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.     

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.     

Molecular forms of cathepsin D in coated vesicle preparations

We have studied the polypeptide pattern of cathepsin D associated with coated vesicle fractions prepared from human placenta. In these fractions cathepsin D was about 35-fold enriched in the precursor polypeptides as compared to the unfractionated tissue extract. The enrichment was more prominent if the vesicles were fractionated in the presence of Triton X-100. Adsorption of exogenously added metabolically labelled cathepsin D precursor to the fractionated material was negligible. It is likely that the precursor and may be also the mature cathepsin D polypeptides are present in the matrix of the coated vesicles. This finding substantiates the idea that coated vesicles participate in the transport of newly synthesized cathepsin D into the lysosomes.     

Synthesis, transport and processing of cathepsin C in Morris hepatoma 7777 cells and rat hepatocytes

The synthesis, transport and processing of cathepsin C was studied in Morris hepatoma 7777 cells by metabolic labelling, immunoprecipitation and characterization of labelled polypeptides by gel electrophoresis and fluorography. The largest detectable precursor of cathepsin C was a polypeptide of Mr = 92 500. Even 3 min after synthesis this precursor was accompanied by four polypeptides with Mr values ranging from 63 000 to 54 000, indicating cleavage of the precursors within the endoplasmic reticulum. The early forms of cathepsin C were associated with low-buoyant-density organelles containing the markers of endoplasmic reticulum and Golgi complex. About 30% of these early forms were secreted within 3 h after synthesis. The remaining 70% were transferred into dense lysosomes and processed between 2 and 3 h after synthesis to a mixture of the least five major and nine minor polypeptides with Mr values ranging from 73 000 to 12 000. These forms remained stable for at least 3 days. In freshly isolated hepatocytes cathepsin C was processed to forms closely related to those found in the hepatoma cells. Cathepsin C was synthesized in Morris hepatoma 7777 cells as a glycoprotein with mannose-6-phosphate residues that mediated mannose-6-phosphate-specific receptor-dependent uptake in human skin fibroblasts. In contrast to hepatocytes, synthesis of mannose-6-phosphate receptors in Morris hepatoma 7777 cells was below the limit of detection. The hepatoma cells did not express at the cell surface these or other receptors mediating endocytosis of lysosomal enzymes. Further, processing and transport of newly synthesized cathepsin C was largely resistant to NH4Cl. Apparently, cathepsin C is transferred in Morris hepatoma 7777 cells by a mechanism independent of mannose-6-phosphate-specific receptors.     

In vitro biosynthesis of two human galactosyltransferase polypeptides

HeLa cell galactosyltransferase is synthesized as two precursor polypeptides of Mr = 45,000 and Mr = 47,000. The enzyme is present in the Golgi complex as a (mature) Mr = 54,000 glycoprotein. If cells are treated with tunicamycin, two precursor polypeptides are synthesized without N-linked oligosaccharides with molecular weights of 42,000 and 44,000, respectively. To investigate whether the two precursor polypeptides are synthesized on different mRNAs total RNA from HeLa cells was translated in a wheat germ cell-free system. Galactosyltransferase polypeptides were isolated by immunoprecipitation and compared to the polypeptides synthesized in vivo in the presence of tunicamycin. The two in vitro translated polypeptides co-migrate exactly with the polypeptides made in the cells in the presence of tunicamycin, indicating two different mRNAs for galactosyltransferase. The results also indicate that translocation of galactosyltransferase through the membrane of the rough endoplasmic reticulum is not followed by signal peptide cleavage.     

Molecular forms of beta-hexosaminidase and cathepsin D in serum and urine of healthy subjects and patients with elevated activity of lysosomal enzymes.

A procedure is described that allows the characterization of the molecular forms of beta-hexosaminidase and cathepsin D in controls and pathological specimens of human serum and human urine. The following observations were made. (1) In human serum, beta-hexosaminidase (alpha- and beta-chain) and cathepsin D are present predominantly in their high-molecular-weight precursor forms. In human urine, these enzymes exist as both precursor and mature forms. (2) Cathepsin D precursor from serum and urine differs in the number of oligosaccharides that are sensitive to endo-beta-N-acetylglucosaminidase H. Therefore the urine enzyme is not likely to originate from the serum. (3) The presence exclusively of precursors of beta-hexosaminidase and of cathepsin D in the sera of patients with hepatitis suggests that in hepatitis secretion of lysosomal enzymes is elevated, rather than the enzymes leaking from damaged cells. (4) In the urine of patients with nephrotic syndrome, beta-hexosaminidase and cathepsin D are present in grossly elevated amounts, but do not differ in the polypeptide patterns from controls. (5) In urine from a patient with mucolipidosis II, the elevated activity of beta-hexosaminidase is accounted for mainly by the precursor forms. Mature beta-chain of beta-hexosaminidase is lacking, and incompletely processed beta-hexosaminidase polypeptides are present. Both the precursor and the mature forms of cathepsin D are increased. They contain only complex oligosaccharides.     

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.     

Sulfated N-linked oligosaccharides affect secretion but are not essential for the transport, proteolytic processing, and sorting of lysosomal enzymes in Dictyostelium discoideum

Although previous studies have indicated that N-linked oligosaccharides on lysosomal enzymes in Dictyostelium discoideum are extensively phosphorylated and sulfated, the role of these modifications in the sorting and function of these enzymes remains to be determined. We have used radiolabel pulse-chase, subcellular fractionation, and immunofluorescence microscopy to analyze the transport, processing, secretion, and sorting of two lysosomal enzymes in a mutant, HL244, which is almost completely defective in sulfation. [3H]Mannose-labeled N-linked oligosaccharides were released from immunoprecipitated alpha-mannosidase and beta-glucosidase of HL244 by digestion with peptide: N-glycosidase. The size, Man9-10GlcNAc2, and processing of the neutral species were similar to that found in the wild type, but the anionic oligosaccharides were less charged than those from the wild-type enzymes. All of the negative charges on the oligosaccharides for HL244 were due to the presence of 1, 2, or 3 phosphodiesters and not to sulfate esters. The rate of proteolytic processing of precursor forms of alpha-mannosidase and beta-glucosidase to mature forms in HL244 was identical to wild type. The precursor polypeptides in the mutant and the wild type were membrane associated until being processed to mature forms; therefore, sulfated sugars are not essential for this association. Furthermore, the rate of transport of alpha-mannosidase and beta-glucosidase from the endoplasmic reticulum to the Golgi complex was normal in the mutant as determined by the rate at which the newly synthesized proteins became resistant to the enzyme, endo-beta-N-acetylglucosaminidase H. There was no increase in the percentage of newly synthesized mutant precursors which escaped sorting and were secreted, and the intracellularly retained lysosomal enzymes were properly localized to lysosomes as determined by fractionation of cell organelles on Percoll gradients and immunofluorescence microscopy. However, the mutant secreted lysosomally localized mature forms of the enzymes at 2-fold lower rates than wild-type cells during both growth and during starvation conditions that stimulate secretion. Furthermore, the mutant was more resistant to the effects of chloroquine treatment which results in the missorting and oversecretion of lysosomal enzymes. Together, these results suggest that sulfation of N-linked oligosaccharides is not essential for the transport, processing, or sorting of lysosomal enzymes in D. discoideum, but these modified oligosaccharides may function in the secretion of mature forms of the enzymes from lysosomes.     

Processing of human cathepsin D in lysosomes in vitro

The proteolytic maturation of cathepsin D polypeptides was studied in lysosomes isolated from metabolically labeled fibroblasts. In lysosomes isolated from fibroblasts labeled with [35S]methionine, 70-95% of labeled cathepsin D polypeptides were represented by a Mr = 47,000 polypeptide after a 20-min pulse and 75-min chase. When these lysosomes were incubated in vitro, up to 70% of the Mr = 47,000 polypeptide was processed to mature cathepsin D polypeptides. The processing was dependent on the integrity of the lysosomes, had an optimum between pH 6 and 7, and could be stimulated by dithiothreitol and ATP. The noncleavable ATP analogue, adenosine 5'-(beta, gamma-imido)triphosphate, and GTP, CTP, and UTP could not substitute for ATP. The ATP-dependent stimulation was associated with an acidification of lysosomes. It was inhibited by agents that dissipate the lysosomal pH gradient (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, N,N'-dicyclohexylcarbodiimide, nigericin, NH4Cl). A stimulatory effect of ATP was observed also at pH 5.5. The stimulation at pH 5.5 was not associated with acidification of lysosomes and was resistant to protonophores. Inhibitors of lysosomal cysteine proteinases and N-ethylmaleimide inhibited the processing. In the presence of ATP the processing activity was partially protected from inhibition by N-ethylmaleimide. In conclusion, the maturation of cathepsin D in lysosomes depends on cysteine proteinases and is stimulated by the ATP-driven acidification of lysosomes. In addition, ATP stimulates maturation at pH 5.5 by a mechanism not involving the proton pump.     

Identification of subcellular compartments involved in biosynthetic processing of cathepsin D.

We have assigned the biosynthetic processing steps of cathepsin D to intracellular compartments which are involved in its transport to lysosomes in HepG2 cells. Cathepsin D was synthesized as a 51-kDa proenzyme. After formation of 51-55-kDa intermediates due to processing of N-linked oligosaccharides, procathepsin D was proteolytically processed to an intermediate 44-kDa and the mature 31-kDa enzyme. The intersection of the biosynthetic pathway of cathepsin D with the endocytic pathway was labeled with horseradish peroxidase and monitored biochemically by 3,3'-diaminobenzidine cytochemistry. Horseradish peroxidase was used either as a fluid-phase marker to label the entire endocytic pathway or conjugated to transferrin (Tf) to label endosomes only. Directly after biosynthesis cathepsin D was accessible neither to horseradish peroxidase nor Tf-horseradish peroxidase. Newly synthesized 51-55-kDa species of cathepsin D present in the trans-Golgi reticulum were accessible to both horseradish peroxidase and Tf-horseradish peroxidase. The accessibility of trans-Golgi reticulum to both endocytosed horseradish peroxidase and Tf-horseradish peroxidase was monitored by colocalization with a secretory protein, alpha 1anti-trypsin. The proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurred in compartments which were fully accessible to fluid-phase horseradish peroxidase. Tf-horseradish peroxidase had access to only 20% of 44-kDa cathepsin D while it had no access to 31-kDa cathepsin D. In contrast, the 31-kDa species was completely accessible to fluid-phase horseradish peroxidase. We conclude that proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurs in endosomes, whereas the processing of 44-31-kDa cathepsin D takes place in lysosomes.     

Calnexin acts as a molecular chaperone during the folding of glycoprotein B of human cytomegalovirus.

Human cytomegalovirus glycoprotein B (gB) is synthesized as a 105-kDa nonglycosylated polypeptide and cotranslationally modified by addition of N-linked oligosaccharides to a 160-kDa precursor in the endoplasmic reticulum (ER). It is then transported to the Golgi complex, where it is endoproteolytically cleaved to form the disulfide-linked mature gp55-116 complex. Pulse-chase experiments demonstrate that the 160-kDa gB precursor was transiently associated with calnexin, a membrane-bound chaperone, in the ER. The association was maximal immediately after synthesis, and they dissociated with a half-time of 15 min. Complete inhibition of binding by tunicamycin or castanospermine indicates the importance of N-linked oligosaccharides for it. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that during an initial stage in the biogenesis, the 160-kDa gB precursor was first synthesized as a fully reduced form and rapidly converted to an oxidized form, with a half-time of 18 min. Both forms of the gB precursor could bind to calnexin. The kinetics of the conversion from the fully reduced to the oxidized form coincided with that of dissociation of the 160-kDa gB precursor from calnexin, suggesting that the two steps are closely related.     

Lysosomal enzymes in Dictyostelium discoideum are transported to lysosomes at distinctly different rates

We are investigating the molecular mechanisms involved in the localization of lysosomal enzymes in Dictyostelium discoideum, an organism that lacks any detectable mannose-6-phosphate receptors. The lysosomal enzymes alpha-mannosidase and beta-glucosidase are both initially synthesized as precursor polypeptides that are proteolytically processed to mature forms and deposited in lysosomes. Time course experiments revealed that 20 min into the chase period, the pulse-labeled alpha-mannosidase precursor (140 kD) begins to be processed, and 35 min into the chase 50% of the polypeptides are cleaved to mature 60 and 58-kD forms. In contrast, the pulse-labeled beta-glucosidase precursor (105 kD) begins to be processed 10 min into the chase period, and by 30 min of the chase all of the precursor has been converted into mature 100-kD subunits. Between 5 and 10% of both precursors escape processing and are rapidly secreted from cells. Endoglycosidase H treatment of immunopurified radioactively labeled alpha-mannosidase and beta-glucosidase precursor polypeptides demonstrated that the beta-glucosidase precursor becomes resistant to enzyme digestion 10 min sooner than the alpha-mannosidase precursor. Moreover, subcellular fractionation studies have revealed that 70-75% of the pulse-labeled beta-glucosidase molecules move from the rough endoplasmic reticulum (RER) to the Golgi complex less than 10 min into the chase. In contrast, 20 min of chase are required before 50% of the pulse-labeled alpha-mannosidase precursor exits the RER. The beta-glucosidase and alpha-mannosidase precursor polypeptides are both membrane associated along the entire transport pathway. After proteolytic cleavage, the mature forms of both enzymes are released into the lumen of lysosomes. These results suggest that beta-glucosidase is transported from the RER to the Golgi complex and ultimately lysosomes at a distinctly faster rate than the alpha-mannosidase precursor. Thus, our results are consistent with the presence of a receptor that recognizes the beta-glucosidase precursor more readily than the alpha-mannosidase precursor and therefore more quickly directs these polypeptides to the Golgi complex.     

Myeloperoxidase is synthesized as larger phosphorylated precursor.

Synthesis and processing of myeloperoxidase were examined in metabolically labeled cells of the human promyelocyte line HL-60 and in an in vitro rabbit reticulocyte lysate system directed with HL-60 mRNA. Radioactivity labeled products were isolated by immunoprecipitation and analyzed by gel electrophoresis and fluorography. In vivo, myeloperoxidase was labeled initially as a 85-K glycosylated polypeptide (75 K after treatment with endo-beta-N-acetylglucosaminidase H). This polypeptide was soon processed to an 81-K intermediate and to smaller mature fragments of 60 K and 13 K within approximately 1 day. A minor portion of the precursor was converted to fragments of 40 K and 43 K. The pattern of labeled polypeptides of mature myeloperoxidase was similar to that of the enzyme purified from human leucocytes. The modifications of the polypeptide and of the oligosaccharide side chains in myeloperoxidase resembled those known to occur during the processing of lysosomal enzymes. In the absence or presence of dog pancreas membranes, myeloperoxidase was synthesized in vitro as a 76-K polypeptide or a 87-K glycosylated polypeptide, respectively. In HL-60 cells [32P]phosphate was incorporated into endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides. The presence of phosphorylated oligosaccharides was inferred from the fact that endocytosis of leucocyte myeloperoxidase in fibroblasts was sensitive to mannose 6-phosphate. It is suggested that myeloperoxidase is synthesized in the rough endoplasmic reticulum as a precursor of larger molecular mass and that the oligosaccharide side chains in the precursor are modified to contain mannose 6-phosphate residues which may be involved in the segregation and transport of the precursor.     

Isolation and characterization of a stable activation intermediate of the lysosomal aspartyl protease cathepsin D.

Procathepsin D is the intracellular aspartyl protease precursor of cathepsin D, a major lysosomal enzyme. Procathepsin D is rapidly processed inside the cell, and, thus, examination of its proteolyic activation and structure has been difficult. To study this proenzyme, a nonglycosylated form of the human fibroblast procathepsin D was expressed in Escherichia coli, refold in vitro, and purified by affinity chromatography on pepstatinyl agarose. Sequence analysis of the refolded, autoactivated enzyme allowed determination of the autoproteolytic cleavage site. The sequence surrounding this cleavage site between residues LeuP26 and IleP27 (in the "pro" region) resembled the first cleavage site found during activation of other aspartyl proteases. Thus, the autoactivated procathepsin D is analogous to the pepsin activation intermediate, which has been termed pseudopepsin. The enzymatic activity, thermal and pH stability, and fluorescence spectra of pseudocathepsin D were compared to mature, predominantly two-chain, cathepsin D isolated from human placenta. The results indicated that pseudocathepsin D and mature enzyme have a similar Km toward a peptide substrate and cleave a protein substrate at identical sites. Temperature stability of the recombinant enzyme was similar to that of the tissue-derived enzyme. However, the recombinant enzyme had increased stability at low pH when compared to the glycosylated tissue-derived two-chain cathepsin D. Fluorescence spectra of the recombinant and tissue-derived enzymes were identical. Thus, the absence of asparagine-linked oligosaccharides and the presence of the remaining segment of propeptide did not significantly alter the structural and enzymatic properties of the enzyme.     

Mannose 6-phosphate-independent targeting of cathepsin D to lysosomes in HepG2 cells.

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.     

Human lysosomal protective protein. Glycosylation, intracellular transport, and association with beta-galactosidase in the endoplasmic reticulum.

In lysosomes beta-galactosidase and neuraminidase acquire a stable and active conformation through their association with the protective protein. The latter is homologous to serine carboxypeptidases and has cathepsin A-like activity which is distinct from its protective function towards the two glycosidases. To define signals in the human protective protein important for its intracellular transport, and to determine the site of its association with beta-galactosidase, we have generated a set of mutated protective protein cDNAs carrying targeted base substitutions. These mutants were either singly transfected into COS-1 cells or cotransfected together with wild type human beta-galactosidase. We show that all point mutations cause either a complete or partial retention of the protective protein precursor in the endoplasmic reticulum. This abnormal accumulation leads to degradation of the mutant proteins probably in this compartment. Only the oligosaccharide chain on the 32-kDa subunit acquires the mannose 6-phosphate recognition marker, the one on the 20-kDa subunit seems to be merely essential for the stability of the mature protein. In cotransfection experiments, wild type beta-galactosidase and protective protein appear to assemble already as precursors, soon after synthesis, in the endoplasmic reticulum. Mutated protective protein precursors that are retained in the endoplasmic reticulum or pre-Golgi complex interact with and withhold normal beta-galactosidase molecules in the same compartments, thereby preventing their normal routing.     

Expression of human cathepsin D in Xenopus oocytes: phosphorylation and intracellular targeting

We have obtained expression of a cDNA clone for human cathepsin D in Xenopus laevis oocytes. Biosynthetic studies with [35S]methionine labeling demonstrated that most of the cathepsin D remained intracellular and underwent proteolytic cleavage, converting a precursor of Mr 47,000 D to a mature form of Mr 39,000 D with processing intermediates of Mr 43,000-41,000 D. greater than 90% of the cathepsin D synthesized by oocytes bound to a mannose 6-phosphate (Man-6-P) receptor affinity column, indicating the presence of phosphomannosyl residues. An analysis of [2-3H]mannose-labeled oligosaccharides directly demonstrated phosphomannosyl residues on cathepsin D. Sucrose-gradient fractionation, performed to define the membranous compartments that cathepsin D traversed during its biosynthesis, demonstrated that cathepsin D is targeted to a subpopulation of yolk platelets, the oocyte equivalent of a lysosome. Xenopus oocytes were able to endocytose lysosomal enzymes from the medium and this uptake was inhibited by Man-6-P, thus demonstrating the presence of Man-6-P receptors in these cells. Therefore, the entire Man-6-P dependent pathway for targeting of lysosomal enzymes is present in the oocytes. Xenopus oocytes should be a useful system for examining signals responsible for the specific targeting of lysosomal enzymes to lysosomes.     

The identification of N-linked oligosaccharides on the human CR2/Epstein-Barr virus receptor and their function in receptor metabolism, plasma membrane expression, and ligand binding

Human complement receptor type 2 (CR2) was biosynthetically labeled by pulsing SB B lymphoblastoid cells for 25 min with [35S]methionine followed by chase in the presence of excess unlabeled methionine. An Mr 134,000 polypeptide represented the major form of the receptor at the end of the pulse period, and within 1 h of chase this disappeared coincident with the appearance of the Mr 145,000 mature form of CR2. Precursor, but not mature, CR2 was sensitive to endoglycosidase H, indicating that maturation of CR2 represented processing of N-linked high mannose oligosaccharides to the complex type. The processing of precursor CR2 was impaired by monensin. In the presence of tunicamycin an Mr 111,000 form of CR2 was synthesized by SB cells, and this did not chase into either precursor or mature CR2. This Mr 111,000 form of CR2 did not incorporate [3H]glucosamine, indicating that it lacked both N- and O-linked oligosaccharide. The half-lives of mature CR2 and nonglycosylated CR2 pulse-labeled in the presence of tunicamycin were 13.8 and 2.8 h, respectively; the turnover rate of B1, a membrane protein normally lacking carbohydrate, was unaffected by the presence of the antibiotic. The percentage of pulse-labeled, nonglycosylated CR2 that was expressed at the cell surface after 1 h of chase in the presence of tunicamycin was 30%, identical to that of mature CR2 in cells chased in the absence of the antibiotic. However, after 6 h of chase there was no additional net accumulation of nonglycosylated CR2 at the plasma membrane, while the proportion of pulse-labeled mature CR2 at this site had risen to 81%. Therefore, N-linked oligosaccharides are essential for the stability of CR2 and have some role in its plasma membrane expression. In contrast, the observation that all three forms of CR2 bound to Sepharose C3 indicates that oligosaccharides are not necessary for the interaction between CR2 and its complement ligand.     

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.     

Identification of Cellular Compartments Involved in Processing of Cathepsin E in Primary Cultures of Rat Microglia

Abstract: Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome-like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome-like vacuoles and partly in the vesicles located in the trans-Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [³⁵S]methionine, >95% of labeled cathepsin E were represented by a 46-kDa polypeptide (reduced form) after a 30-min pulse. Most of it was proteolytically processed via a 44-kDa intermediate to a 42-kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A₁, a specific inhibitor of vacuolar-type H⁺-ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse-labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high-mannose oligosaccharides and processed to the active mature enzyme with complex-type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome-like acidic compartments.