Biomedical subjects
J Ezaki
Publications and source records attributed to J Ezaki.
Accumulation of mitochondrial ATP synthase subunit c in muscle in a patient with neuronal ceroid lipofuscinosis (late infantile form).
We report a case late infantile neuronal ceroid lipofuscinosis (NCL). Abnormal granules were found in the skeletal muscle fibers, Schwann cells, perineurial cells, endothelial cells, fibroblasts, and perivascular smooth muscle cells in the sural nerve. Electron microscopy revealed that these granules showed fingerprint profiles, curvilinear profiles or membrane-bound membranous structures. Acid phosphatase reaction was increased in these cells. Immunohistochemical studies for mitochondrial ATP synthase subunit c showed a strong reaction in these cells, suggesting abnormal accumulation of subunit c. Immunohistochemistry for subunit c in muscle may be useful in the diagnosis of late infantile NCL.
Decreased lysosomal subunit c-degrading activity in fibroblasts from patients with late infantile neuronal ceroid lipofuscinosis.
We investigated in in-vitro cell-free incubation experiments which factor, lysosomal proteolytic dysfunction or structural alteration of subunit c, is responsible for the specific delay in the degradation of subunit c in patient cells with the late infantile form of neuronal ceroid lipofuscinosis. Experiments using substrates and soluble lysosomal fractions isolated separately from control and patient cells indicated that lysosomes from control cells are able to degrade mitochondrial subunit c either from control or patient cells at much faster rate than lysosomes from patient cells. Subunit c stored in patient cell lysosomes showed much more resistance to proteolytic attack than mitochondrial subunit c, suggesting that conformation of subunit c as well as lysosomal proteolytic dysfunction both participate in the specific lysosomal accumulation of subunit c in the late infantile disease.
Lysosomal proteinosis based on decreased degradation of a specific protein, mitochondrial ATP synthase subunit C: Batten disease.
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Specific delay in the degradation of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid lipofuscinosis is derived from cellular proteolytic dysfunction rather than structural alteration of subunit c.
Previously we indicated that a specific delay in subunit c degradation causes the accumulation of mitochondrial ATP synthase subunit c in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (NCL). To explore the mechanism of lysosomal storage of subunit c in patient cells, we investigated the mechanism of the lysosomal accumulation of subunit c both in cultured normal fibroblasts and in in vitro cell-free incubation experiments. Addition of pepstatin to normal fibroblasts causes the marked lysosomal accumulation of subunit c and less accumulation of Mn(2+)-superoxide dismutase (SOD). In contrast, E-64-d stimulates greater lysosomal storage of Mn(2+)-SOD than of subunit c. Incubation of mitochondrial-lysosomal fractions from control and diseased cells at acidic pH leads to a much more rapid degradation of subunit c in control cells than in diseased cells, whereas other mitochondrial proteins, including Mn(2+)-SOD, beta subunit of ATP synthase, and subunit i.v. of cytochrome oxidase, are degraded at similar rates in both control and patient cells. The proteolysis of subunit c in normal cell extracts is inhibited markedly by pepstatin and weakly by E-64-c, as in the cultured cell experiments. However, there are no differences in the lysosomal protease levels, including the levels of the pepstatin-sensitive aspartic protease cathepsin D between control and patient cells. The stable subunit c in mitochondrial-lysosomal fractions from patient cells is degraded on incubation with mitochondrial-lysosomal fractions from control cells. Exchange experiments using radiolabeled substrates and nonlabeled proteolytic sources from control and patient cells showed that proteolytic dysfunction, rather than structural alterations such as the posttranslational modification of subunit c, is responsible for the specific delay in the degradation of subunit c in the late infantile form of NCL.
Abnormal degradative pathway of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid-lipofuscinosis (Batten disease).
Subunit c is normally present as an inner mitochondrial membrane component of the F0 sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid-lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. The rate of degradation of subunit c as measured by pulse-chase and immunoprecipitation showed a marked delay of degradation in patients' fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes through an autophagic process. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes.
New insight into lysosomal protein storage disease: delayed catabolism of ATP synthase subunit c in Batten disease.
Subunit c is normally present as an inner mitochondrial membrane component of the Fo sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. Mechanism for specific accumulation of subunit c in lysosomes is not known. The rate of degradation of subunit c as measured by pulsechase and immunoprecipitation showed a marked delay of degradation in patients fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes, through an autophagic process. There were no large differences of various lysosomal protease activities between control and patient cells. In patient cells sucrose loading caused a marked shift of lysosomal density, but did not a shift of subunit c containing storage body. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes.
Specific delay of degradation of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid lipofuscinosis (Batten disease).
Subunit c is normally present as an inner mitochondrial membrane component of the F0 section of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. To explore the mechanism of storage of subunit c, the rates of degradation and synthesis of subunit c were measured in fibroblast cell types from controls and patients with the late infantile form of NCL. The radiolabel from subunit c decreased with time in control cells, whereas no apparent loss of radioactivity of subunit c was found in patients' cells. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. A combination of pulse-chase and subcellular fractionation analysis showed that a delay of intramitochondrial loss from prelabeled subunit c was seen in all diseased cells tested. Lysosomal appearance of labeled subunit c could be detected after chase for more than 1 week and its radioactivities were variable among diseased cell types. The biosynthetic rate of subunit c was almost the same in both control and patient cells. Northern blotting analyses showed that mRNAs for P1 and P2 genes had no significant difference in lengths and amounts between control and patient cells. Results suggest a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes. This is the first direct evidence to show a delay of subunit c degradation in the cells from the late infantile form of NCL.
Defect of proteolysis of mitochondrial ATP synthase subunit C in neuronal ceroid lipofuscinosis.
Mechanism of lysosomal storage of mitochondrial ATP synthase subunit c in late infantile form of NCL was studied. Morphological and biochemical examinations with patient fibroblasts showed that subunit c, not other mitochondrial proteins was specifically localized in lysosomes. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed a specific delay of degradation of subunit c in patient cells with late infantile form of NCL and lysosomal transfer of radioactive mitochondrial subunit c after chase for 1-2 weeks, suggesting that subunit c is transfered to lysosomes through an autophagic process and accumulated as a consequence of abnormal catabolism in lysosomes.
[Batten disease (Neuronal ceroid lipofuscinoses)--accumulation of ATP synthase subunit c caused by the delay of lysosomal degradation].
The neuronal ceroid lipofuscinoses (NCLs) represent a group of recessively inherited neurogenerative diseases of infants, children, and young adults that leads to blindness, seizures, dementia, and premature death. These diseases are pathologically characterized by a massive lysosomal storage of autofluorescent lipopigments in neurons and a wide variety of extraneuronal cells. Linkage studies have shown localization of the infantile disease to chromosome region 1p32 the juvenile onset disease to chromosome 16p12.1-p11.2 and a variant form of late infantile form to chromosome 13q21.1-q32. Recently, protein sequencing and immunochemical studies have identified subunit c of the mitochondrial ATP synthase as a major component of the storage material in the late infantile and juvenile types of NCL, and SAPs in infantile type of NCL. Immunolocalization studies demonstrated a dot-like staining of subunit c in the cells with NCL and the staining pattern of subunit c was similar to that of a lysosomal membrane marker, 1gp120. Pulse-chase experiments revealed that a specific failure occurs in the degradation of subunit c in lysosomes whereas its transport into mitochondria and subsequent sequestration into lysosomes are apparently normal.
Molecular basis of lysosomal accumulation of subunit c of mitochondrial ATP synthase in neuronal ceroid-lipofuscinosis.
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Purification and characterization of flavine-adenine dinucleotide phosphohydrolase from rat liver lysosomal membranes.
An enzyme hydrolyzing flavine-adenine dinucleotide (FAD) to flavine mononucleotide (FMN) and adenosine monophosphate (AMP) was purified about 460-fold over the isolated lysosomal membranes with 9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and the absence of SDS. Purification procedures included: preparation of crude lysosomal membranes, solubilization with Triton X-100, WGA-Sepharose, Con A-Sepharose, hydroxylapatite chromatography, gel filtration with Superdex 200, DEAE ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme, estimated by gel filtration with Superdex 200, was approximately 560 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular weight of 140,000. The pH optimum for FAD hydrolysis was 8.5 with an apparent Km of 0.1 mM and the isoelectric point was pH 7.3. The activity was inhibited by o-phenanthroline, EDTA, DTT, and NEM and was slightly stimulated by Zn ion, but was not affected by Ca or Mg ions. The purified FADase contained N-linked complex type oligosaccharide chains lacking neuraminic acids. The NH2 terminal 21 amino acid residues of the purified FADase were Ser-Pro-Cys-Val-Cys-Asp-Pro-Val-Val-Val-Cys-Lys-Val-Val-Pro-Cys-Thr-Leu- Ala-Leu .
Specific storage of subunit c of mitochondrial ATP synthase in lysosomes of neuronal ceroid lipofuscinosis (Batten's disease).
Immunochemical studies demonstrated the specific accumulation of subunit c of mitochondrial ATP synthase in the brain homogenates of late infantile and juvenile forms of Batten's disease. It is not stored in the infantile form. Storage of subunit alpha of mitochondrial ATP synthase and cytochrome c oxidase subunit IV, an inner membrane protein of mitochondria was not detected in the brains. There was also no difference in the levels of cathepsin B between the two forms of Batten's disease and controls. In cultured skin fibroblasts subunit c accumulates in the late infantile form, whereas it does not in other lysosomal storage diseases. Crude mitochondrial lysosomal preparations of control fibroblasts were separated into high-density fractions rich in a lysosomal marker and low-density fractions rich in a mitochondrial marker on Percoll density gradients. Subunit c was mostly recovered in low-density mitochondrial fractions, but in cells from the late infantile disease a part of subunit c was recovered in the high-density lysosomal fractions. Immunolocalization studies demonstrated a dot-like staining of storage materials for subunit c in the cells from late infantile patients and the staining pattern of subunit c is similar to that of a lysosomal membrane marker, lgp120. Immunostaining failed to detect subunit c in control cells. These results indicate a specific accumulation of subunit c in lysosomes, and suggest that the two forms of Batten's disease are caused by a specific failure in the degradation of subunit c.
Purification and characterization of an 85 kDa sialoglycoprotein in rat liver lysosomal membranes.
Sialoglycoprotein with a molecular mass of 85 kDa (LGP85) was purified from rat liver lysosomal membranes with a 0.9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included: preparation of lysosomal membranes, elimination of LGP107 and LGP96 with immunoaffinity columns, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. LGP85 contains about 22.8% carbohydrate and the carbohydrate moiety is composed of mannose, galactose, fucose, glucosamine, galactosamine, and neuraminic acid, in a molar ratio of 40:20:2:23:3:13. Susceptibility to neuraminidase and immunoreactivity of the protein in intact tritosomes were examined to study the topology of the protein in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the protein were not observed in intact tritosomes until the tritosomes had been disrupted by osmotic shock. These observations suggest that both oligosaccharide chains and the main protein portion of the protein are located on the interior surface of the tritosomal membranes. Subcellular localization of LGP85 was determined using enzyme immunoassay. The lysosomes seem to be the major location. LGP85 in the lysosomes was divided into the membrane bound form (90%) and the soluble form (10%). Immunoelectron microscopy clearly confirmed that the localization of LGP85 is mainly confined to lysosomes.
Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.
A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.
Isolation and sequencing of a cDNA clone encoding 85kDa sialoglycoprotein in rat liver lysosomal membranes.
We used the oligonucleotide probe corresponding to the internal amino acid sequence of a lysosomal membrane glycoprotein with a molecular weight of 85 K (LGP85) and isolated and characterized cDNA clones containing the entire coding region. The isolated cDNA comprised 2065 nucleotides. The predicted amino acid sequences of LGP85 consisted of 478 amino acid residues (Mr.54,090) and the protein has 11 potential N-glycosylation sites. Since the NH2 terminal sequence determined from purified LGP85 was identical to the NH2 terminal sequence deduced from the nucleotide sequence of the cDNA, except for the lack of initiator methionine which is likely to be cleaved off posttranslationally, it is likely that LGP85 has an uncleavable signal peptide at the NH2 terminus. Hydropathy plots show that LGP85 possesses two strong hydrophobic regions at the NH2 terminus (residues 4-26) and near the COOH terminus (residues 433-457), respectively. Either one or both of the domains might be used for membrane anchoring. A comparison of the sequences of the other lysosomal membrane glycoproteins with that of LGP85 revealed no homology. Glycine-tyrosine residues (so-called GY motif) which are thought an important signal for delivery of lysosomal membrane glycoproteins to lysosomes were not contained in the cytoplasmic tail of LGP85 (residues 458-478). LGP85 appears to be an unique lysosomal membrane glycoprotein that does not require tyrosine residues for targeting to lysosomes. Tyrosine residue may not be an essential signal for delivering newly synthesized lysosomal membrane glycoproteins to lysosomes.
[Clinical significance of urinary transferrin in the diagnosis of diabetes mellitus].
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