pepstatin and Neuronal-Ceroid-Lipofuscinoses

A recent study has shown mutations in CLN2 gene, that encodes a novel lysosomal pepstatin-insensitive proteinase (LPIP), in the pathophysiology of late-infantile neuronal ceroid lipofuscinosis (LINCL). We have measured the LPIP activities in brains from various forms of human neuronal ceroid lipofuscinoses (NCL), canine ceroid lipofuscinosis and other neurodegenerative disorders with a highly sensitive assay using a tetrapeptide Gly-Phe-Phe-Leu-amino-trifluoromethyl coumarin (AFC) as substrate. Brain LPIP has a pH optimum of 3.5 and an apparent km of 100 microM for the crude enzyme. The enzyme activity is totally absent in LINCL patients. Pronounced increase in the LPIP activity was seen in patients suffering from infantile (INCL), juvenile (JNCL) and adult (ANCL) forms of neuronal ceroid lipofuscinoses. LPIP activity was also found to be increased about two-fold in Alzheimer's disease when compared with normal or age-matched controls, while in globoidal-cell leukodystrophy (Krabbe's disease) it was similar to the normal controls. Although mannose-6-phosphorylated LPIP is increased 13-fold in brains of patients with JNCL, this form of LPIP did not have any enzyme activity. The mechanism by which LPIP activities are increased in a wide range of neurodegenerative diseases is unknown, although neuronal loss, followed by gliosis are common characteristics of these diseases.

Topics: Adult; Aged; Aged, 80 and over; Aging; Alzheimer Disease; Aminopeptidases; Animals; Brain; Child; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Dog Diseases; Dogs; Endopeptidases; Female; Humans; Leukodystrophy, Globoid Cell; Male; Mannose; Middle Aged; Neurodegenerative Diseases; Neuronal Ceroid-Lipofuscinoses; Pepstatins; Peptide Hydrolases; Reference Values; Serine Proteases; Tripeptidyl-Peptidase 1

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.

Topics: Amino Acid Sequence; Aminopeptidases; Chromosome Mapping; Chromosomes, Human, Pair 11; Codon; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Female; Glycosylation; Humans; Isoelectric Point; Lysosomes; Male; Mannosephosphates; Molecular Sequence Data; Molecular Weight; Mutation; Neuronal Ceroid-Lipofuscinoses; Pepstatins; Peptide Hydrolases; Polymerase Chain Reaction; Serine Proteases; Tripeptidyl-Peptidase 1

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.

Topics: Cathepsin B; Cell Fractionation; Cell-Free System; Cells, Cultured; Electron Transport Complex IV; Fibroblasts; Humans; Infant; Kinetics; Lysosomes; Macromolecular Substances; Mitochondria; Neuronal Ceroid-Lipofuscinoses; Pepstatins; Protease Inhibitors; Proton-Translocating ATPases; Reference Values; Skin