We have raised specific polyclonal immunoglobulin G (IgG) against a major lysosomal membrane sialoglycoprotein (LGP107) taken from rat liver and have prepared a conjugate of its Fab’ fragment with ...horseradish peroxidase (HRP-anti LGP107 Fab) as a probe for the subcellular antigen. Electron immunocytochemistry in primary cultured rat hepatocytes showed that LGP107 resided primarily within lysosomes and was associated with luminal amorphous materials as well as limiting membranes. In addition, LGP107 was shown to be substantially distributed throughout the endocytic vacuolar system. The glycoprotein was found clustered in coated pits at the cell surface and localized along the surrounding membranes in endocytic vesicles. When cultured cells were exposed to HRP-anti LGP107 Fab‘, the antibody which was bound to its antigen within the coated pits was internalized via a system of endocytic vesicles and transported to lysosomes. During 20 min of incubation at 37’C, the HRP tracer appeared at an early stage in small vesicles and moved progressively to larger vesicles, including multivesicular bodies. After 1 h, the tracer could be clearly seen in lysosomes heterogenous in shape and size. The existence of LGP107 in endocytic compartments and the uptake of anti LGP107 antibody by hepatocytes were not blocked by prior treatment of the cells with cycloheximide and excess amounts of anti LGP107 IgG. These data suggest that LGP107 circulates between the cell surface and lysosomes through the endocytic membrane traffic in hepatocytes.
Our recent studies with pulse-chase kinetic analysis in primary cultures of rat hepatocytes suggest that newly synthesized lysosomal cathepsins H and L are initially synthesized as larger proform ...enzymes, and then the precursor molecules are subsequently converted to the mature enzymes by limited proteolysis during the intracellular sorting process. This proteolytic maturation of procathepsins appears to proceed within an acidic environment, and these processing events are closely connected with the activation of enzymes. To further characterize the intracellular processing site for lysosomal cathepsins H and L, the pulse-chase kinetic study was carried out at 20 degrees C in cultured rat hepatocytes, because the transport of the procathepsins was expected to be blocked at the trans-Golgi compartment at 20 degrees C. We show here that the newly synthesized procathepsins are accumulated intracellularly and the processing for lysosomal cathepsins is completely arrested at 20 degrees C along the sorting pathway. The procathepsins thus accumulated in the cell are presumed to be transported to the Golgi complex, since the oligosaccharide moieties of these polypeptides appear to be phosphorylated. When the cells were shifted to 37 degrees C after an incubation for 4 h at 20 degrees C, a gradual increase of the mature forms was found. However, the processing kinetics generating the mature enzymes were slow compared to those in control cells at 37 degrees C. When the NH4Cl was present in the cells after the temperature shift to 37 degrees C, the intracellular processing of procathepsins was considerably retarded and the release of intracellular procathepsins into the extracellular medium was observed. These results indicate that NH4Cl might exert the inhibitory effect on the mannose 6-phosphate receptor-mediated intracellular targeting mechanism for the lysosomal cathepsins. Hence, the intracellular location of procathepsins accumulated at 20 degrees C is considered to be in proximity to the trans-Golgi compartment. Taken together, the present observations suggest that the propeptide-processing step for procathepsins, which is a critical step for generating the active enzymes, proceeds within the prelysosomal compartment or the lysosomes after the enzymes leave the trans-Golgi compartment.
Fabry disease is a lysosomal storage disorder caused by an α-galactosidase A (α-Gal A) deficiency and resulting in the accumulation of glycosphingolipids, predominantly globotriaosylceramide (Gb3). A ...transgenic mouse expressing the human α-Gal A R301Q mutant in an α-Gal A-knockout background (TgM/KO) should be useful for studying active-site-specific chaperone (ASSC) therapy for Fabry disease. However, the Gb3 content in the heart tissue of this mouse was too low to detect an ASSC-induced effect. To increase the Gb3 levels in mouse organs, we created transgenic mice (TgG3S) expressing human α1,4-galactosyltransferase (Gb3 synthase). High levels of Gb3 were observed in all major organs of the TgG3S mouse. A TgG3S (+/−)M(+/−)/KO mouse was prepared by cross-breeding the TgG3S and TgM/KO mice and the Gb3 content in the heart of the TgG3S(+/−)M(+/−)/KO mouse was 1.4 µg/mg protein, higher than in the TgM(+/−)/KO (<0.1 µg/mg protein). Treatment with an ASSC, 1-deoxygalactonojirimycin, caused a marked induction of α-Gal A activity and a concomitant reduction of the Gb3 content in the TgG3S(+/−) M(+/−)/KO mouse organs. These data indicated that the TgG3S(+/−) M(+/−)/KO mouse was suitable for studying ASSC therapy for Fabry disease, and that the TgG3S mouse would be useful for studying the effect of high Gb3 levels in mouse organs.
Precursor forms of lysosomal cathepsins B, H and L in the hepatic endoplasmic lumen were identified as having a molecular weight of 39-, 41-, and 39-kDa, respectively, by immunoblotting analysis. The ...proenzymes were then concentrated by applying the microsomal contents to a concanavalin A-Sepharose chromatography. The concanavalin A-adsorbed fractions containing the proenzymes showed no appreciable activities of cathepsins B, H and L. When those fractions were incubated at pH 3.0, the enzymatic activities markedly increased. Immunoblotting analysis showed that after 36 hr incubation the proenzymes disappeared and the mature enzymes increased. Thus the proenzymes were processed to the mature enzymes under acidic conditions of pH 3.0. The marked increased of enzymatic activities and the conversion of the proenzymes to the mature forms were completely blocked with pepstatin which is a potent inhibitor of aspartic proteinases. The results strongly suggested that a processing proteinase for procathepsins B, H, and L might be cathepsin D, a major lysosomal aspartic proteinase. Indeed, lysosomal cathepsin D could convert the immunoaffinitypurified microsomal procathepsin B to the mature enzyme in vitro. Therefore, procathepsins B, H, and L seem to be firstly synthesized as the enzymatically inactive forms in endoplasmic reticulum and may successively be converted into the active forms by cathepsin D in lysosomal compartments.
Fabry disease is a lysosomal storage disorder caused by an α-galactosidase A (α-Gal A) deficiency and resulting in the accumulation of glycosphingolipids, predominantly globotriaosylceramide (Gb3). A ...transgenic mouse expressing the human α-Gal A R301Q mutant in an α-Gal A-knockout background (TgM/KO) should be useful for studying active-site-specific chaperone (ASSC) therapy for Fabry disease. However, the Gb3 content in the heart tissue of this mouse was too low to detect an ASSC-induced effect. To increase the Gb3 levels in mouse organs, we created transgenic mice (TgG3S) expressing human α1,4-galactosyltransferase (Gb3 synthase). High levels of Gb3 were observed in all major organs of the TgG3S mouse. A TgG3S (+/-)M(+/-)/KO mouse was prepared by cross-breeding the TgG3S and TgM/KO mice and the Gb3 content in the heart of the TgG3S(+/-)M(+/-)/KO mouse was 1.4 µg/mg protein, higher than in the TgM(+/-)/KO (<0.1 µg/mg protein). Treatment with an ASSC, 1-deoxygalactonojirimycin, caused a marked induction of α-Gal A activity and a concomitant reduction of the Gb3 content in the TgG3S(+/-) M(+/-)/KO mouse organs. These data indicated that the TgG3S(+/-) M(+/-)/KO mouse was suitable for studying ASSC therapy for Fabry disease, and that the TgG3S mouse would be useful for studying the effect of high Gb3 levels in mouse organs.
Human herpes virus 6 (HHV-6) is known to cause primary encephalitis in the frontal lobes/cerebral hemisphere or reactivated encephalitis in the hippocampus, but the pathogenesis remains unclear. ...HHV-6B has also been detected in hippocampal samples in patients with mesial temporal lobe epilepsy. A 1 year and 3 months old female, who had been clinically diagnosed with exanthema subitum and febrile convulsion, was found dead on the third day after onset. Macroscopic findings showed massive brain edema. Microscopic examination revealed gemistocytic astrocytes and ballooned oligodendrocytes in the frontal white matter, along with neuronal cell death with microglial infiltration in the frontal cortex. Polymerase chain reaction detected HHV-6B in the cerebrospinal fluid and necropsy brain samples. The hippocampus showed a 4-5-fold increase in virus copy number of HHV-6B compared to samples from other brain sites. Immunostaining indicated that HHV-6B had infected vascular endothelial cells, neurons and oligodendrocytes but not astrocytes or microglia. Hippocampal neurons were infected with highly concentrated HHV-6B, but the hippocampus had neither neuronal loss nor reactive glial response. Silent and abundant HHV-6B infection in the hippocampus might be associated with latent infection, reactivation and some hippocampus-oriented disorders, including mesial temporal lobe epilepsy.
Assessment was made of the thermal stability of long-chain alkyl phosphate by thermal analysis (TG/DTA/DSC). Several monoalkyl phosphates (Cn-MAP) differing in chain length (C12, C14, C16) showed ...essentially the same thermal stability. The decomposition temperature of didodecyl phosphate (C12-DAP) was 257.8°C 40-50°C higher than that of monododecyl phosphate (C12-MAP). C12-MAP·2 Na salt was thermally more stable than C12-MAP, but C12-MAP·Na salt was less stable than this. Gas chromatographic analysis of the decomposition gas indicated dodecene to be mainly formed from C12-MAP, whereas 1-dodecanol from C12-MAP·Na salt and C12-MAP·2 Na salt. 31P-NMR analysis of C12-MAP decomposition mixtures revealed that the disproportionation of C12-MAP to phosphoric acid (PA) and C12-DAP and condensed phosphate formation fromC12-MAP were competetive with the elimination of olefin from C12-MAP. The elimination of olefins from monoalkyl pyrophosphate Py- (1.0) and dialkyl pyrophosphate Py- (1, 1) besides from C12-MAP thus must be taken into consideration for assessement of the thermostability of long-chain alkyl phoshates. The formation of PA+ Py- (0, 0) from MAP+DAP +Py- (1, 0) +Py- (1, 1) appears to be a zero order reaction with an activation energy of 26.2kcal/mol.
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