Hepatocyte growth factor stimulates root growth during the development of mouse molar teeth.

BACKGROUND AND OBJECTIVE: It is well known that tooth root formation is initiated by the development of Hertwig's epithelial root sheath (HERS). However, relatively little is known about the regulatory mechanisms involved in root development. As hepatocyte growth factor (HGF) is one of the mediators of epithelial-mesenchymal interactions in rodent tooth, the objective of this study was to examine the effects of HGF on the root development of mouse molars. MATERIAL AND METHODS: The HERS of mouse molars and HERS01a, a cell line originated from HERS, were used in this study. For detection of HGF receptors in vivo and in vitro, we used immunochemical procedures. Root development was assessed by implanting molar tooth germs along with HGF-soaked beads into kidney capsules, by counting cell numbers in HERS01a cell cultures and by performing a 5'-bromo-2'-deoxyuridine (BrdU) assay in an organ-culture system. RESULTS: HGF receptors were expressed in the enamel epithelium of molar germs as well as in HERS cells. HGF stimulated root development in the transplanted tooth germs, the proliferation of HERS01a cells in culture and HERS elongation in the organ-culture system. Examination using BrdU revealed that cell proliferation in HERS was increased by treatment with HGF, especially that in the outer layer of HERS. This effect was down-regulated when antibody against HGF receptor was present in the culture medium. CONCLUSION: Our results raise the possibility that HGF signaling controls root formation via the development of HERS. This study is the first to show that HGF is one of the stimulators of root development.

Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport.

The fate of free cholesterol released after endocytosis of low-density lipoproteins remains obscure. Here we report that late endosomes have a pivotal role in intracellular cholesterol transport. We find that in the genetic disease Niemann-Pick type C (NPC), and in drug-treated cells that mimic NPC, cholesterol accumulates in late endosomes and sorting of the lysosomal enzyme receptor is impaired. Our results show that the characteristic network of lysobisphosphatidic acid-rich membranes contained within multivesicular late endosomes regulates cholesterol transport, presumably by acting as a collection and distribution device. The results also suggest that similar endosomal defects accompany the anti-phospholipid syndrome and NPC.

Protective protein gene mutations in galactosialidosis.

Four different protective protein cDNA mutations, 146A-->G (Q49R), 193T-->C (W65R), 268-269TC-->CT (S90L), and 1184A-->G (Y395C), were identified in six Japanese galactosialidosis patients with various phenotypic manifestations, and another mutation, 746T-->A (Y249N), in a patient of French-German origin with an atypical clinical course. Y395C was a common mutation in four Japanese patients in infancy and childhood; two juvenile patients were compound heterozygotes of Y395C and another common mutation, SpDEx7, and the other two infants were compound heterozygotes of Y395C and mutant alleles other than SpDEx7. We confirmed these mutations in genomic DNA by direct-sequence analysis or restriction-site analysis. The mutant cDNA clones, transiently expressed in a transformed galactosialidosis cell line, did not restore the secondarily deficient beta-galactosidase or alpha-neuraminidase activity except for the Y249N mutation that expressed some carboxypeptidase activity and restored the two lysosomal enzyme activities. Pulse-chase analysis detected a small amount of the mature form, as well as the precursor, in the cells transfected with the Y249N cDNA. Only precursor proteins were detected, mature proteins not appearing for the other mutant cDNAs.

Fabry disease: six gene rearrangements and an exonic point mutation in the alpha-galactosidase gene.

Fabry disease, an X-linked recessive disorder of glycosphingolipid catabolism, results from the deficient activity of the lysosomal hydrolase, alpha-galactosidase. Southern hybridization analysis of the alpha-galactosidase gene in affected hemizygous males from 130 unrelated families with Fabry disease revealed six with different gene rearrangements and one with an exonic point mutation resulting in the obliteration of an Msp I restriction site. Five partial gene deletions were detected ranging in size from 0.4 to greater than 5.5 kb. Four of these deletions had breakpoints in intron 2, a region in the gene containing multiple Alu repeat sequences. A sixth genomic rearrangement was identified in which a region of about 8 kb, containing exons 2 through 6, was duplicated by a homologous, but unequal crossover event. The Msp I site obliteration, which mapped to exon 7, was detected in an affected hemizygote who had residual enzyme activity. Genomic amplification by the polymerase chain reaction and sequencing revealed that the obliteration resulted from a C to T transition at nucleotide 1066 in the coding sequence. This point mutation, the first identified in Fabry disease, resulted in an arginine356 to tryptophan356 substitution which altered the enzyme's kinetic and stability properties. The detection of these abnormalities provided for the precise identification of Fabry heterozygotes, thereby permitting molecular pedigree analysis in these families which revealed paternity exclusions and the first documented new mutations in this disease.

The functional role of glutamine-280 and threonine-282 in human alpha-galactosidase.

Our previous study on chimeric mutants of alpha-galactosidase suggested that two peptide regions encoded by exons 1-2 and 6 of the enzyme gene contribute to substrate recognition (Ishii, S. et al. (1994) Biochim. Biophys. Acta 1204, 265-270). In this study, we constructed five single amino acid substitutions for functional analysis of the amino acid residues around glutamine-279, the mutation site detected in an atypical Fabry disease patient. Two mutants, Q280S (Gln280-->Ser; CAA-->TCA) and T282A (Thr282-->Ala; ACT-->GCT), showed increased Km and decreased thermostability as compared with normal enzyme. Circular dichroism spectrum was not modified. An additional chimeric mutation in the exon 1-2 region by substitution with the homologous sequence of alpha-N-acetylgalactosaminidase cDNA restored catalytic activity and thermostability in both mutants. These data indicated the functional significance of glutamine-280 and threonine-282 for expressing the activity and stability of alpha-galactosidase molecule, and also the presence of an intramolecular interaction between the two peptide regions encoded by exons 1-2 and 6.

The NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum: cloning, sequencing, and expression of the enzyme gene(1).

The NAD-dependent glutamate dehydrogenase (GluDH) gene from the hyperthermophilic archaeon Pyrobaculum islandicum was cloned and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1266 bp encoding a protein of 421 amino acids with a molecular weight of 46,905. In the alignment of the amino acid sequence with those of mesophilic Clostridium symbiosum NAD-dependent GluDH and hyperthermophilic NADP-dependent enzymes from Thermococcus profundus and Pyrococcus furiosus, substitutions in the residues involved in dinucleotide binding were observed. On the other hand, the residues involved in glutamate binding were well conserved. This is the first description of the primary structure of NAD-dependent GluDH in hyperthermophilic archaea.

Human alpha-galactosidase gene expression: significance of two peptide regions encoded by exons 1-2 and 6.

Two proteins with alpha-galactosidase activity, alpha-galactosidase A (alpha-GalA) and alpha-galactosidase B (alpha-GalB, or alpha-N-acetylgalactosaminidase; alpha-NAGA) have a high homology of amino-acid sequence. Point mutations of the alpha-GalA gene have been reported only in the exons 1, 2 or 6. In this study, the exon 1-2 and/or 6 sequences of alpha-GalA cDNA were partly substituted by the corresponding regions of alpha-GalB cDNA, and three chimeric proteins were prepared by the baculovirus expression system: CMB12 with substitution at the exon 1-2 region, CMB6 at the exon 6 region, and CMB126 at both regions. They all preserved alpha-GalA antigenicity. Their kinetic properties toward 4-methylumbelliferyl alpha-galactopyranoside were compared with those of alpha-GalA. The catalytic activity was slightly low in CMB12, decreased to 1/10 in CMB6, and restored to a significant degree in CMB126. Km was more than 4-fold higher for CMB6 and CMB126 than for alpha-GalA. The pH optimum was 4.0 for both CMB12 and alpha-GalA, 4.8 for CMB6, and 4.6 for CMB126 and alpha-GalB. The catalytic activity was inhibited most by galactosamine in CMB6, and less in alpha-GalB, CMB126, alpha-GalA and CMB12 in decreasing order. The 50% inhibition concentrations of melibiose (Gal alpha 1-6Glc) and methyl alpha-galactopyranoside were 2.5- to 3-fold higher for CMB126 than for alpha-GalA. These results indicate that the low affinity of CMB126 to the substrate was caused by a reduced affinity to terminal alpha-linked galactose. We conclude that (1) the two regions encoded by exons 1-2 and 6 contribute to the alpha-galactosidic cleavage, and (2) an increase in Km of CMB6 or CMB126, with chimeric substitutions at the exon 6 region, was caused by a loss of affinity toward terminal alpha-linked galactose.

Characterization of two alpha-galactosidase mutants (Q279E and R301Q) found in an atypical variant of Fabry disease.

The mutant products Q279E ((279)Gln to Glu) and R301Q ((301)Arg to Gln) of the X-chromosomal inherited alpha-galactosidase (EC 3.2.1. 22) gene, found in unrelated male patients with variant Fabry disease (late-onset cardiac form) were characterized. In contrast to patients with classic Fabry disease, who have no detectable alpha-galactosidase activity, atypical variants have residual enzyme activity. First, the properties of insect cell-derived recombinant enzymes were studied. The K(m) and V(max) values of Q279E, R301Q, and wild-type alpha-galactosidase toward an artificial substrate, 4-methylumbelliferyl-alpha-D-galactopyranoside, were almost the same. In order to mimic intralysosomal conditions, the degradation of the natural substrate, globotriaosylceramide, by the alpha-galactosidases was analyzed in a detergent-free-liposomal system, in the presence of sphingolipid activator protein B (SAP-B, saposin B). Kinetic analysis revealed that there was no difference in the degradative activity between the mutants and wild-type alpha-galactosidase activity toward the natural substrate. Then, immunotitration studies were carried out to determine the amounts of the mutant gene products naturally occurring in cells. Cultured lymphoblasts, L-57 (Q279E) and L-148 (R301Q), from patients with variant Fabry disease, and L-20 (wild-type) from a normal subject were used. The 50% precipitation doses were 7% (L-57) and 10% (L-148) of that for normal lymphoblast L-20, respectively. The residual alpha-galactosidase activity was 3 and 5% of the normal level in L-57 and L-148, respectively. The quantities of immuno cross-reacting materials roughly correlated with the residual alpha-galactosidase activities in lymphoblast cells from the patients. Compared to normal control cells, fibroblast cells from a patient with variant Fabry disease, Q279E mutation, secreted only small amounts of alpha-galactosidase activity even in the presence of 10 mM NH(4)Cl. It is concluded that Q279E and R301Q substitutions do not significantly affect the enzymatic activity, but the mutant protein levels are decreased presumably in the ER of the cells.

Immunohistochemical characterization of transgenic mice highly expressing human lysosomal alpha-galactosidase.

Human lysosomal alpha-galactosidase predominantly hydrolyzes ceramide trihexoside. A transgenic mouse line, C57BL/6CrSIc-TgN(GLA) 1951 Rin, highly expressing human alpha-galactosidase, has been established and investigated biochemically and immunohistochemically in order to clarify the distribution of the expressed enzyme proteins and to evaluate it as a donor model of organ transplantation therapy for Fabry disease caused by a genetic defect of alpha-galactosidase. In these transgenic mice, about five copies of the transgene were integrated, and alpha-galactosidase activity was expressed in liver, kidney, heart, spleen, small intestine, submaxillary gland, skeletal muscle, cerebrum, cerebellum, bone marrow cells and serum. The enzyme activity was about 22 to 11,080-fold higher than that in non-transgenic mice. In liver, heart and kidney tissues, which are important organs for transplantation studies, sufficient amounts of alpha-galactosidase mRNAs were transcribed, and the expressed enzymes, with molecular weights of 54-60 kDa, are abundant in the liver (enzyme activity: 53,965 nmol h-1 mg-1 protein) and heart (39,906 nmol h-1 mg-1 protein), followed by in the kidney tissue (9177 nmol h-1 mg-1 protein), respectively. An immunohistochemical microscopic study clearly demonstrated the distribution of the expressed enzyme proteins in kidney and liver tissues. Highly expressed alpha-galactosidase was detected in glomerular cells, tubular cells and hepatocytes. These transgenic mice will be useful as a donor model for experimental organ transplantation, and also it will enable recurrent biopsies and long-term observation. The organ transplantation data on mice will provide us with important information.

Cloning and sequence analysis of a full length cDNA encoding human mitochondrial 3-oxoacyl-CoA thiolase.

The cDNA sequence of human mitochondrial 3-oxoacyl-CoA thiolase was determined. The nucleotide sequence contains an open reading frame of 1191 base pairs and encodes an amino acid sequence of 397 residues which exhibits 86.6% homology with that of the rat enzyme. Northern blot analysis gave a single mRNA species of 1.6 kb in the human liver, fibroblasts and intercostal muscle.

Late onset variants in Fabry disease: Results in high risk population screenings in Argentina.

BACKGROUND: Screening for Fabry disease (FD) in high risk populations yields a significant number of individuals with novel, ultra rare genetic variants in the GLA gene, largely without classic manifestations of FD. These variants often have significant residual α-galactosidase A activity. The establishment of the pathogenic character of previously unknown or rare variants is challenging but necessary to guide therapeutic decisions. OBJECTIVES: To present 2 cases of non-classical presentations of FD with renal involvement as well as to discuss the importance of high risk population screenings for FD. RESULTS: Our patients with non-classical variants were diagnosed through FD screenings in dialysis units. However, organ damage was not limited to kidneys, since LVH, vertebrobasilar dolichoectasia and cornea verticillata were also present. Lyso-Gb3 concentrations in plasma were in the pathologic range, compatible with late onset FD. Structural studies and in silico analysis of p.(Cys174Gly) and p.(Arg363His), employing different tools, suggest that enzyme destabilization and possibly aggregation could play a role in organ damage. CONCLUSIONS: Screening programs for FD in high risk populations are important as FD is a treatable multisystemic disease which is frequently overlooked in patients who present without classical manifestations.

Retrovirus-mediated transfer of human alpha-galactosidase A gene to human CD34+ hematopoietic progenitor cells.

Fabry disease, caused by a deficiency of lysosomal enzyme alpha-galactosidase A (alpha-gal A), is one of the inherited disorders potentially treatable by gene transfer to hematopoietic stem cells. In this study, a high-titer amphotropic retroviral producer cell line, MFG-alpha-gal A, was established. CD34+ cells from normal umbilical cord blood were transduced by centrifugal enhancement. The alpha-gal A activity in transduced cells increased 3.6-fold above the activity in nontransduced cells. Transduction efficiency measured by PCR for the integrated alpha-gal A cDNA in CFU-GM colonies was in the range of 42-88% (average, 63%). The expression of functional enzyme in TFI erythroleukemia was sustained for as long as cells remained in culture (84 days) and for 28 days in LTC-IC cultures of CD34+ cells. The ability of the transduced CD34+ cells to secrete the enzyme and to correct enzyme-deficient Fabry fibroblasts was assessed by cocultivation of these cells. The enzyme was secreted into the medium from transduced CD34+ cells and taken up by Fabry fibroblasts through mannose 6-phosphate receptors. These findings suggest that genetically corrected hematopoietic stem/progenitor cells can be an enzymatic source for neighboring enzyme-deficient cells, and can potentially be useful for gene therapy of Fabry disease.

Generation and characterization of transgenic mice expressing a human mutant alpha-galactosidase with an R301Q substitution causing a variant form of Fabry disease.

Transgenic mice expressing a human mutant alpha-galactosidase with an R301Q substitution, which was found in a patient with a variant form of Fabry disease, were established. The mice transcribed a sufficient amount of alpha-galactosidase mRNA, but the steady-state levels of the enzyme protein were decreased in liver, kidney and heart, only residual activity being detected in these tissues. The mice will be useful for the clarification of the defective regulation of the structurally altered enzyme protein expressed by the mutant gene at the organ or individual level as well as for the evaluation of drugs that stabilize and/or activate the mutant alpha-galactosidase.

Only sphingolipid activator protein B (SAP-B or saposin B) stimulates the degradation of globotriaosylceramide by recombinant human lysosomal alpha-galactosidase in a detergent-free liposomal system.

The degradation of globotriaosylceramide (GbO-se3Cer) by insect-cell derived recombinant human alpha-galactosidase (EC 3.2.1.22) was carried out in a detergent-free liposomal system in order to mimic intralysosomal conditions. GbOse3Cer incorporated into unilamellar liposomes was used as the substrate, and naturally occurring sphingolipid activator proteins, rather than detergents, were used to stimulate the enzyme reaction. The degradation of GbOse3Cer was dependent on the presence of both alpha-galactosidase and sphingolipid activator protein B (SAP-B or saposin B). It proceeded optimally at pH 4.6, and was enhanced by increasing amounts of both alpha-galactosidase (0.24-24 mU/50 microliters assay) and SAP-B (0-5 micrograms/50 microliters assay). The enzyme reaction was not affected by SAP-A, SAP-C, or SAP-D. Therefore, our results indicate that only SAP-B is essential for the degradation of GbOse3Cer by alpha-galactosidase.

GM2 gangliosidosis AB variant: clinical and biochemical studies of a Japanese patient.

OBJECTIVE: To determine the clinical features and biochemical basis of the first Japanese patient with the GM2 gangliosidosis AB variant. METHODS: The clinical manifestations and laboratory findings in the patient were investigated. Cultured fibroblasts from the patient were analyzed by means of immunofluorescence staining with an anti-GM2 ganglioside monoclonal antibody and thin-layer chromatography and immunostaining. GM1 ganglioside catabolism in cultured cells was analyzed by pulse labeling, and the amount of GM2 activator in cells was determined by Western blot analysis. Gene analysis was performed according to standard protocols. RESULTS: The patient showed progressive neurologic manifestations of quite early onset. Muscular weakness and hypotonia became evident by 1 month of age, and the patient then developed a startle reaction, severe psychomotor retardation, and myoclonic seizures. Immunocytochemical analysis clearly revealed the accumulation of GM2 ganglioside in cultured fibroblasts from the patient, and thin-layer chromatography confirmed it. Western blot and metabolic studies showed a complete deficiency of GM2 activator. Gene analysis did not reveal any mutations in the protein coding region of the GM2 activator gene. CONCLUSION: The clinical features and biochemical basis of this Japanese patient with GM2 gangliosidosis AB variant were determined. Immunocytochemical analysis using cultured fibroblasts as samples is available for the diagnosis of this disease.

Immunofluorescence analysis of trihexosylceramide accumulated in the hearts of variant hemizygotes and heterozygotes with Fabry disease.

An immunofluorescence method was applied to detect trihexosylceramide accumulated in the cardiac tissues from a variant hemizygote and a heterozygous female with Fabry disease, the incidence of which had been suspected to be high.

Detection of donor lymphocytes in the cerebrospinal fluid of a patient with metachromatic leukodystrophy following bone marrow transplantation.

An unrelated donor bone marrow transplant (UD-BMT) was carried out on a 10-year-old patient with metachromatic leukodystrophy (MLD). We collected cerebrospinal fluid (CSF) on day +168 and cultured it with recombinant IL-2 and PHA-P to examine the origin of cells in the CSF. Analysis on variable number of tandem repeat (VNTR) of lymphocytes in the CSF amplified by PCR revealed that lymphocytes in the CSF were of donor origin. These data support that BMT at an early stage may prevent deterioration in MLD. Although the patient developed grade III acute GVHD with rash and diarrhea, we successfully treated acute GVHD using rabbit anti-human thymocyte immunoglobulin (ATG). UD-BMT may be an alternative treatment for patients with MLD in the absence of an HLA matched family donor.

Beta-galactosidase-neuraminidase deficiency: restoration of beta-galactosidase activity by protease inhibitors.

Beta-Galactosidase was partially restored by protease inhibitors, leupeptin, chymostatin and E-64 in cultured fibroblasts from three patients with beta-galactosidase-neuraminidase deficiency. Pepstatin did not activate this enzyme. Neuraminidase was not affected by any of these compounds in the culture medium. It was concluded that the activating effect was produced by a specific inhibition of thiol proteases.

Biochemical characterization, cloning, and sequencing of ADP-dependent (AMP-forming) glucokinase from two hyperthermophilic archaea, Pyrococcus furiosus and Thermococcus litoralis.

The ADP-dependent (AMP-forming) glucokinases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis catalyze the phosphorylation of glucose using ADP as the essential phosphoryl group donor. Both enzymes were purified to homogeneity and characterized with regard to each other. The enzymes had similar enzymological properties as to substrate specificity, coenzyme specificity, optimum pH, and thermostability. However, a difference was observed in the subunit composition; while the T. litoralis enzyme is a monomer with a molecular mass of 52 kDa, the P. furiosus enzyme has a molecular mass of about 100 kDa and consists of two subunits with identical molecular masses of 47 kDa. The genes encoding these enzymes were cloned and sequenced. The gene for the P. furiosus enzyme contains an open reading frame for 455 amino acids with a molecular weight of 51,265, and that for the T. litoralis enzyme contains an open reading frame for 467 amino acids with a molecular weight of 53,621. About 59% similarity in amino acid sequence was observed between these two enzymes, whereas they did not show similarity with any ATP-dependent kinases that have been reported so far. In addition, two phosphate binding domains, and adenosine and glucose binding motifs commonly conserved in the eukaryotic hexokinase family were not observed.

Lysosomal enzyme replacement using alpha 2-macroglobulin as a transport vehicle.

Improvement of the delivery of exogenous enzymes is essential to achieve effective enzyme replacement therapy in lysosomal storage diseases. To test whether alpha 2-macroglobulin, an endogenous plasma protein, could serve as a transport vehicle of therapeutic agents to cells, alpha 2-macroglobulin and acid alpha-glucosidase or alpha-galactosidase A were coupled using two heterobifunctional cross-linking reagents. The alpha-glucosidase-alpha 2-macroglobulin conjugate was internalized and transported into lysosomes of acid alpha-glucosidase-deficient fibroblasts. The enzyme activity was stable after being taken up by the cells. Uptake of the conjugate resulted in the degradation of glycogen accumulated in lysosomes. The alpha-galactosidase A-alpha 2-macroglobulin conjugate was also internalized into the lysosomes of alpha-galactosidase A-deficient fibroblasts. Internalized alpha-galactosidase A-conjugate degraded globotriaosylceramide accumulated in lysosomes. The endocytosis of both conjugate was inhibited by alpha 2-macroglobulin-trypsin complex, indicating that the conjugates were endocytosed by an alpha 2-macroglobulin receptor system. These results showed the usefulness of alpha 2-macroglobulin as a transport vehicle of lysosomal enzymes for effective enzyme replacement.