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...

Antibodies against acetylcholine receptors (AChRs) cause pathogenicity in myasthenia gravis (MG) patients through complement pathway-mediated destruction of postsynaptic membranes at neuromuscular junctions (NMJs). However, antibodies against muscle-specific kinase (MuSK), which constitute a major subclass of antibodies found in MG patients, do not activate the complement pathway. To investigate the pathophysiology of MuSK-MG and establish an experimental autoimmune MG (EAMG) model, we injected MuSK protein into mice deficient in complement component five (C5). MuSK-injected mice simultaneously developed severe muscle weakness, accompanied by an electromyographic pattern such as is typically observed in MG patients. In addition, we observed morphological and functional defects in the NMJs of EAMG mice, demonstrating that complement activation is not necessary for the onset of MuSK-MG. Furthermore, MuSK-injected mice exhibited acetylcholinesterase (AChE) inhibitor-evoked cholinergic hypersensitivity, as is observed in MuSK-MG patients, and a decrease in both AChE and the AChE-anchoring protein collagen Q at postsynaptic membranes. These findings suggest that MuSK is indispensable for the maintenance of NMJ structure and function, and that disruption of MuSK activity by autoantibodies causes MG. This mouse model of EAMG could be used to develop appropriate medications for the treatment of MuSK-MG in humans.

Muscle-specific kinase (MuSK) is critical for the synaptic clustering of nicotinic acetylcholine receptors (AChRs) and plays multiple roles in the organization and maintenance of neuromuscular junctions (NMJs). MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering at the postsynaptic membrane. Although autoantibodies against the ectodomain of MuSK have been found in a proportion of patients with generalized myasthenia gravis (MG), it is unclear whether MuSK autoantibodies are the causative agent of generalized MG. In the present study, rabbits immunized with MuSK ectodomain protein manifested MG-like muscle weakness with a reduction of AChR clustering at the NMJs. The autoantibodies activated MuSK and blocked AChR clustering induced by agrin or by mediators that do not activate MuSK. Thus MuSK autoantibodies rigorously inhibit AChR clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of MG.

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Senescence marker protein-30 (SMP30) was originally identified as a novel protein of which expression decreases in an androgen-independent manner with aging in the rat liver and functions to protect cells from apoptosis. By reverse transcription-polymerase chain reaction analysis, SMP30 mRNA transcripts were found in the mouse lung, liver, kidney, testis and cerebrum. We examined SMP30 expression in the mouse liver, kidney and lung during aging and a distinct temporal profile of SMP30 expression was found in each tissue; the SMP30 mRNA level peaked at 1-3 months of age and decreased thereafter in the liver (the highest at 1 month of age followed by a rapid decline and consistently low thereafter in the kidney), and peaked at 12 months of age in the lung. To investigate the physiological role of SMP30 in the lung, immunohistochemical studies of wild-type (SMP30Y/+) mice and histopathological examinations of SMP30 knockout (SMP30Y/-) mice were performed. Immunoreactivity against anti-SMP30 antibody was mainly detected in bronchial epithelial cells and strongly detected at 6-12 months of age. Morphometric analysis was performed to measure the mean linear intercept and destructive index, and found peripheral airspace enlargement without alveolar destruction in SMP30Y/- mice at 1, 3 and 6 months of age compared with the SMP30Y/+ mice. Our results strongly suggest that SMP30Y/- mice could be a novel model for a senile lung and further examinations of SMP30Y/- mice may offer clues to elucidate the mechanisms of the development of pulmonary diseases in the elderly.