The processing of a cathepsin L precursor in vitro

Subcultured rat fibroblasts secreted a cathepsin L precursor when maintained for 24 h in serum-free medium containing 20 mM ammonium ions. The precursor was identified by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using polyclonal antibodies to cathepsin L. The molecular mass of the precursor was found to be approximately 39 kDa, which confirms the result originally reported by Y. Nishimura et al. (1988, Arch. Biochem. Biophys. 263, 107-116). Treatment of the precursor containing medium with cathepsin D at pH values ranging from 3.5 to 5.5 caused a limited cleavage of the precursor molecule. The resultant polypeptides are an unstable intermediate form with Mr 35,000 and a stable single chain form of cathepsin L showing a Mr about 32,500. The cathepsin D-mediated conversion was strongly accelerated by Hg2+ ions. A further proteolytic cleavage of the 32.5-kDa polypeptide has not been observed. The enzymatic activity toward Z-Phe-Arg-NHMec at pH 5.5 increased during the conversion, indicating that active cathepsin L was formed from an inactive precursor molecule.     

Endocytosis and the recycling of plasma membrane

For study of the time order of glycosylation, formation of complex oligosaccharides and proteolytic maturation as well as the site of proteolytic maturation of cathepsin D, fibroblasts were subjected to pulse-chase labeling, and cathepsin D was isolated from either total cell extracts or subcellular fractions by immune precipitation and analyzed for its molecular forms and sensitivity to endo-beta-N- acetylglucosaminidase H. After a 10-min pulse, cathepsin D was detected in its glycosylated precursor form, indicating an early, probably a cotranslational, N-glycosylation of cathepsin D. Conversion of the high- mannose oligosaccharide side chains into forms resistant to endo-beta-N- acetylglucosaminidase H started after approximately 40 min, indicating that transport of cathepsin D from the endoplasmic reticulum to the trans-Golgi apparatus requires approximately 40 min. Processing of the 53-kdalton precursor polypeptide of cathepsin D to a 47-kdalton intermediate followed about 20 min after the formation of complex oligosaccharides, and, another 30 min later, 31-kdalton mature forms of cathepsin D were detected. Processing of cathepsin D was first observed in light membranes as a partial conversion of the 53-kdalton precursor into the 47-kdalton intermediate. Both the precursor and the intermediate are transferred into the high density-class lysosomes. After 8 h, the processing to the mature 31-kdalton form of cathepsin D is mostly completed.     

Identification of a precursor form of cathepsin D in microsomal lumen: characterization of enzymatic activation and proteolytic

A precursor form of cathepsin D with 45 kDa was demonstrated in the rat liver microsomal lumen by immunoblotting analysis. The microsomal fraction containing procathepsin D which passed through a pepstatin-Sepharose resin showed no appreciable activity of cathepsin D. The in vitro incubation of this fraction at pH 3.0 resulted in a gradual increase of proteolytic activity toward hemoglobin as substrate and also, the proteolytic conversion of procathepsin D to the mature form was concomitantly observed. The proteolytic processing step was sensitive to pepstatin. These results suggest that procathepsin D is inactive in the endoplasmic reticulum and may be converted to the active forms by autoproteolytic processing mechanism at acidic pH during biosynthesis.     

Progestin and estrogen control of cathepsin D expression and processing in rat uterine luminal epithelium and stroma-myometrium.

Progestins increase the activity and rate of synthesis of cathepsin D, a lysosomal aspartyl protease, in the uterine luminal epithelium in ovariectomized rats. Western blot analysis of luminal epithelial proteins determined that the progestin, medroxyprogesterone acetate (MPA) increased the 43-kDa form of cathepsin D by 7-fold in 24 hr, whereas estradiol increased the amount of the same form by only 2-fold. To examine the precursor-product relationship between cathepsin D proteins in the luminal epithelium and stroma-myometrium after progestin or estradiol treatment, uterine proteins were prelabeled by incubation with [35S]methionine in vitro, cathepsin D was isolated by immunoprecipitation, and equal amounts of labeled cathepsin D were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After each hormonal treatment in each uterine tissue, a 48-kDa precursor was processed into a 44-kDa cathepsin D product. Endoglycosidase H digestion of [35S]methionine-labeled cathepsin D from the luminal epithelium and stroma-myometrium of medroxyprogesterone-treated rats shifted the molecular masses of the cathepsin D proteins by approximately 5.7 kDa. To examine the contribution of increased mRNA to increased rates of cathepsin D synthesis, we measured levels of cathepsin D mRNA in uterine tissues after progestin and estrogen treatment. Total RNA was isolated from the uterine luminal epithelium and from the stroma-myometrium. Northern blot analysis identified a single 2.2-kb RNA band corresponding to the size expected for cathepsin D mRNA. Medroxyprogesterone increased levels of cathepsin D mRNA in the luminal epithelium (greater than 17-fold) and in the stroma myometrium (3-fold), with maximum increases at 9 hr after treatment. Estradiol also increased cathepsin D mRNA levels in both uterine tissues, but by only 2-fold. No hormonal effects on liver cathepsin D mRNA were observed. Increases in cathepsin D synthesis and activity in uterine tissues in response to progestin and estrogen appear to depend in part upon increased levels of mRNA.     

Biosynthesis of the multiple forms of rabbit cardiac cathepsin D

Rabbit cardiac cathepsin D exists as multiple isomeric forms of Mr = 48,000 within cardiac tissue. Their mechanism of formation and their functional role in cardiac protein degradation are unknown. We have previously demonstrated that cathepsin D is initially synthesized as an Mr = 53,000 precursor that is processed by limited proteolysis within cardiac lysosomes to the Mr = 48,000 active forms of the enzyme. To determine if the multiple forms of active cathepsin D originate from a common precursor, isolated perfused Langendorff rabbit hearts were labeled in pulse (15 or 30 min) and pulse-chase (30 or 150 min) experiments with [35S]methionine. Newly synthesized cathepsin D was isolated by butanol/Triton X-100 extraction and immunoadsorption with anti-cathepsin D IgG-Sepharose, and the isomeric forms were separated by two-dimensional electrophoresis and fluorography. After 15- and 30-min pulse perfusions, 35S-labeled cathepsin D appeared as a single precursor form (Mr = 53,000, pI = 6.6). After 30-min pulse and 30-min chase, the precursor was modified to yield multiple precursor forms, all with molecular weight 53,000, but with differing pI values (6.6-6.0). After 30-min pulse and 150-min chase perfusion, multiple forms of both precursor and proteolytically processed active cathepsin D (Mr = 48,000, pI = 6.2-5.6) were detected. The 35S-labeled, proteolytically processed forms of active cathepsin D co-migrated with the major cathepsin D forms present in cardiac tissue. Subcellular fractionation and perfusions in the presence of chloroquine demonstrated that the multiple precursor forms of cathepsin D originated in a nonlysosomal intracellular compartment. Thus, the multiple forms of active cathepsin D originate from a common high molecular weight precursor, and their synthesis occurs prior to the limited proteolysis of the precursor in cardiac lysosomes.     

A large cathepsin D-derived fragment from the central part of the fibronectin subunit chains

A mild cathepsin D digest of fibronectin only contained single-chain peptides of 200, 140 and 70 kDa and double-chain fragments of about 300 and 140 kDa containing the C-terminal disulfide link. Among the single-chain fragments the 200 kDa peptide was a precursor of the 140 kDa and 70 kDa peptides. The latter was correlated to the N-terminal and the former to the central region of the fibronectin subunit chains.     

Cathepsin D precursors in clathrin-coated organelles from human fibroblasts

Coated vesicles were isolated from metabolically labeled human fibroblasts with the aid of affinity-purified antibodies against human brain clathrin and Staphylococcus aureus cells. The material adsorbed to the S. aureus cells was enriched in clathrin. When the S. aureus cells bearing the immunoadsorbed material were treated with 0.5% saponin, extracts containing the precursor form of cathepsin D were obtained. The extraction of the precursor was promoted in the presence of mannose 6-phosphate. Material adsorbed to S. aureus cells coated with control immunoglobulins was nearly free of clathrin and contained a small amount of the cathepsin D precursor (less than 20% of that adsorbed with anti-clathrin antibodies). The extraction of this cathepsin D precursor was independent of mannose 6-phosphate and was complete after a brief exposure to saponin. The amount of cathepsin D precursor in coated membranes varied between 0.4 and 2.5% of total precursor. Analysis of pulse chase-labeled fibroblasts revealed that cathepsin D was only transiently associated with coated membranes. The mean residence time of cathepsin D precursor in coated membranes was estimated to be 2 min. These observations support the view that coated membranes participate in the transfer of precursor forms of endogenous lysosomal enzymes to lysosomes.     

Association of the precursor of cathepsin D with coated membranes. Kinetics and carbohydrate processing

Specific anti-chlathrin antibodies were used to isolate clathrin-coated membranes from homogenates of metabolically labelled fibroblasts. The isolated membranes were extracted with detergents and cathepsin D was isolated from the extracts. The 53-kDa precursor of cathepsin D was transiently associated with the coated membranes with a maximum approximately 60 min after synthesis. At maximum about 4.0% of the precursor was recovered with the coated membranes and the associated precursor contained complex oligosaccharides. The proportion of complex oligosaccharides in the coated membrane-associated precursor did not differ from that in the total precursor. These data support the view that coated membranes are involved in the transport of cathepsin D between trans Golgi and a prelysosomal organelle.     

A cathepsin B-like enzyme from mackerel white muscle is a precursor of cathepsin B

A cathepsin B-like enzyme from the white muscle of common mackerel Scomber japonicus was a cysteine protease that hydrolyzed Z-Arg-Arg-MCA, the substrate for cathepsin B. In a partial purified cathepsin B-like enzyme preparation at 4 degrees C left over time, a converted enzyme that hydrolyzes Z-Arg-Arg-MCA and Z-Phe-Arg-MCA appeared in the preparation. The converted enzyme was purified from the cathepsin B-like enzyme, characterized and was identified as mackerel cathepsin B. These results suggested that the mackerel cathepsin B-like enzyme was a precursor of cathepsin B. Mackerel cathepsin B formed in the purified cathepsin B-like enzyme preparation by adding of a small amount of the purified cathepsin B to the preparation. Therefore, mackerel cathepsin B-like enzyme was converted to the mature form of cathepsin B by autoactivation. The conversion of the cathepsin B-like enzyme (molecular mass 60 kDa) to cathepsin B (molecular mass 23 kDa) was detected by immunoblotting by using human anti-(cathepsin B) antibody. The intermediate forms of 40 kDa and 38 kDa were also detected during the conversion.     

Cathepsin D is membrane-associated in macrophage endosomes

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.     

Cathepsin D targeted by acid sphingomyelinase-derived ceramide.

Ceramide has been recognized as a common intracellular second messenger for various cytokines, growth factors and other stimuli, such as CD95, chemotherapeutic drugs and stress factors. To understand the role of ceramide during apoptosis and other cellular responses, it is critically important to characterize direct targets of ceramide action. In this paper, we show that ceramide specifically binds to and activates the endosomal acidic aspartate protease cathepsin D. Direct interaction of ceramide with cathepsin D results in autocatalytic proteolysis of the 52 kDa pre-pro cathepsin D to form the enzymatically active 48/32 kDa isoforms of cathepsin D. Acid sphingomyelinase (A-SMase)-deficient cells show decreased cathepsin D activity, which could be reconstituted by transfection with A-SMase cDNA. The results of our study identify cathepsin D as the first endosomal ceramide target that colocalizes with and may mediate downstream signaling effects of A-SMase.     

Evaluation of Cathepsins D and G and EC 3.4.24.15 as Candidate β-Secretase Proteases Using Peptide and Amyloid Precursor Protein Substrates

Abstract: No single protease has emerged that possesses all the expected properties for β-secretase, including brain localization, appropriate peptide cleavage specificity, and the ability to cleave amyloid precursor protein exactly at the amino-terminus of β-amyloid peptide. We have isolated and purified a brain-derived activity that cleaves the synthetic peptide substrate SEVKMDAEF between methionine and aspartate residues, as required to generate the amino-terminus of β-amyloid peptide. Its molecular size of 55–60 kDa and inhibitory profile indicate that we have purified the metalloprotease EC 3.4.24.15. We have compared the sequence specificity of EC 3.4.24.15, cathepsin D, and cathepsin G for their ability to cleave the model peptide SEVKMDAEF or related peptides that contain substitutions reported to modulate β-amyloid peptide production. We have also tested the ability of these enzymes to form carboxy-terminal fragments from full-length, membrane-embedded amyloid precursor protein substrate or amyloid precursor protein that contains the Swedish KM → NL mutation. The correct cleavage was tested with an antibody specific for the free amino-terminus of β-amyloid peptide. Our results exclude EC 3.4.24.15 as a candidate β-secretase. Although cathepsin G cleaves the model peptide correctly, it displays poor ability to cleave the Swedish KM → NL peptide and does not generate carboxy-terminal fragments that are immunoreactive with amino-terminal-specific antiserum. Cathepsin D does not cleave the model peptide or show specificity for wild-type amyloid precursor protein; however, it cleaves the Swedish "NL peptide" and "NL precursor" substrates appropriately. Our results suggest that cathepsin D could act as β-secretase in the Swedish type of familial Alzheimer's disease and demonstrate the importance of using full-length substrate to verify the sequence specificity of candidate proteases.     

Cell type dependent inhibition of transport of cathepsin D in HepG2 cells and fibroblasts exposed to deoxy-manno-nojirimycin and deoxynojirimycin

The synthesis, transport and processing of lysosomal enzymes was examined in human hepatoma HepG2 cells and in human fibroblasts exposed to the Golgi alpha-mannosidase I inhibitor 1-deoxy-manno-nojirimycin. In HepG2 cells cathepsin D, beta-hexosaminidase and arylsulfatase B synthesized in the presence of 5 mM 1-deoxy-manno-nojirimycin contained exclusively endo-beta-N-acetylglucosaminidase H-cleavable oligosaccharides, indicating that alpha-mannosidase I had been inhibited efficiently. The proteolytic processing of intracellularly retained cathepsin D was retarded and the fraction of secreted cathepsin D was increased two-fold. In fibroblasts neither segregation nor maturation of cathepsin D were affected by 1-deoxy-manno-nojirimycin in spite of the inhibition of oligosaccharide processing. In the presence of the glucosidase I inhibitor 1-deoxynojirimycin, the precursor of cathepsin D (larger by about 1 kDa than the secreted form) accumulated transiently in light membranes in HepG2 cells. Release from the site of accumulation was accompanied by a decrease in size by about 1 kDa. This change was attributed to the removal of glucose residues. In fibroblasts the transient accumulation of larger precursors in the presence of 1-deoxynojirimycin was more pronounced than in HepG2 cells. The differential effects of alpha-mannosidase I and glucosidase I inhibitors on the transport of cathepsin D in HepG2 cells and fibroblasts may indicate that different intermediates in the biosynthetic pathway of asparagine-linked oligosaccharides participate in the transport of lysosomal enzymes in the two cell types.     

Characterisation of xenopsin immunoreactivity derived from pepsinised human skin and possible mechanism of in vivo generation

Human skin was subjected to a variety of extraction and enzymatic digestion procedures. Extracts and digests were subjected to neurotensin and xenopsin radioimmunoassays of known specificity. No neurotensin immunoreactivity was detected in any preparation with any region-specific antiserum. C-terminal xenopsin immunoreactivity was present in skin homogenates following incubation with both soluble and solid-phase pepsin and in those incubated with a leucocyte lysate or purified cathepsin D. The generation of xenopsin immunoreactivity was dependent on low pH and enzymes of pepsin-type specificity acting on a tissue precursor of approximately 30 kDa. Gel permeation chromatography of skin-derived xenopsin immunoreactivity identified a single molecular species larger than synthetic xenopsin which was resolved into two components by reverse-phase HPLC with retention times similar to synthetic xenopsin and kinetensin. Human skin thus contains a high-molecular-weight precursor protein and an endogenous acid protease, cathepsin D, capable of generating a peptide of similar size and C-terminal structure to amphibian xenopsin under acidic conditions such as might occur locally in wounds or at sites of inflammation.     

Mechanisms of a conversion from membrane associated lysosomal acid phosphatase to content forms

We reported that membrane-associated APase (M-APase) is anchored in the lipid bilayer through its hydrophobic sequence close to the COOH-terminus [Biochem. Biophys. Res. Commun. (1989) 162, 1044-1053] and is released from lysosomal membranes into the lysosomal contents by limited proteolysis with cathepsin D [J. Biochem. (1990) 108, 287-291]. We here report the conversion process of M-APase to three forms of the content enzyme (C-APase I, II, and III) by assigning the COOH-terminus of each APase in lysosomes. The purified M-APase (67 kDa) was subjected to COOH-terminal determination after digestion with cathepsin D. The COOH-terminus of cathepsin D-digested M-APase (65 kDa) ended at the position of the 382nd leucine residue. The COOH-termini of C-APase I (48 kDa) and III (64 kDa) were also determined. Since the two enzymes ended at the same position of the 373rd alanine residue, this COOH-terminal is 9 amino acid residues shorter than that of cathepsin D-digested M-APase. Then, we compared NH2-terminal sequences of the three enzymes, and found that those of three enzymes are exactly the same. Therefore, protein portions of C-APase I and III proved to be identical. The above results indicate that in lysosomes M-APase is first hydrolyzed between amino acid residues 382 and 383 by cathepsin D, and after solubilization, the enzyme is converted to C-APase III by losing 9 amino acid residues by lysosomal carboxypeptidase(s). Molecular weight differences among three C-APases (III, 64 kDa; II, 55 kDa; I, 48 kDa) probably are due to different degrees of carbohydrate chain degradations as reported previously [J. Biochem. (1989) 105, 449-456].     

Synthesis and intracellular distribution of cathepsins E and D in differentiating murine Friend erythroleukemia cells.

The synthesis, accumulation, and cellular distribution of cathepsins E and D during the dimethyl sulfoxide (DMSO)-induced differentiation of Friend erythroleukemia cells were investigated. The cellular levels of cathepsins E and D rapidly increased within 1 day of DMSO induction and then sharply decreased over the next 7 days. Since the cells during 1 day of differentiation were morphologically the same as uninduced cells, the results suggest the importance of these enzymes in more cellular proteolysis for the following committed differentiation. While cathepsin D was present mostly in the sedimentable fraction of cells throughout the differentiation period, the distribution of cathepsin E varied to the stage of differentiation. The ratio of the soluble/sedimentable cathepsin E content was 1.1, 1.4, 0.9, and 0.7 in cells after 0, 1, 4, and 7 days of DMSO treatment, respectively. The maturation of reticulocytes to erythrocytes was accompanied by complete loss of the soluble cathepsin E and of all of the cellular cathepsin D. Immunoblotting analyses revealed that both uninduced and induced cells contained two forms of cathepsin E; a high molecular weight form (82 kDa) which was mainly associated with the sedimentable fraction and a low molecular weight form (74 kDa) which was found largely in the soluble fraction. The distribution of these two forms was not significantly changed throughout the differentiation period, but the 74-kDa protein was completely eliminated with maturation of reticulocytes to erythrocytes. Cathepsin D also appeared in two forms in both uninduced and induced cells; a minor (46 kDa) and a major (42 kDa) form which appear to have a precursor-product relationship.     

Lysosomal enzyme precursors in human fibroblasts. Activation of cathepsin D precursor in vitro and activity of beta-hexosaminidase A precursor towards ganglioside GM2

Precursors of cathepsin D and beta-hexosaminidase were isolated from secretions of human fibroblasts and their activity was studied with natural substrates. The immunoprecipitated precursor of cathepsin D, Mr 53000, was inactive with radioactive hemoglobin as substrate. At pH 3.8-4.2 an activation of the precursor took place, which was correlated by a reduction in size to Mr 51500. The observed cleavage of cathepsin D precursor in vitro resembles the autocatalytic activation of pepsinogen. The precursor of beta-hexosaminidase A is able to cleave the natural substrate GM2 ganglioside. This reaction, like that of the mature enzyme, depends on the presence of a protein activator, which interacts with the substrate and the enzyme.     

Identification and characterization of cathepsin D in a highly purified sialidase from starfish A. pectinifera

A sialidase [EC 3.2.1.18] from the ovary of starfish Asterina pectinifera was isolated and highly purified by preparative PAGE. The SDS-PAGE separation of the purified enzyme revealed two natures of protein bands, upper (50 kDa) and a lower (47 kDa). To identify the protein, N-terminal amino acid sequence of the upper band was done. The sequence matched with the N-terminal amino acid sequence of human lysosomal mature cathepsin D and cathepsin D activity was also found in all the preparation steps. Protease inhibitor pepstatin A inhibited the proteolysis activity of cathepsin D against a synthetic substrate. The two enzymes sialidase and cathepsin D were separated from each other by using high-performance gel-filtration chromatography. The Western blot analysis and isoelectric focusing showed the co-purified cathepsin D is a 50 kDa protein with a PI value of 4.2.     

Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.     

Cathepsin D and beta-hexosaminidase synthesized in the presence of 1-deoxynojirimycin accumulate in the endoplasmic reticulum

Biosynthesis, transport, and maturation of cathepsin D and beta-hexosaminidase was examined in fibroblasts exposed to 1-deoxynojirimycin, a glucose analogue known to inhibit trimming glucosidases (Saunier, B., Kilker, R. D., Jr., Tkacz, J. S., Quaroni, A., and Herscovics, A. (1982) J. Biol. Chem. 257, 14155-14161; Hettkamp, H., Bause, E., and Legler, G. (1982) Biosci. Rep. 2, 899-906). Cells treated with 1-deoxynojirimycin contained precursors of cathepsin D and beta-hexosaminidase larger by about 1-2 kDa than control cells. The shift in molecular size was probably due to glucose residues that were rapidly removed from the precursors in the absence but not in the presence of 1-deoxynojirimycin. In addition, 1-deoxynojirimycin inhibited the glycosylation of the beta-chain precursor of beta-hexosaminidase and the synthesis of glycoproteins, including that of cathepsin D. The proteolytic processing of the larger precursors was retarded by several hours. The delay in proteolytic maturation was secondary to the accumulation of the larger precursors in organelles, which fractionated with membranes of the endoplasmic reticulum and Golgi complex. The accumulated cathepsin D precursor contained neither mannose 6-phosphate residues nor complex type oligosaccharides, which are formed in the cis and trans aspects of the Golgi complex. Cathepsin D precursors eventually released from the site of accumulation were apparently deglucosylated, acquired mannose 6-phosphate residues and complex type oligosaccharides, and were transferred into lysosomes as efficiently as in control cells. Our results suggest that transport of cathepsin D from the endoplasmic reticulum to the Golgi complex depends on removal of glucose residues from its carbohydrate.