required for the biosynthesis of this vitamin. Isolation of a cDNA for rat L-gulono-gamma-lactone oxidase allowed us to study the basic defect underlying this deficiency at the gene level and led to isolation of a human genomic clone related to L-gulono-gamma-lactone oxidase as well as three overlapping clones covering the entire coding region of the rat L-gulono-gamma-lactone oxidase cDNA. Sequence analysis study indicated that the human L-gulono-gamma-lactone oxidase gene has accumulated a large number of mutations since it stopped being active and that it now exists as a pseudogene in the human genome.

No abstract