pepstatin and mannose-6-phosphate

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

We have studied the role of N-linked oligosaccharides and proteolytic processing on the targeting of cathepsin D to the lysosomes in the human hepatoma cell line HepG2. In the presence of tunicamycin cathepsin D was synthesized as an unglycosylated 43-kDa proenzyme which was proteolytically processed via a 39-kDa intermediate to a 28-kDa mature form. Only a small portion was secreted into the culture medium. During intracellular transport the 43-kDa procathepsin D transiently became membrane-associated independently of binding to the mannose 6-phosphate receptor. Subcellular fractionation showed that unglycosylated cathepsin D was efficiently targeted to the lysosomes via intermediate compartments similar to the enzyme in control cells. The results show that in HepG2 cells processing and transport of cathepsin D to the lysosomes is independent of mannose 6-phosphate residues. Inhibition of the proteolytic processing of 53-kDa procathepsin D by protease inhibitors caused this form to accumulate intracellularly. Subcellular fractionation revealed that the procathepsin D was transported to lysosomes, thereby losing its membrane association. Procathepsin D taken up by the mannose 6-phosphate receptor also transiently became membrane-associated, probably in the same compartment. We conclude that the mannose 6-phosphate-independent membrane-association is a transient and compartment-specific event in the transport of procathepsin D.

Topics: Amidohydrolases; Carcinoma, Hepatocellular; Cathepsin D; Cell Fractionation; Cell Line; Centrifugation, Density Gradient; Glycosylation; Humans; Liver Neoplasms; Lysosomes; Mannosephosphates; Pepstatins; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Protein Processing, Post-Translational; Tunicamycin

An estrogen-induced 52-kDa glycoprotein secreted by human breast cancer cells and able to autostimulate the growth of MCF7 cells has been purified, using monoclonal antibodies, and characterized. The protein contains mannose 6-phosphate signals on its N-linked high-mannose chains, suggesting that it is a lysosomal enzyme. Both the secreted 52-kDa protein and its processed cellular forms (52-, 48- and 34-kDa) were identified as carboxyl proteinases having an optimal activity at pH 3.5 and being specifically inhibited by pepstatin. This protease is characterized by its inducibility by estrogens and its high concentration in proliferative benign and malignant mammary tissue, when detected by immunohistochemistry. The estrogen-induced secretion of this protease may help to understand how estrogens stimulate mammary tumor growth and/or invasion.

Topics: Antibodies, Monoclonal; Aspartic Acid Endopeptidases; Breast Neoplasms; Cathepsin D; Cell Line; Electrophoresis, Paper; Endopeptidases; Estrogens; Female; Humans; Hydrogen-Ion Concentration; Lysosomes; Mannosephosphates; Molecular Weight; Pepstatins; Protease Inhibitors