Inhibition of intracellular sorting and processing of lysosomal cathepsins H and L at reduced temperature in primary cultures of rat hepatocytes

Abstract

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 °C in cultured rat hepatocytes, because the transport of the procathepsins was expected to be blocked at the trans-Golgi compartment at 20 °C. We show here that the newly synthesized procathepsins are accumulated intracellularly and the processing for lysosomal cathepsins is completely arrested at 20 °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 °C after an incubation for 4 h at 20 °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 °C. When the NH 4Cl was present in the cells after the temperature shift to 37 °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 NH 4Cl 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 °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.