Role of LAMP-2 in lysosome biogenesis and autophagy

In LAMP-2-deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2-deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2-deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation.     

Acid hydrolases in early and late endosome fractions from rat liver.

We have examined the distribution of the cation-independent mannose 6-phosphate receptor and five acid hydrolases in early and late endosomes and a receptor-recycling fraction isolated from livers of estradiol-treated rats. Enrichment of mannose 6-phosphate receptor mass relative to that of crude liver membranes was comparable in membranes of early and late endosomes but was even greater in membranes of the receptor-recycling fraction. Enrichment of acid hydrolase activities (aryl sulfatase, N-acetyl-beta-glucosaminidase, tartrate-sensitive acid phosphatase, and cholesteryl ester acid hydrolase) and cathepsin D mass was also comparable in early and late endosomes but was considerably lower in the receptor-recycling fraction. The enrichment of two acid hydrolases, acid phosphatase and cholesteryl ester acid hydrolase, in endosomes was severalfold greater than that of the other three examined, about 40% of that found in lysosomes. Acid phosphatase and cholesteryl ester acid hydrolase were partially associated with endosome membranes, whereas cathepsin D was found entirely in the endosome contents. These findings raise the possibility that lysosomal enzymes traverse early endosomes during transport to lysosomes in rat hepatocytes and suggest that the greater enrichment of some acid hydrolases in endosomes is related to their association with endosome membranes. Despite the substantial enrichment of lysosomal enzymes in hepatocytic endosomes, we found that two, cholesteryl ester acid hydrolase and cathepsin D, did not degrade cholesteryl esters and apolipoprotein B-100 of endocytosed low density lipoproteins in vivo, presumably because they are inactive at the pH within endosomes.     

Enzymic studies of sulphatases in tissues of the normal human and in metachromatic leukodystrophy with multiple sulphatase deficiencies: arylsulphatases A, B and C, cerebroside sulphatase, psychosine sulphatase and steroid sulphatases

Abstract— Several sulphatases (arylsulphatases A, B and C, cholesterol sulphatase, dehydroepiandroster-one sulphatase, cerebroside sulphatase and psychosine sulphatase) were deficient in various tissues from two patients with a variant form of metachromatic leukodystrophy. Deficient activities of cerebroside sulphatase and psychosine sulphatase, using physiological substrates, in tissues from metachromatic leukodystrophy with multiple sulphatase deficiencies provided another example that these enzymes may be identical to arylsulphatase A. β-Galactosidase activity was reduced to about 30-50 per cent of normal in brain and liver. Other lysosomal enzyme activities were found to be normal or elevated five to eight times. Arylsulphatase B isolated from the liver of one patient was abnormal, with respect to pi (70) and enzyme kinetics. In mixing experiments with normal enzymes the reduced activities of arylsulphatases A. B and C, cerebroside sulphatase and steroid sulphatases were shown not to be due to the presence of endogenous inhibitors. No arylsulphatase A or B activity in the brain specimen from the patient with multiple sulphatase deficiencies could be detected on isoelectric focussing. In normal brain tissue arylsulphatase A had a pi of 4-6-4-8 while arylsulphatase B had a pi of 7-8 and 8-1. When 4-methylumbelliferyl sulphate was used as a substrate the elution patterns of normal brain and liver arylsulphatase B were more heterogeneous and showed more variation than that when p-nitrocatechol sulphate was used. Arylsulphatase C and steroid sulphatases (cholesterol sulphatase, dehydroepiandrosterone sulphatase and oes-trone sulphatase I were solubilized by the addition of lysolecithin and Triton X-100 and subjected to isoelectric focussing. The pi of cholesterol sulphatase, oestrone sulphatase and arylsulphatase C was 6-8, and the elution patterns of the activities of these enzymes were similar. The pattern of dehydroepiandrosterone sulphatase was more heterogeneous and two major peaks were observed at pi 6 5 and 70. Residual enzyme activities of arylsulphatase C and steroid sulphatases from the brain of the patient with multiple sulphatase activities were not detectable by isoelectric focussing. Simultaneous deficiencies of arylsulphatase C and steroid sulphatases plus isoelectric focussing findings in tissues suggest that these enzymes are closely related in regard to their function. The nature of the genetic defect in metachromatic leukodystrophy with multiple sulphatase deficiencies is discussed.     

Transport of surface mannose 6-phosphate receptor to the Golgi complex in cultured human cells

The cation-independent mannose 6-phosphate receptor (MPRCI) functions in the packaging of both newly made and extracellular lysosomal enzymes into lysosomes. The subcellular location of MPRCI reflects these two functions; receptor is found in the Golgi complex, in endosomes, and on the cell surface. To learn about the intracellular pathway followed by surface receptor and to study the relationship between the receptor pools, we examined the entry of the surface MPRCI into Golgi compartments that contain sialyltransferase. Sialic acid was removed from surface-labeled K562 cultured human erythroleukemia cells by neuraminidase treatment. When the cells were returned to culture at 37 degrees C, surface MPRCI was resialylated by the cells with a half-time of 1-2 h. Resialylation was inhibited by reduced temperature, a treatment that allows surface molecules to reach endosomes but blocks further transport. These results indicate that surface MPRCI is transported to the sialyltransferase compartment in the Golgi complex. After culture at 37 degrees C, a small fraction (10-20%) of the resialylated receptor was found on the cell surface. Because a similar fraction of the total receptor pool is found on the cell surface, it is likely that cell surface MPRCI mixes with the cellular pool after resialylation. These data also support the idea that extracellular and newly made lysosomal enzymes are transported to lysosomes through a common compartment.     

Phosphatidylinositol 3-kinase is not required for recycling of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network.

Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from the trans-Golgi network to endosomes, then return to the trans-Golgi network for another round of enzyme delivery. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized lysosomal enzymes to lysosomes. We used two independent assays of mannose 6-phosphate receptor trafficking to determine the precise step that is blocked by wortmannin. Using an assay that monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transport to the trans-Golgi network were not inhibited by 2 microM wortmannin. In addition, this concentration of drug had no effect on the transport of the mannose 6-phosphate receptor from late endosomes to the trans-Golgi network using a system that reconstitutes this transport process in cell extracts. Under the same conditions, wortmannin significantly inhibited the generation of mature cathepsin D. In addition, the structurally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi network transport reactions. These experiments demonstrate that the interruption in lysosomal enzyme targeting is most likely due to a wortmannin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosphatidylinositol 3-kinase in the equivalent transport process in Saccharomyces cerevisiae.     

The mannose 6-phosphate receptor and the biogenesis of lysosomes

Localization of the 215 kd mannose 6-phosphate receptor (MPR) was studied in normal rat kidney cells. Low levels of receptor were detected in the trans Golgi network, Golgi stack, plasma membrane, and peripheral endosomes. The bulk of the receptor was localized to an acidic, reticular-vesicular structure adjacent to the Golgi complex. The structure also labeled with antibodies to lysosomal enzymes and a lysosomal membrane glycoprotein (lgp120). While lysosome-like, this structure is not a typical lysosome that is devoid of MPRs. The endocytic marker alpha 2 macroglobulin-gold entered the structure at 37 degrees C, but not at 20 degrees C. With prolonged chase, most of the marker was transported from the structure into lysosomes. We propose that the MPR/lgp-enriched structure is a specialized endosome (prelysosome) that serves as an intermediate compartment into which endocytic vesicles discharge their contents, and where lysosomal enzymes are released from the MPR and packaged along with newly synthesized lysosomal glycoproteins into lysosomes.     

Differences in the endosomal distributions of the two mannose 6- phosphate receptors

Multiple immunolabeling of cryosections was performed to compare the subcellular distributions of the two mannose 6-phosphate receptors (MPRs) involved in the intracellular targeting of lysosomal enzymes: the cation-dependent (CD) and cation-independent (CI) MPR. In two cell types, the human hepatoma cell line HepG2 and BHK cells double transfected with cDNA's encoding for the human CD-MPR and CI-MPR, we found the two receptors at the same sites: the trans-Golgi reticulum (TGR), endosomes, electron-dense cytoplasmic vesicles, and the plasma membrane. In the TGR the two receptors colocalized and were concentrated to the same extent in the same HA I-adaptor positive coated buds and vesicles. Endosomes were identified by the presence of exogenous tracers. The two MPR codistributed to the same endosomes, but semiquantitative analysis showed a relative enrichment of the CI-MPR in endosomes containing many internal vesicles. Two endosomal subcompartments were discerned, the central vacuole and the associated tubules and vesicles (ATV). We found an enrichment of CD-MPR over CI- MPR in the ATV. Lateral segregation of the two receptors within the plane of membranes was also detected on isolated organelles. Double immunolabeling for the CD-MPR and the asialoglycoprotein receptor, which mainly recycles between endosomes and the plasma membrane, revealed that these two receptors were concentrated in different subpopulations of endosomal ATV. The small GTP-binding protein rab4, which has been shown to mediate recycling from endosomes to the plasma membrane, was localized at the cytosolic face of many endosomal ATV. Quantitative analysis of double-immunolabeled cells revealed only a limited codistribution of the MPRs and rab4 in ATV. These data suggest that the two MPRs exit the TGR via the same coated vesicles, but that upon arrival in the endosomes CD-MPR is more rapidly than CI-MPR, segregated into ATV which probably are destined to recycle MPRs to TGR.     

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.     

Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines

Phosphomannosyl residues present on lysosomal enzymes are specifically recognized by the mannose 6-phosphate receptor protein. This interaction results in the selective targeting of lysosomal enzymes to lysosomes. While this pathway is operative in many cell types, we have found four cultured cell lines that are deficient in the ability to bind lysosomal enzymes containing phosphomannosyl residues to their intracellular or surface membranes (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). These cells appear to segregate lysosomal enzymes by an alternate intracellular pathway. To determine the basis for the lack of mannose 6-phosphate receptor activity in these cell lines, we studied the biosynthesis of the receptor in receptor-positive (BW5147) and receptor-deficient (P388D1 and MOPC 315) cells. The cells were labeled with [2-3H]mannose or [35S]methionine and the receptor was immunoprecipitated with an antireceptor antiserum. BW5147 cells synthesize a receptor protein whose size increases after translation/glycosylation. MOPC 315 cells produce an apparently normal receptor and degrade it rapidly. P388D1 cells fail to synthesize any detectable receptor. The receptor from BW5147 and MOPC 315 cells is a glycoprotein with both high mannose and complex asparagine-linked oligosaccharides. The complex-type units become fully sialylated and remain so during long periods of chase.     

Mannose-6-phosphate receptors for lysosomal enzymes cycle between the Golgi complex and endosomes

We have examined the distribution of mannose-6-phosphate (Man6P) receptors (215 kD) for lysosomal enzymes in cultured Clone 9 hepatocytes at various times after the addition or removal of lysosomotropic weak bases (chloroquine or NH4Cl). Our previous studies demonstrated that after treatment with these agents, Man6P receptors are depleted from their sorting site in the Golgi complex and accumulate in dilated vacuoles that could represent either endosomes or lysosomes (Brown, W. J., E. Constantinescu, and M. G. Farquhar, 1984, J. Cell Biol., 99:320-326). We have now investigated the nature of these vacuoles by labeling NH4Cl-treated cells simultaneously with anti-Man6P receptor IgG and lysosomal or endosomal markers. The structures in which the immunolabeled receptors are found were identified as endosomes based on the presence of endocytic tracers (lucifer yellow and cationized ferritin). The lysosomal membrane marker, lgp120, was associated with a separate population of swollen vacuoles that did not contain detectable Man6P receptors. When cells were allowed to recover from weak base treatment, the receptors reappeared in the Golgi cisternae of most cells (approximately 90%) within approximately 20 min, indicating that as the intra-endosomal pH drops and lysosomal enzymes dissociate, the entire population of receptors rapidly recycles to Golgi cisternae. When NH4Cl-treated cells were allowed to endocytose Man6P, a competitive inhibitor of lysosomal enzyme binding, the receptors also recycled to the Golgi cisternae, suggesting that lysosomal enzymes can dissociate from the receptors under these conditions (high pH + presence of competitive inhibitor). From these results it can be concluded that the intracellular itinerary of the 215-kD Man6P receptor involves its cycling via coated vesicles between the Golgi complex and endosomes, ligand dissociation is both necessary and sufficient to trigger the recycling of Man6P receptors to the Golgi complex, and endosomes rather than secondary lysosomes represent the junction where endocytosed material and primary lysosomes carrying receptor-bound lysosomal enzymes meet.(ABSTRACT TRUNCATED AT 400 WORDS)     

2-difluoromethyloestrone 3-O-sulphamate, a highly potent steroid sulphatase inhibitor

Steroid sulphatase is a target enzyme of growing therapeutic importance. The synthesis and in vitro biological evaluation of three novel 2-substituted analogues of oestrone 3-O-sulphamate (EMATE), an established steroid sulphatase inhibitor, are described. One inhibitor, 2-difluoromethyloestrone 3-O-sulphamate (6), was found to have an IC50 of 100 pM and be some 90-fold more potent than EMATE in inhibiting steroid sulphatase activity in a placental microsomal preparation, rendering this agent the most potent steroidal STS inhibitor in vitro reported to date. Lowering of the pKa value of the leaving parent steroid phenol by the 2-difluoromethyl group during irreversible enzyme sulphamoylation most likely facilitates the potent inactivation of steroid sulphatase by (6). However, our preliminary molecular docking studies using the X-ray crystal structure of steroid sulphatase suggest that F.......H interactions between the 2-difluoromethyl group of (6) and hydrogen bond donor residues lining the catalytic site of STS might also contribute to the high potency observed for (6).     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.     

Polarized secretion of lysosomal enzymes: co-distribution of cation- independent mannose-6-phosphate receptors and lysosomal enzymes along the osteoclast exocytic pathway

The osteoclast is a polarized cell which secretes large amounts of newly synthesized lysosomal enzymes into an apical extracellular lacuna where bone resorption takes place. Using immunocytochemical techniques, we have localized the cation-independent mannose-6-phosphate (Man6P) receptor and lysosomal enzymes in this cell type in order to determine the expression and distribution of this receptor and its ligands. The results demonstrate that the osteoclast expresses large amounts of immunoreactive cation-independent Man6P receptors, despite the fact that most of the lysosomal enzymes it synthesizes are secreted. The lysosomal enzymes and the receptors are co-distributed along the exocytic pathway, i.e., the endoplasmic reticulum, including the perinuclear envelope, the Golgi stacks as well as numerous small transport vesicles that appear to fuse with the ruffled border membrane. Within the Golgi complex, the receptors and lysosomal enzymes were found distributed in two predominant patterns; (a) in all the cisternae, from cis to trans, or (b) predominantly in cis- and trans-Golgi cisternae, with the middle Golgi cisternae being unstained or depleted in antigen. This pattern suggests that enzymes and receptors traverse the Golgi from cis to trans and preferentially accumulate in cis- and in trans-cisternae. This study therefore suggests that, in the osteoclast, Man6P receptors are involved in the vectorial transport and targeting of newly synthesized lysosomal enzymes, presumably via a constitutive pathway, to the apical membrane where they are secreted into the bone-resorbing compartment. This mechanism could insure polarized secretion of lysosomal enzymes into the bone-resorbing lacuna.     

78 kDa receptor for Man6P-independent lysosomal enzyme targeting: biosynthetic transport from endoplasmic reticulum to "high-density vesicles"

Recent work has shown that the cation-independent mannose 6-phosphate and the 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments. While the mannose 6-phosphate receptor is enriched in the Percoll fractions that contain Golgi apparatus, most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient. This report presents the biosynthetic transport of the 78 kDa receptor. Newly synthesized 78 kDa receptor was transported to Golgi from endoplasmic reticulum with a half life of 5 min. From the Golgi apparatus, the receptor takes two routes; about 15-25% is transported to the plasma membrane, and the rest migrates to late endosomes, subsequently to prelysosomes and finally to the dense vesicles. The 78 kDa receptor starts appearing at the dense vesicles 120 min after biosynthesis and reaches a maximum of 40-50% of the total receptor. Treatment of cells with NH4Cl causes depletion of the receptor from the dense vesicles and prelysosomes and corresponding augmentation in endosomes and plasma membrane. These results suggest that the 78 kDa receptor cycles between compartments and that the dense vesicles seem to represent the most distal compartment in the biosynthetic pathway of this receptor.     

Identification of mannose 6-phosphate receptors in rabbit alveolar macrophages

Mannose 6-phosphate is an important recognition site involved in transport of newly synthesized lysosomal enzymes from the endoplasmic reticulum to lysosomes. The current study is the first demonstration of functional mannose phosphate receptors in macrophages. The receptor appears to be similar in many respects to that expressed in fibroblasts. Binding at 4 degrees C of a mannose-6-P-containing ligand, alpha-mannosidase from Dictyostelium discoideum, was specific and saturable (KD = 1.6 nM). In the presence of permeabilizing agents (saponin and digitonin), macrophage mannose-6-P receptors gave a distribution of 15-20% on the surface and 80-85% inside. Uptake studies gave a Kuptake value of 4.9 nM. Mannose-6-P, Hansenula holstii phosphomannan, and fructose 1-phosphate were effective inhibitors of alpha-mannosidase uptake. Inhibitors of mannose uptake, such as beta-glucuronidase, mannose-bovine serum albumin, fucose-bovine serum albumin, or mannan had no effect on alpha-mannosidase uptake. Likewise, an inhibitor (fucoidin) of the macrophage receptor which recognizes negatively charged proteins did not inhibit alpha-mannosidase uptake. Uptake was linear over 90 min and inhibited by chloroquine, suggesting that surface receptors recycle. These data demonstrate that macrophages contain receptors which specifically recognize mannose-6-P units and are distinct from the well characterized mannose receptors. The finding that the mannose-6-P receptors play a role at the surface, together with the fact that most of the receptors are intracellular (similar to the mannose receptor) suggests that both carbohydrate receptors play a regulatory role at the surface and intracellularly in transport of lysosomal enzymes.     

Subcellular Trafficking and Activity of Hyal‐1 and Its Processed Forms in Murine Macrophages

The hyaluronidase Hyal‐1 is an acid hydrolase that degrades hyaluronic acid (HA), a component of the extracellular matrix. It is often designated as a lysosomal protein. Yet few data are available on its intracellular localization and trafficking. We demonstrate here that in RAW264.7 murine macrophages, Hyal‐1 is synthesized as a glycosylated precursor that is only weakly mannose 6‐phosphorylated. Nevertheless, this precursor traffics to endosomes, via a mannose 6‐phosphate‐independent secretion/recapture mechanism that involves the mannose receptor. Once in endosomes, it is processed into a lower molecular mass form that is transported to lysosomes, where its activity could be detected using native gel zymography. Indeed, this activity co‐distributed with lysosomal hydrolases in the densest fraction of a self‐forming Percoll^TM density gradient. Moreover, it shifted toward the lower density region, in parallel with those hydrolases, when a decrease of lysosomal density was induced by the endocytosis of sucrose. Interestingly, the activity of the processed form of Hyal‐1 was largely underestimated when assayed by zymography after SDS‐PAGE and subsequent renaturation of the proteins, by contrast to the full‐length protein that could efficiently degrade HA in those conditions. These results suggest that noncovalent associations support the lysosomal activity of Hyal‐1.      

Identification of a membrane glycoprotein found primarily in the prelysosomal endosome compartment

Cells contain an intracellular compartment that serves as both the "prelysosomal" delivery site for newly synthesized lysosomal enzymes by the mannose 6-phosphate (Man6P) receptor and as a station along the endocytic pathway to lysosomes. We have obtained mAbs to a approximately 57-kD membrane glycoprotein, (called here plgp57), found predominantly in this prelysosomal endosome compartment. This conclusion is supported by the following results: (a) plgp57 was primarily found in a population of late endosomes that were located just distal to the 20 degrees C block site in the endocytic pathway to lysosomes (approximately 83% of the prelysosomes were positive for plgp57 but less than 5% of the early endosomes had detectable amounts of this marker); (b) plgp57 and the cation-independent (CI) Man6P receptor were located in many of the same intracellular vesicles; (c) plgp57 was found in the membranes of an acidic compartment; (d) immunoelectron microscopy showed that plgp57 was located in characteristic multilamellar- and multivesicular-type vacuoles believed to be prelysosomal endosomes; and (e) cell fractionation studies demonstrated that plgp57 was predominantly found in low density organelles which comigrated with late endosomes and CI Man6P receptors, and only approximately 10-15% of the antigen was found in high density fractions containing the majority of secondary lysosomes. These results indicate that plgp57 is a novel marker for a unique prelysosomal endosome compartment that is the site of confluence of the endocytic and biosynthetic pathways to lysosomes.     

Alzheimer's disease-related overexpression of the cation-dependent mannose 6-phosphate receptor increases Abeta secretion: role for altered lysosomal hydrolase distribution in beta-amyloidogenesis

Prominent endosomal and lysosomal changes are an invariant feature of neurons in sporadic Alzheimer's disease (AD). These changes include increased levels of lysosomal hydrolases in early endosomes and increased expression of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is partially localized to early endosomes. To determine whether AD-associated redistribution of lysosomal hydrolases resulting from changes in CD-MPR expression affects amyloid precursor protein (APP) processing, we stably transfected APP-overexpressing murine L cells with human CD-MPR. As controls for these cells, we also expressed CD-MPR trafficking mutants that either localize to the plasma membrane (CD-MPRpm) or to early endosomes (CD-MPRendo). Expression of CD-MPR resulted in a partial redistribution of a representative lysosomal hydrolase, cathepsin D, to early endosomal compartments. Turnover of APP and secretion of sAPPalpha and sAPPbeta were not altered by overexpression of any of the CD-MPR constructs. However, secretion of both human Abeta40 and Abeta42 into the growth media nearly tripled in CD-MPR- and CD-MPRendo-expressing cells when compared with parental or CD-MPRpm-expressing cells. Comparable increases were confirmed for endogenous mouse Abeta40 in L cells expressing these CD-MPR constructs but not overexpressing human APP. These data suggest that redistribution of lysosomal hydrolases to early endocytic compartments mediated by increased expression of the CD-MPR may represent a potentially pathogenic mechanism for accelerating Abeta generation in sporadic AD, where the mechanism of amyloidogenesis is unknown.     

Antibody to mannose 6-phosphate specific receptor induces receptor deficiency in human fibroblasts.

Polyclonal antibodies to the mannose 6-phosphate specific receptor from human liver inhibited the endocytosis of lysosomal enzymes in fibroblasts by greater than 95% and enhanced 3-20-fold the secretion of precursors of lysosomal enzymes in these cells. Exposing fibroblasts for 4 h to antibody resulted in loss of greater than 90% of the membrane-bound receptors. If fibroblasts were treated with the antibody in the presence of CBZ-Phe-Ala-CHN2, an inhibitor of lysosomal cysteine proteinases, the receptor and smaller degradation products are recovered in dense lysosomes. In treated cells 18-58% of total receptor-related polypeptides were recovered in dense lysosomes. In control cells less than 4% of the receptor was found in the lysosomal fraction. We conclude from these results that normally the receptor is spared from lysosomal degradation. When tagged with antibody, however, the receptor is transported into lysosomes and degraded. The loss of intracellular receptors involved in segregation of newly synthesized lysosomal enzymes indicates an exchange between the former and the plasma membrane-bound receptors.     

Lectins and traffic in the secretory pathway

Evidence is accumulating that intracellular animal lectins play important roles in quality control and glycoprotein sorting along the secretory pathway. Calnexin and calreticulin in conjunction with associated chaperones promote correct folding and oligomerization of many glycoproteins in the endoplasmic reticulum (ER). The mannose lectin ERGIC-53 operates as a cargo receptor in transport of glycoproteins from ER to Golgi and the homologous lectin VIP36 may operate in quality control of glycosylation in the Golgi. Exit from the Golgi of lysosomal hydrolases to endosomes requires mannose 6-phosphate receptors and exit to the apical plasma membrane may also involve traffic lectins. Here we discuss the features of these lectins and their role in glycoprotein traffic in the secretory pathway.