The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5′ end. It has a 2,278‐nucleotide sequence including a 2,004‐nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG → TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6‐kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3′‐untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.

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Fabry disease is an X-linked recessive inborn metabolic disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (EC 3.2. throughout the 7 exons of the α-galactosidase A gene (GLA). Mutation hotspots for Fabry disease do not exist. We examined 62 Fabry patients in Japan and found 24 GLA mutations, including 11 novel ones. A potential treatment reported for Fabry disease is active site specific chaperone (ASSC) therapy using 1-deoxygalactonojirimycin (DGJ), an inhibitor of α-galactosidase A, at subinhibitory concentrations. We transfected COS-7 cells with the 24 mutant GLAs and analyzed the α-galactosidase A activities. We then treated the transfected COS-7 cells with DGJ and analyzed its effect on the mutant enzyme activities. The activity of 11 missense mutants increased significantly with DGJ. Although ASSC therapy is useful only for misfolding mutants and therefore not applicable to all cases, it may be useful for treating many Japanese patients with Fabry disease. © 2007 Wiley-Liss, Inc.KEY WORDS: α-galactosidase A; GLA; lysosomal storage disease; 1-deoxygalactonojirimycin; chaperone therapy INTRODUCTIONFabry disease (FD; MIM# 301500) is a pan-ethnic, X-linked, lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-galactosidase A (EC 3.2.1.22) (Brady et al., 1967). Human α-galactosidase A is a homodimeric glycoprotein with three N-linked oligosaccharide chains on each subunit. Enzyme deficiency results in a systemic lysosomal accumulation of glycolipids, primarily globotriosylceramide (Gb3), in the vascular endothelium and other tissues. In the classical form of the disease, the patient develops angiokeratoma, hypohidrosis, and episodic pain crises in the extremities during childhood or adolescence. With advancing age, the morbidity of renal failure, cardiac disease, and early onset of stroke increases. The severity of the clinical manifestations depends on the amount of residual α-galactosidase A activity. Hemizygous male patients with no or very low α-galactosidase A activity usually have severe clinical symptoms and die as young adults. Heterozygous female Fabry patients exhibit a wide range of severity, from a virtually symptom-free course (Marguery et al., 1993) to one comparable to that of their male counterparts (Whybra et al., 2001), although they usually have no symptoms or very mild manifestations.We examined Fabry patients in Japan and sequenced the patients' α-galactosidase A gene (GLA) (MIM# 300644). To analyze the mutant α-galactosidase A activities, we transfected COS-7 cells with the mutant GLAs. In addition, since 1-deoxygalactonojirimycin (DGJ) stabilizes the α-galactosidase A conformation and improves its stability (Asano et al., 2000;Ishii et al., 2000;Yam et al., 2006), we added DGJ to the incubation medium and examined its effect on the mutant α-galactosidase A activities. MATERIALS AND METHODS PatientsWe examined 62 Fabry patients from 31 unrelated families. Diagnosis was based on reduced or absent α-galactosidase A activity and typical signs an...

Bone morphogenetic proteins (BMPs) are highly conserved signaling molecules that are part of the transforming growth factor (TGF)-beta superfamily, and function in the patterning and morphogenesis of many organs including development of the dentition. The functions of the BMPs are controlled by certain classes of molecules that are recognized as BMP antagonists that inhibit BMP binding to their cognate receptors. In this study we tested the hypothesis that USAG-1 (uterine sensitization-associated gene-1) suppresses deciduous incisors by inhibition of BMP-7 function. We learned that USAG-1 and BMP-7 were expressed within odontogenic epithelium as well as mesenchyme during the late bud and early cap stages of tooth development. USAG-1 is a BMP antagonist, and also modulates Wnt signaling. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Using explant culture and subsequent subrenal capsule transplantation of E15 USAG-1 mutant maxillary incisor tooth primordia supplemented with BMP-7 demonstrated in USAG-1+/− as well as USAG-1−/− rescue and supernumerary tooth development. Based upon these results, we conclude that USAG-1 functions as an antagonist of BMP-7 in this model system. These results further suggest that the phenotypes of USAG-1 and BMP-7 mutant mice reported provide opportunities for regenerative medicine and dentistry.