RESUMO
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.
Assuntos
Mitocôndrias/enzimologia , Lipofuscinoses Ceroides Neuronais/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Endopeptidases/metabolismo , Expressão Gênica , Humanos , Lactente , Membranas Intracelulares/enzimologia , Cinética , Lisossomos/enzimologia , Substâncias Macromoleculares , Lipofuscinoses Ceroides Neuronais/genética , ATPases Translocadoras de Prótons/biossíntese , Valores de ReferênciaRESUMO
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.
Assuntos
ATPase de Ca(2+) e Mg(2+)/isolamento & purificação , Membranas Intracelulares/enzimologia , Fígado/enzimologia , Lisossomos/enzimologia , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , ATPase de Ca(2+) e Mg(2+)/química , Cálcio/farmacologia , Cromatografia em Gel , Concanavalina A , Durapatita , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Hidrólise , Hidroxiapatitas , Membranas Intracelulares/química , Ponto Isoelétrico , Cinética , Fígado/química , Lisossomos/química , Magnésio/farmacologia , Masculino , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Desnaturação Proteica , Ratos , Ratos Wistar , Sefarose/análogos & derivados , Especificidade por SubstratoRESUMO
The specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of NCL (LINCL) is caused by a defect in the CLN2 gene product, tripeptidyl peptidase I (TPP-I). The data here show that TPP-I is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c. The inclusion of a specific inhibitor of TPP-I, Ala-Ala-Phe-chloromethylketone (AAF-CMK), in the culture medium of normal fibroblasts induced the lysosomal accumulation of subunit c. In an in vitro incubation experiment the addition of AAF-CMK to mitochondrial-lysosomal fractions from normal cells inhibited the proteolysis of subunit c, but not the b-subunit of ATP synthase. The use of two antibodies that recognize the aminoterminal and the middle portion of subunit c revealed that the subunit underwent aminoterminal proteolysis, when TPP-I, purified from rat spleen, was added to the mitochondrial fractions. The addition of both purified TPP-I and the soluble lysosomal fractions, which contain various proteinases, to the mitochondrial fractions resulted in rapid degradation of the entire molecule of subunit c, whereas the degradation of subunit c was markedly delayed through the specific inhibition of TPP-I in lysosomal extracts by AAF-CMK. The stable subunit c in the mitochondrial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I. The presence of TPP-I led to the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding to the amino terminal sequence of subunit c.
Assuntos
Grupo dos Citocromos c/metabolismo , Endopeptidases/metabolismo , Lisossomos/enzimologia , Lipofuscinoses Ceroides Neuronais/enzimologia , Peptídeo Hidrolases/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Aminopeptidases , Animais , Dipeptidil Peptidases e Tripeptidil Peptidases , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/enzimologia , Expressão Gênica , Humanos , Lactente , Membranas Intracelulares/enzimologia , Cinética , Mitocôndrias/enzimologia , Lipofuscinoses Ceroides Neuronais/genética , Peptídeos/metabolismo , ATPases Translocadoras de Prótons/isolamento & purificação , Ratos , Serina Proteases , Inibidores de Serina Proteinase/farmacologia , Baço/enzimologia , Tripeptidil-Peptidase 1RESUMO
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 .
Assuntos
Fígado/enzimologia , Lisossomos/enzimologia , Pirofosfatases/isolamento & purificação , Sequência de Aminoácidos , Animais , Cromatografia , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Masculino , Metais/farmacologia , Dados de Sequência Molecular , Peso Molecular , Pirofosfatases/química , Pirofosfatases/metabolismo , Ratos , Ratos Wistar , Coloração e Rotulagem , Especificidade por SubstratoRESUMO
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.
Assuntos
Fígado/metabolismo , Lisossomos/metabolismo , Sialoglicoproteínas/isolamento & purificação , Animais , Carboidratos/análise , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Fígado/ultraestrutura , Masculino , Proteínas de Membrana/isolamento & purificação , Peso Molecular , Ratos , Ratos Endogâmicos , Sialoglicoproteínas/química , Sialoglicoproteínas/metabolismoRESUMO
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.
Assuntos
Mitocôndrias/enzimologia , Lipofuscinoses Ceroides Neuronais/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Western Blotting , Encéfalo/enzimologia , Eletroforese em Gel de Poliacrilamida , Fibroblastos/enzimologia , Humanos , Lisossomos/enzimologiaRESUMO
A 24-week study was conducted to evaluate the effects of the dihydropyridine calcium channel blocker nilvadipine on urinary albumin excretion in eight microalbuminuric hypertensive patients with non-insulin-dependent (type II) diabetes mellitus. Blood pressure and urinary albumin excretion measurements before the administration of nilvadipine (8 mg) were compared with those after 4, 8, 12 and 24 weeks of treatment. No significant changes were observed in the mean values of haemoglobin A1C. Systolic blood pressure was significantly reduced from 174 +/- 23 mmHg before treatment to 144 +/- 13 mmHg after 24 weeks of treatment (P < 0.02). Diastolic blood pressure was significantly reduced from 93 +/- 11 mmHg at baseline to 79 +/- 8 mmHg after 24 weeks of treatment (P < 0.05). Urinary albumin excretion was significantly reduced from 65.4 +/- 37.4 mg/g creatinine at baseline to 51.6 +/- 41.1 mg/g creatinine (P < 0.05) after 4 weeks, and to 39.1 +/- 26.9 mg/g creatinine (P < 0.02) after 24 weeks of treatment. These data suggest that in hypertensive microalbuminuric patients with non-insulin-dependent diabetes mellitus, treatment of hypertension with the calcium blocker nilvadipine may slow the progression of diabetic nephropathy.
Assuntos
Albuminúria/urina , Bloqueadores dos Canais de Cálcio/farmacologia , Diabetes Mellitus Tipo 2/fisiopatologia , Nefropatias Diabéticas/prevenção & controle , Hipertensão/fisiopatologia , Nifedipino/análogos & derivados , Idoso , Albuminúria/complicações , Albuminúria/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/urina , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/urina , Feminino , Hemoglobinas Glicadas/análise , Hemoglobinas Glicadas/efeitos dos fármacos , Humanos , Hipertensão/complicações , Hipertensão/urina , Masculino , Pessoa de Meia-Idade , Nifedipino/farmacologia , Nifedipino/uso terapêutico , Fatores de TempoRESUMO
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.
Assuntos
Lisossomos/metabolismo , Lipofuscinoses Ceroides Neuronais , ATPases Translocadoras de Prótons/metabolismo , Adulto , Idade de Início , Animais , Criança , Humanos , Lactente , Peroxidação de Lipídeos , Lipofuscina/metabolismo , Lipofuscinoses Ceroides Neuronais/etiologia , Lipofuscinoses Ceroides Neuronais/metabolismoAssuntos
Extração de Catarata/métodos , Adulto , Idoso , Humanos , Lentes Intraoculares , Pessoa de Meia-Idade , Terapia por UltrassomRESUMO
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.
Assuntos
Mitocôndrias/enzimologia , Lipofuscinoses Ceroides Neuronais/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Pele/enzimologia , Catepsina B/metabolismo , Fracionamento Celular , Sistema Livre de Células , Células Cultivadas , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/enzimologia , Humanos , Lactente , Cinética , Lisossomos/enzimologia , Substâncias Macromoleculares , Mitocôndrias/efeitos dos fármacos , Pepstatinas/farmacologia , Inibidores de Proteases/farmacologia , ATPases Translocadoras de Prótons/isolamento & purificação , Valores de ReferênciaRESUMO
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.
Assuntos
Complexos Multienzimáticos/metabolismo , Lipofuscinoses Ceroides Neuronais/enzimologia , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Complexos de ATP Sintetase , Adolescente , Encéfalo/enzimologia , Sistema Livre de Células , Criança , Pré-Escolar , Fibroblastos , Humanos , Lactente , Lisossomos/enzimologia , Mitocôndrias/enzimologia , Valores de ReferênciaRESUMO
Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH(2) is readily hydrolyzed by Cln2p/TPP-I (K(cat)/K(m) = 7.8 s(-1) mM(-1)). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH(2)Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity.
Assuntos
Endopeptidases/deficiência , Endopeptidases/metabolismo , Lipofuscinoses Ceroides Neuronais/enzimologia , Peptídeo Hidrolases/deficiência , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Aminopeptidases , Animais , Linhagem Celular , Dipeptidil Peptidases e Tripeptidil Peptidases , Endopeptidases/genética , Estabilidade Enzimática , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Lactente , Cinética , Lipofuscinoses Ceroides Neuronais/genética , Oligopeptídeos/química , Peptídeo Hidrolases/genética , Ratos , Serina Proteases , Especificidade por Substrato , Tripeptidil-Peptidase 1RESUMO
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.
Assuntos
Lisossomos/metabolismo , Mitocôndrias/metabolismo , Lipofuscinoses Ceroides Neuronais , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Humanos , Peroxidação de Lipídeos , Lipofuscina/metabolismo , Mitocôndrias/patologia , Lipofuscinoses Ceroides Neuronais/enzimologia , Lipofuscinoses Ceroides Neuronais/patologiaRESUMO
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.
Assuntos
Doenças por Armazenamento dos Lisossomos/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Fragmentos de Peptídeos/metabolismo , ATPases Translocadoras de Prótons/química , Humanos , Lipofuscinoses Ceroides Neuronais/enzimologiaRESUMO
The specific accumulation of the hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolytic dysfunction. The defective gene in LINCL (CLN2 gene) has been identified recently. To elucidate the mechanism of lysosomal storage of subunit c, antibodies against the human CLN2 gene product (Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46-kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from four patients with LINCL. RT-PCR analysis indicated the presence of mRNA for CLN2 in cells from the four different patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable translation products in these patient cells. Pulse-chase analysis showed that Cln2p was synthesized as a 67-kDa precursor and processed to a 46-kDa mature protein (t(1/2) = 1 h). Subcellular fractionation analysis indicated that Cln2p is localized with cathepsin B in the high-density lysosomal fractions. Confocal immunomicroscopic analysis also revealed that Cln2p is colocalized with a lysosomal soluble marker, cathepsin D. The immunodepletion of Cln2p from normal fibroblast extracts caused a loss in the degradative capacity of subunit c, but not the beta subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.
Assuntos
Lisossomos/enzimologia , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Aminopeptidases , Linhagem Celular , DNA Complementar , Dipeptidil Peptidases e Tripeptidil Peptidases , Endopeptidases , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica , Triagem de Portadores Genéticos , Humanos , Cinética , Substâncias Macromoleculares , Mitocôndrias/enzimologia , Lipofuscinoses Ceroides Neuronais/genética , ATPases Translocadoras de Prótons/química , RNA Mensageiro/genética , Valores de Referência , Serina Proteases , Pele/enzimologia , Transcrição Gênica , Tripeptidil-Peptidase 1RESUMO
Neuronal ceroid lipofuscinoses (NCL) form a distinct group of storage diseases where the normal development of the central nervous system is interrupted and neurons of the neocortex begin to degenerate. Mutations in genes encoding three lysosomal enzymes are the causes for three early-onset forms of NCLs: palmitoyl-protein thioesterase 1 (PPT1) is deficient in human infantile NCL, tripeptidyl peptidase 1 (TTP1) in late-infantile NCL, and cathepsin D in congenital ovine NCL. We wanted to compare the developmental expression profiles of these enzymes in rat brain. In conclusion, the PPT1 expression pattern differed from the two other lysosomal enzymes implicated in NCL diseases, thus suggesting a distinctive role for PPT1 in brain development.
Assuntos
Lipofuscinoses Ceroides Neuronais/enzimologia , Aminopeptidases , Animais , Sequência de Bases , Catepsina D/genética , Catepsina D/metabolismo , Sistema Nervoso Central/enzimologia , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/patologia , Primers do DNA/genética , Dipeptidil Peptidases e Tripeptidil Peptidases , Endopeptidases/genética , Endopeptidases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lisossomos/enzimologia , Mutação , Degeneração Neural , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Serina Proteases , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Tripeptidil-Peptidase 1RESUMO
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.