Procathepsin L Self‐Association as a Mechanism for Selective Secretion

Abstract

The lysosomal cysteine pro-protease procathepsin L was enriched in dense vesicles detectable when microsomes prepared from wild-type or transformed mouse fibroblasts were resolved on sucrose gradients. These dense vesicles did not comigrate with proteins characteristic of the endoplasmic reticulum, Golgi, endosomes or lysosomes. When gradient fraction vesicles were lysed at acidic pH in the presence of excess mannose 6-phosphate to prevent binding to mannose phosphate receptors, the majority of the procathepsin L was associated with the membrane, not the soluble, fraction. Immunogold labeling of procathepsin L in thin sections of cells or gradient fractions, using antibodies directed against the propeptide to avoid detection of the mature enzyme in dense lysosomes, revealed that the proenzyme was concentrated in dense cores localized in small vesicles near the plasma membrane and in multivesicular bodies. Consistent with the density of the gradient fraction and the electron density of the cores, yeast two-hybrid assays indicated the proenzyme could bind itself but could not interact with the aspartic proprotease procathepsin D. The data suggest that in mouse fibroblasts procathepsin L may self-associate into aggregates, initiating the formation of dense vesicles that could mediate the selective secretion of procathepsin L independent of mannose phosphate receptors.Key words: Lysosome, Procathepsin L, Secretion, Selfassociation, Transformation Received 4 January 2000, revised and accepted for publication 19 June 2000All newly synthesized soluble lysosomal enzymes are secreted to some extent, although the amount of the enzyme released varies with the enzyme, the cell type, and the metabolic state of the cells. In wild-type fibroblasts, only a small percent of the newly synthesized enzyme is released to the cell culture medium. For proteases, the major biosynthetic form secreted is an inactive precursor, a proenzyme, while most glycosidases are synthesized and secreted as active enzymes. The proenzyme molecules in the culture medium bear mannose 6-phosphate (M6P) recognition markers yet apparently fail to bind mannose phosphate receptors (MPRs) in the trans Golgi network and so are secreted constitutively. Typical of molecules in the secretory pathway, they are generally slightly larger than cellular proforms due to the acquisition of complex carbohydrate on mannose sidechains not modified with phosphate.Under certain physiological conditions, synthesis of a particular lysosomal enzyme increases. An extensive literature documents the increased expression of particular lysosomal proteases in specific tumors. The cysteine protease procathepsin L was initially named the 'major excreted protein' (MEP) because its synthesis and secretion is markedly increased in NIH3T3 fibroblasts upon viral transformation (1). In general, cancers express higher levels of cathepsin L than do normal tissues (2). Similarly, the cysteine protease cathepsin B has been associated with a large number of human and murine malignancies (3), whil...

The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation

“…We found a LAMP1-negative vesicular compartment, defined by expression of the cation-independent mannose-6-phosphate receptor (CI-M6PR) and by dramatic expansion of the proprotein convertase cathepsin L in the cell apex ( Figure 1C). In cells with secretory granule stores, LAMP1-negative, and cathepsin Land CI-M6PR-positive vesicles define a specific late endosomal and/or multivesicular body compartment involved in secretory granule maturation (37)(38)(39)(40)(41). This vesicular compartment was not detectable with these markers in neck cells ( Figure 1C, insets) and parietal cells ( Figure 1C, white arrows).…”

Direct recruitment of H+-ATPase from lysosomes for phagosomal acidification

“…The mutant macrophages accumulated mature forms of the lysosomal proteinases cathepsin-B, -D and -L (Dragonetti et al, 2000;Kominami et al, 1988;Yeyeodu et al, 2000), implying that the steady-state levels of active lysosomal enzymes were not severely changed by the loss of the a3 subunit (Fig. 8).…”

The Novel Drosophila Lysosomal Enzyme Receptor Protein Mediates Lysosomal Sorting in Mammalian Cells and Binds Mammalian and Drosophila GGA Adaptors

“…Human MPR300 was detected either by using a mouse monoclonal antibody (2C2) recognizing a lumenal epitope (28) or by using a polyclonal antiserum raised against the recombinant cytoplasmic tail of the MPR300 kindly provided by Thomas Braulke, Universitaets-Klinikum-Eppendorf, Hamburg, Germany. Cathepsin L was detected using a rabbit antiserum kindly provided by Ann Erickson (29), cathepsin D was detected using a rabbit antiserum (30), HA-tagged LERP was detected by monoclonal antibody 3F10 from rat or monoclonal antibody 12C5 from mouse (Roche Diagnostics), and maltose-binding protein fusion proteins were detected with monoclonal antibody MBP-17 from mouse (Sigma).…”