Mannose 6-phosphate-independent endocytosis of beta-glucuronidase by human fibroblasts. I. Evidence for the existence of a membrane-binding activity

Prior work has shown that endocytosis of bovine beta-glucuronidase by human fibroblasts can be mediated by the existence of a Man6P-independent receptor for the recapture and targeting to lysosomes. In this study, we have isolated a peptide (IIIb2) from pronase digested bovine beta-glucuronidase that behaved as competitive inhibitor of the endocytosis of bovine beta-glucuronidase by human fibroblasts. This peptide contained a Ser-X-Ser sequence, where X is probably a posttranslational modified Trp. Antibodies raised against this peptide impaired the endocytosis of the bovine but not the human beta-glucuronidase, implying that the new recognition marker for the endocytosis of acid hydrolases might reside in a single discrete stretch of amino acid sequence. On the other hand, bovine beta-glucuronidase has been shown to bind specifically to receptors of human fibroblast membranes. The binding was saturable, divalent cation-dependent and was competitively inhibited by the IIIb2 peptide, but not by mannose 6-phosphate. Results presented suggested an interplay between manganese concentrations, temperature and pH on the dissociation of the beta-glucuronidase-receptor complexes. All together, these data reinforce the presence of two endocytic systems for the recapture and targeting of beta-glucuronidase in human fibroblasts.     

Intracellular transport of acid alpha-glucosidase in human fibroblasts: evidence for involvement of phosphomannosyl receptor-independent system

Intracellular transport of two lysosomal enzymes, acid alpha-glucosidase and beta-hexosaminidase, was analyzed in human fibroblasts. The precursors of beta-hexosaminidase in normal fibroblasts were released from the membrane fraction by treatment with mannose 6-phosphate, but the precursor of alpha-glucosidase was not. Percoll density gradient centrifugation revealed a normal amount of acid alpha-glucosidase activity in heavy lysosomes in I-cell disease fibroblasts despite impaired maturation and defective phosphorylation, and beta-hexosaminidase activity was markedly reduced in lysosomes. It was concluded that the membrane-bound precursor of acid alpha-glucosidase is transported to lysosomes by a phosphomannosyl receptor-independent system although the enzyme lacks the recognition marker for the phosphomannosyl receptor and processing of an intermediate form to mature forms does not occur in this disease.     

Biosynthesis and turnover of the phosphomannosyl receptor in human fibroblasts.

The phosphomannosyl receptor mediates intracellular targeting of newly synthesized acid hydrolases to lysosomes, and is also expressed as a pinocytosis receptor on the cell surface of fibroblasts. We have purified the phosphomannosyl receptor from bovine liver and produced rabbit antibodies to the bovine receptor. The antibodies partially blocked pinocytosis of human spleen beta-glucuronidase by fibroblasts, a process mediated by the phosphomannosyl receptor. Affinity-purified antibodies to the phosphomannosyl receptor were used to study the biosynthesis and turnover of the receptor in human fibroblasts. Phosphomannosyl receptor immunoprecipitated after a 15 min pulse-labelling of fibroblasts with [35S]methionine exhibited an identical mobility on sodium dodecyl sulphate/polyacrylamide gels as purified bovine liver phosphomannosyl receptor. Pulse-chase experiments for up to 3 days provided no evidence for changes in molecular weight attributable to post-translational processing of the phosphomannosyl receptor. Turnover studies determined that the half-life of the phosphomannosyl receptor in normal human fibroblasts was 24-29 h. The half-life of the receptor was slightly longer (32 h) in I-cell disease fibroblasts and normal fibroblasts exposed to leupeptin (32 h), slightly shorter in fibroblasts exposed to NH4Cl (23 h) and saturating amounts of ligand (21 h) and unaffected in cells exposed to mannose 6-phosphate (24 h). These studies show that the turnover of the phosphomannosyl receptor in fibroblasts is very slow, in contrast with its rate of internalization in endocytosis, and that its rate of degradation is not greatly altered by a variety of agents that affect lysosomal protein turnover and/or receptor-mediated endocytosis. These results suggest that the degradative activities of the lysosomes do not play an important role in phosphomannosyl receptor turnover in cultured fibroblasts.     

Lysosomal enzyme trafficking in mannose 6-phosphate receptor-positive mouse L-cells: demonstration of a steady state accumulation of phosphorylated acid hydrolases

During their transport from the endoplasmic reticulum to lysosomes, newly synthesized acid hydrolases are phosphorylated in the Golgi apparatus to generate a common recognition marker, mannose 6-phosphate (Man 6-P). The phosphorylated acid hydrolases then bind to the Man 6-P receptor and are transported by an unknown route to lysosomes. To learn more about the delivery pathway, we examined the fate of the phosphorylated oligosaccharides synthesized by a Man 6-P receptor-positive line of mouse L-cells. In contrast to the rapid degradation of the recognition marker previously observed in mouse lymphoma cells (Gabel, C. A., D. E. Goldberg, and S. Kornfield. 1982. J. Cell Biol., 95:536-542), the number of high mannose oligosaccharides phosphorylated by the L-cells after a 30-min pulse labeling with [2-3H]mannose increased continuously during a subsequent 4-h chase period to a maximum of 9.3% of the total cell-associated structures. After 19 h of chase the absolute number of phosphorylated oligosaccharides declined, but the loss was accompanied by a general loss of cellular oligosaccharides such that 7.4% of the cell-associated high mannose oligosaccharides remained phosphorylated. The longevity of the Man 6-P recognition marker in the L-cells was verified by analyzing the ability of an individual acid hydrolase, beta-glucuronidase, to serve as a ligand for the Man 6-P receptor. At least 60% of the steady state beta-glucuronidase molecules isolated from the L-cells could undergo receptor-mediated endocytosis into enzyme-deficient human fibroblasts. Dense lysosomal granules isolated by metrizamide gradient centrifugation from [3H]mannose-labeled L-cells were found to be highly enriched in their content of phosphomonoester-containing oligosaccharides. The data indicate that acid hydrolases may retain their Man 6-P recognition markers within lysosomes, and suggest the possibility that dephosphorylation occurs at a nonlysosomal location through which the newly synthesized enzymes pass en route to lysosomes.     

Mannose 6-phosphate receptor-mediated endocytosis of acid hydrolases: internalization of beta-glucuronidase is accompanied by a limited dephosphorylation

Endocytosis of acid hydrolases via the cell surface mannose 6-phosphate (Man 6-P) receptor results in the delivery of the enzymes to lysosomes. To examine the fate of the ligand-associated phosphorylated high mannose oligosaccharides, we have analyzed the asparagine-linked oligosaccharides attached to beta-glucuronidase after uptake and processing by Man 6-P receptor-positive mouse L cells. beta-Glucuronidase, double-labeled with [2-3H]mannose and [35S]methionine, was isolated from the growth medium of mouse P388D1 cells. 80% of the [3H]mannose associated with the secreted enzyme was recovered as high mannose-type oligosaccharides, and 24-37% of these units were phosphorylated. Three species of phosphorylated oligosaccharides were identified; high mannose-type units containing either one or two phosphomonoesters, and hybrid-type units containing one phosphomonoester and one sialic acid residue. After endocytosis by the L cells, the beta-glucuronidase molecules migrated faster on an SDS gel, suggesting that the enzymes had been processed within lysosomes. Examination of the cell-associated beta-glucuronidase molecules indicated that: (a) the percentage of phosphorylated oligosaccharides remained comparable to the input form of the enzyme, even after a 24-h chase period, (b) the presence of a single species of phosphorylated oligosaccharide that contained one phosphomonoester, and (c) the positioning of the phosphate within the intracellular monophosphorylated species was comparable to the positioning of the phosphate within the two phosphomonoester species originally secreted by the P388D1 cells. Therefore, the internalized beta-glucuronidase molecules undergo a limited dephosphorylation; oligosaccharides containing two phosphomonoesters are converted to monophosphorylated species, but the one phosphomonoester forms are conserved. A comparison of the phosphorylated oligosaccharides recovered from ligands internalized by the L cells at 37 degrees and 20 degrees C indicated that: (a) molecules internalized at 20 degrees C retain a higher percentage of phosphorylated structures; and (b) at both temperatures the predominant phosphorylated oligosaccharide contains a single phosphomonoester group. The results indicate that the Man 6-P recognition marker persists after endocytosis and delivery to lysosomes and support the possibility that the limited dephosphorylation of the oligosaccharides may occur en route to these organelles.     

Human acid ceramidase: processing, glycosylation, and lysosomal targeting.

The biosynthesis of human acid ceramidase (hAC) starts with the expression of a single precursor polypeptide of approximately 53-55 kDa, which is subsequently processed to the mature, heterodimeric enzyme (40 + 13 kDa) in the endosomes/lysosomes. Secretion of hAC by either fibroblasts or acid ceramidase cDNA-transfected COS cells is extraordinarily low. Both lysosomal targeting and endocytosis critically depend on a functional mannose 6-phosphate receptor as judged by the following criteria: (i) hAC-precursor secretion by NH(4)Cl-treated fibroblasts and I-cell disease fibroblasts, (ii) inhibition of the formation of mature heterodimeric hAC in NH(4)Cl-treated fibroblasts or in I-cell disease fibroblasts, and (iii) blocked endocytosis of hAC precursor by mannose 6-phosphate receptor-deficient fibroblasts or the addition of mannose 6-phosphate. The influence of the six individual potential N-glycosylation sites of human acid ceramidase on targeting, processing, and catalytic activity was determined by site-directed mutagenesis. Five glycosylation sites (sites 1-5 from the N terminus) are used. The elimination of sites 2, 4, and 6 has no influence on lysosomal processing or enzymatic activity of recombinant ceramidase. The removal of sites 1, 3, and 5 inhibits the formation of the heterodimeric enzyme form. None of the mutant ceramidases gave rise to an increased rate of secretion, suggesting that lysosomal targeting does not depend on one single carbohydrate chain.     

Lysosomal storage causes cellular dysfunction in mucolipidosis II skin fibroblasts

Mucolipidosis II (ML-II) is a fatal inherited metabolic disease caused by deficiency of GlcNAc-phosphotransferase, which plays a role in generating the mannose 6-phosphate recognition marker on lysosomal enzymes. In ML-II, many lysosomal acid hydrolases are mistargeted out of cells, and lysosomes become filled with undigested substrates, which explains inclusion cell disease as an alternative name for this disease. In this study, we revealed various cellular phenotypes in ML-II skin fibroblasts. We quantitated phospholipid and cholesterol within cells and showed ~2-fold accumulation in ML-II as compared with normal cells. Lysosomal pH of ML-II cells was higher than that of normal cells (5.29 ± 0.08 versus 4.79 ± 0.10, p < 0.001). The proliferated lysosomes in ML-II cells were accumulated ~3-fold in amount as compared with normal cells. Intracellular logistics including endocytosis and mannose 6-phosphate receptor recycling were impaired in ML-II cells. To confirm whether these ML-II cellular phenotypes derive from deficient lysosomal acid hydrolases within lysosomes, we performed supplementation of lysosomal enzymes using a partially purified total enzyme mixture, which was derived from the conditioned culture medium of normal skin fibroblasts after NH(4)Cl treatment. This supplementation corrected all of the previously described ML-II phenotypes. In addition, the autophagic and mitochondrial impairment that we have previously reported improved, and inclusion bodies disappeared on electron micrography following total lysosomal enzyme supplementation. Our results indicate that various cellular phenotypes in ML-II are caused by the deficiency of many lysosomal enzymes and massive accumulation of undigested substrates.     

Biosynthesis and endocytosis of lysosomal enzymes in human colon carcinoma SW 1116 cells: impaired internalization of plasma membrane-associated cation-independent mannose 6-phosphate receptor.

The human colon adenocarcinoma cell lines SW 948, SW 1116, and SW 1222 were tested for their ability to sort and internalize lysosomal enzymes. The biosynthesis of the lysosomal enzymes cathepsin B, arylsulfatase A, and beta-hexosaminidase in these cell lines exhibits no significant differences to that in human fibroblasts. The intracellular targeting of newly synthesized hydrolases to the lysosomes relies in colon carcinoma cells on the mannose 6-phosphate receptor system. Both the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor are expressed in all colon carcinoma cell lines investigated. Endocytosis of lysosomal enzymes via mannose 6-phosphate receptors is reduced in colon carcinoma cells as compared with human fibroblasts. SW 1116 cells were shown to be deficient in receptor-mediated endocytosis of mannose 6-phosphate containing ligands. Ligands of other endocytic receptors as well as the fluid-phase marker horseradish peroxidase were internalized at normal rates. While antibodies against CI-MPR bind to the surface of SW 1116 cells, these antibodies cannot be internalized. These data suggest that the cycling of CI-MPR is specifically impaired in SW 1116 cells.     

The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.     

Binding of phosphorylated oligosaccharides to immobilized phosphomannosyl receptors

We have purified phosphomannosyl-enzyme receptors from bovine liver on an affinity column composed of glycoproteins isolated from Dictyostelium discoideum secretions. Binding of human fibroblast beta-hexosaminidase B to receptors reconstituted into phosphatidylcholine liposomes was 1) specifically inhibited by mannose 6-phosphate, but not mannose 1-phosphate or glucose 6-phosphate, and 2) had properties similar to the previously reported binding of enzyme to receptors on cell surfaces and isolated membranes. In order to determine the structural features of the phosphomannosyl recognition marker required for receptor recognition, we covalently coupled purified receptor to an agarose gel bead support for affinity chromatography of phosphorylated, high mannose-type oligosaccharides isolated from fibroblast secretions radiolabeled with [2-3H]mannose. Neutral oligosaccharides and oligosaccharides containing one or two phosphates in phosphodiester linkage were not retained by the receptor column. By contrast, oligosaccharides bearing one phosphomonoester moiety were retarded on the column; those bearing two phosphomonoesters were bound to the column and were eluted with 10 mM mannose 6-phosphate. The binding of the oligosaccharides to the immobilized receptor correlates with their ability to be pinocytosed by fibroblasts and shows that the preferred recognition marker for the phosphomannosyl-enzyme receptor is a high mannose-type oligosaccharide chain bearing two uncovered phosphomannosyl groups.     

Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling

Adsorptive pinocytosis of acid hydrolases by fibroblasts depends on phosphomannosyl recognition markers on the enzymes and high-affinity pinocytosis receptors on the cell surface. In this study, beta- glucuronidase binding to the cell surface of attached fibroblasts was found to be saturable and inhibitable by mannose-6-phosphate (Man-6-P). Dissociation of cell-bound beta-glucuronidase occurred very slowly at neutral pH, but was greatly accelerated by lowering the pH below 6.0, or by exposure to Man-6-P. Comparison of the maximal cell surface binding and the observed rate of enzyme pinocytosis suggests that the pinocytosis receptors are replaced or reused about every 5 min. Enzyme pinocytosis was not affected by inhibition of new protein synthesis for several hours, suggesting a large pool of internal receptors and/or reuse of internalized receptors. Chloroquine treatment of normal human fibroblasts had three effects: (a) greatly enhanced secretion of newly synthesized acid hydrolases bearing the recognition marker for uptake, (b) depletion of enzyme-binding sites from the cell surface, and (c) inhibition of pinocytosis of exogenous enzyme. Only the third effect was seen in I-cell disease fibroblasts, which were also less sensitive than control cells to this effect. These observations are consistent with a model for transport of acid hydrolases that proposes that delivery of newly synthesized acid hydrolases to lysosomes requires the phosphomannosyl recognition marker on the enzymes, and intracellular receptors that segregate receptor-bound enzymes into vesicles for transport to lysosomes. This model explains how chloroquine, which raises intralysosomal pH, can disrupt both the intracellular pathway for newly synthesized acid hydrolases, and the one for uptake of exogenous enzyme by cell surface pinocytosis receptors.     

Annexin VI is a mannose-6-phosphate-independent endocytic receptor for bovine β-glucuronidase

Endocytosis and transport of bovine liver β-glucuronidase to lysosomes in human fibroblasts are mediated by two receptors: the well-characterized cation-independent mannose 6-phosphate receptor (IGF-II/Man6PR) and an IGF-II/Man6PR-independent receptor, which recognizes a Ser-Trp*-Ser sequence present on the ligand. The latter receptor was detergent extracted from bovine liver membranes and purified. LC/ESI–MS/MS analysis revealed that this endocytic receptor was annexin VI (AnxA6). Several approaches were used to confirm this finding. First, the binding of bovine β-glucuronidase to the purified receptor from bovine liver membranes and His-tagged recombinant human AnxA6 protein was confirmed using ligand-blotting assays. Second, western blot analysis using antibodies raised against IGF-II/Man6PR-independent receptor as well as commercial antibodies against AnxA6 confirmed that the receptor and AnxA6 were indeed the same protein. Third, double immunofluorescence experiments in human fibroblasts confirmed a complete colocalization of the bovine β-glucuronidase and the AnxA6 receptor on the plasma membrane. Lastly, two cell lines were stably transfected with a plasmid containing the cDNA for human AnxA6. In both transfected cell lines, an increase in cell surface AnxA6 and in mannose 6-phosphate-independent endocytosis of bovine β-glucuronidase was detected. These results indicate that AnxA6 is a novel receptor that mediates the endocytosis of the bovine β-glucuronidase.     

Human beta-glucuronidase pinocytosis and binding to the immobilized phosphomannosyl receptor. Effects of treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase

beta-D-Glucuronidase (EC 3.2.1.31) was purified to homogeneity from human spleen, and enzyme fractions from CM-Sephadex were examined for uptake by fibroblasts and retention by a column of immobilized phosphomannosyl receptor. Uptake and binding were enhanced by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase, greatly reduced by prior treatment with alkaline phosphatase, and restored by subsequent treatment with alpha-N-acetylglucosaminyl phosphodiesterase. Immobilized phosphomannosyl receptor was used to separate high and low uptake enzyme forms. About 25% of the total beta-glucuronidase was retained by the receptor column and eluted with mannose 6-phosphate. The rate of uptake of retained enzyme was 2.5-3.0-fold greater than that of the enzyme applied to the receptor column. The fraction retained by the column was reduced to 5-10% by prior treatment of the enzyme with alkaline phosphatase. This phosphatase-resistant, receptor-retained fraction was taken up at only 24% the rate of non-phosphatase-treated, receptor-retained enzyme. However, its uptake was increased 7-fold by treatment with alpha-N-acetylglucosaminyl phosphodiesterase. The enhanced rate of pinocytosis conferred by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase was destroyed by a subsequent treatment with alkaline phosphatase. These studies demonstrate that although most of the "high uptake" enzyme in beta-glucuronidase from human spleen binds to receptors through phosphomonoesters of mannose, a significant fraction can interact with immobilized phosphomannosyl receptor and be taken up by fibroblasts through interactions involving mannose 6-phosphate in diester linkage with N-acetyl-D-glucosamine.     

Uptake and stability of human and bovine acid alpha-glucosidase in cultured fibroblasts and skeletal muscle cells from glycogenosis type II patients

Acid alpha-glucosidase (EC 3.2.1.20) was purified from human placenta and bovine testis by affinity chromatography using concanavalin A (conA) and Sephadex G 200. When added to the culture medium of human fibroblasts, the enzyme purified from bovine testis is taken up with a 200-fold higher efficiency than the enzyme from human placenta. Uptake of acid alpha-glucosidase from bovine testis is mediated by the mannose-6-phosphate receptor, whereas only a minor fraction of placental enzyme appears to be equipped with the mannose-6-phosphate recognition marker. Once internalized, both human and bovine acid alpha-glucosidase demonstrate a half-life of about 10 days in fibroblasts from control individuals and patients with different clinical forms of glycogenosis type II (Pompe's disease, acid alpha-glucosidase deficiency). Evidence is presented that the mannose-6-phosphate receptor is also present on the plasma membrane of the clonal myogenic skeletal muscle cell lines G8-1 and L6J1 (respectively from mouse and rat origin) and on cultured human skeletal muscle cells derived from a muscle biopsy. Addition of bovine testis acid alpha-glucosidase to skeletal muscle cell cultures from an adult patient with glycogenosis type II leads to complete correction of the enzyme deficiency.     

Effects of antimycin A and 2-deoxyglucose on secretion in human platelets. Differential inhibition of the secretion of acid hydrolases and adenine nucleotides

Adsorptive endocytosis of alpha-N-acetylglucosaminidase from human urine by isolated rat hepatocytes is inhibited by glycoproteins, polysaccharides and sugars that are known to bind to cell-surface receptors specific for either terminal galactose/N-acetylgalactosamine residues, terminal mannose residues or mannose 6-phosphate residues. Recognition of alpha-N-acetylglucosaminidase by a cell-surface receptor specific for terminal galactose/N-acetylgalactosamine residues is supported by the observations (a) that neuraminidase pretreatment of the enzyme enhances endocytosis, (b) that beta-galactosidase treatment decreases endocytosis and (c) that neuraminidase pretreatment of hepatocytes decreases alpha-N-acetylglucosaminidase endocytosis. Recognition of alpha-N-acetylglucosaminidase via receptors recognizing mannose 6-phosphate residues is lost after treatment of the enzyme with alkaline phosphatase and endoglucosaminidase H. The effect of endoglucosaminidase H supports the view that the mannose 6-phosphate residues reside in N-glycosidically linked oligosaccharide side chains of the high-mannose type. The weak inhibition of endocytosis produced by compounds known to interact with cell-surface receptors specific for mannose residues suggests that this recognition system plays only a minor role in the endocytosis of lysosomal alpha-N-acetylglucosaminidase by hepatocytes.     

Epithelial rat liver cells have cell surface receptors recognizing a phosphorylated carbohydrate on lysosomal enzymes

Receptor-mediated endocytosis of alpha-N-acetylglucosaminidase by cultured epithelial rat liver cells is inhibited by mannose, L-fucose and most effectively by mannose 6-phosphate. Endocytosis of alpha-N-acetylglucosaminidase is lost after treatment of the enzyme with alkaline phosphatase. These findings indicate that epithelial rat liver cells possess cell surface receptors that recognize a phosphorylated carbohydrate on alpha-N-acetylglucosaminidase, as was previously reported for cell surface receptors of human skin fibroblasts. Inhibition of alpha-mannosidase endocytosis by epithelial rat liver cells in the presence of mannose 6-phosphate and loss of enzyme endocytosis after treatment with alkaline phosphatase suggest that this enzyme is recognized by the same receptor.     

Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? evidence from I-cell disease

We have examined frozen liver tissue for N-acetylglucosamine-l-phosphotransferase, an enzyme required for the formation of the mannose 6-phosphate recognition marker of lysosomal enzymes. Using [β³²P]-UDPGlcNAc and placental β-hexosaminidase B as N-acetylglucosamine l-phosphate donor and acceptor, respectively, we were unable to find activity of the transferase in 100,000 × g membranes prepared from livers of patients with I-cell disease, whereas activity was readily observed in membranes from control livers stored under the same conditions. Yet the activity of several lysosomal enzymes (β-N-acetylglucosaminidase, β-glucuronidase, α-mannosidase and α-$\text{L}$-iduronidase) was comparable in liver tissue of I-cell patients and controls, and only β-galactosidase activity showed a marked reduction. These results suggest that in contrast to cultured skin fibroblasts, liver may be able to introduce into lysosomes acid hydrolases that lack the mannose 6-phosphate recognition marker.     

Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts

Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.     

Cation-independent mannose 6-phosphate and 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments

The distribution of the cation-independent mannose 6-phosphate and 78 kDa receptors was studied in postnuclear subcellular fractions from two rat liver cell lines. ELISA assays revealed that the mannose 6-phosphate receptor is enriched in the light buoyant Percoll fractions that contain Golgi structures and early endosomes. Most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient and smaller amounts in the endosomal fractions. The high-density compartment is denser than lysosomes, contains LAMP2 but not LIMPII or acid hydrolases, and is not disrupted with glycyl-l-phenylalanine 2-naphthylamide, a substrate for cathepsin C that selectively disrupts lysosomes. Immunofluorescence microscopy studies indicate no colocalization of the 78 kDa receptor with the mannose 6-phosphate receptor or LIMPII. Mannose 6-phosphate-independent endocytosed beta-glucuronidase was found in the lysosomal, the early and late endosomal fractions. These fractions were immunoadsorbed in columns containing antibodies against the 78 kDa receptor. Only the endocytosed beta-glucuronidase present in the early and late endosomal fractions is associated to immunoadsorbed vesicles. In these vesicles, LAMP2 was detected but no LIMPII or the mannose 6-phosphate receptor. Results obtained suggest that the 78 kDa receptor is found along the endocytic pathway, but in vesicles different from the cation-independent mannose 6-phosphate receptor.     

Mannose 6-phosphate-independent endocytosis of beta-glucuronidase. II. Purification of a cation-dependent receptor from bovine liver

A new binding protein, which recognizes a specific peptide sequence from pronase digested bovine beta-glucuronidase, has been isolated from bovine liver membranes. Prior work has shown that this peptide (IIIb2) contains a Ser-X-Ser sequence, where X might be a posttranslational modified Trp. This receptor was detergent-extracted from total bovine liver membranes and purified by affinity chromatography on a bovine beta-glucuronidase-Sepharose and a IIIb2 peptide-Sepharose column. Binding of bovine beta-glucuronidase to the isolated receptor requires divalent cations, and their presence was necessary to maintain the receptor-ligand complex. Only the peptide sequence containing the fraction IIIb2 was able to impair the binding of the bovine enzyme to the receptor, no other peptide from bovine beta-glucuronidase had an effect on binding. When analyzed by SDS-PAGE under reducing conditions, two bands were observed, a major band of 78 kDa and a faint band of 72 kDa. Rabbit antibodies against this binding protein revealed the presence of the 78 kDa protein in membranes from bovine liver, human and bovine fibroblasts. These antibodies impaired human fibroblasts endocytosis of the bovine but not of the human beta-glucuronidase, which is taken up by a 300 kDa receptor that recognizes phosphomannosyl moieties in the enzyme.