Studies of the biosynthesis of the cation-dependent mannose 6-phosphate receptor in murine cell lines

We have studied the biosynthesis of the cation-dependent mannose 6-phosphate receptor in murine BW5147 lymphoma cells and MOPC 315 plasmacytoma cells. The cells were labeled with [35S]methionine or [2-3H]mannose and the receptor immunoprecipitated with an anti-receptor antiserum. The receptor was first detected as a glycoprotein with an apparent molecular mass of 40 kDa. This intermediate was rapidly processed to a mature form which was stable during 22 h of chase. In these cells, the mature receptor has an apparent molecular mass of 43 kDa. The 3-kDa increase occurs as a result of processing of Asn-linked high-mannose oligosaccharides to complex-type units.     

Expression of human cation-independent mannose 6-phosphate receptor cDNA in receptor-negative mouse P388D1 cells following gene transfer

We recently reported the cDNA cloning, sequence, and expression of the human cation-independent mannose 6-phosphate receptor (hCI-MPR) (Oshima, A., Nolan, C. M., Kyle, J. W., Grubb, J. H., and Sly, W. S. (1988) J. Biol. Chem. 263, 2553-2562). The sequence of the hCI-MPR was virtually identical to that of the human insulin-like growth factor II receptor cDNA (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). To test the role of the putative bifunctional receptor in intracellular sorting of acid hydrolases, we studied its effect on lysosomal enzyme transport following gene transfer to receptor-negative cells. Receptor-negative mouse P388D1 cells were transfected with a cDNA construct containing the entire coding sequence of hCI-MPR under the control of the mouse metallothionine I promoter. Stable transformants were isolated and characterized. The expressed hCI-MPR was localized in membranes including the plasma membrane, bound mannose 6-phosphate containing ligands, and mediated endocytosis which could be specifically blocked by mannose 6-phosphate. We next measured the effect of the expressed hCI-MPR on intracellular and secreted acid hydrolases. The intracellular activity of the lysosomal marker enzymes beta-glucuronidase and beta-hexosaminidase increased up to 2-fold following transformation. In addition, expression of the receptor greatly reduced the fraction of acid hydrolases secreted. These phenotypic changes in the transformed cell lines support the proposed role of the cation-independent mannose 6-phosphate receptor in intracellular sorting and targeting of lysosomal enzymes.     

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.     

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.     

Receptor-mediated phagocytosis of zymosan is unaffected by some conditions which reduce macrophage lysosomal enzyme secretion

We have previously shown that several agents which interfere with binding of ligands to the mannose-glycoprotein receptor on macrophages can inhibit zymosan-induced lysosomal enzyme secretion. Here we show that mannose only reduces the association of zymosan with macrophages during the first hour of exposure; after longer periods of uptake no effect is detectable. We have previously shown that mannose reduces surface binding of zymosan, probably by interfering selectively with binding to the mannose receptor. The present inhibition of association of zymosan with macrophages during short exposures can be entirely explained by this reduction of binding. Macrophages must therefore internalize zymosan at sites in addition to the mannose receptor. In contrast to macrophages the murine macrophage-like cell line P388D1 is lacking the mannose-glycoprotein receptor. Accordingly we find that binding of zymosan to P388D1 is much slighter than to macrophages and is unaffected by mannose or mannose-6-phosphate. The spontaneous lysosomal enzyme secretion of P388D1 is also unaffected by mannose. The data on macrophages confirm our previous suggestion that agents interfering with the mannose receptor inhibit the induction of lysosomal enzyme secretion by acting directly on the receptor. The data on P388D1 cells support this assertion by excluding effects at later steps in the secretory pathway.     

Formation of lipid-linked oligosaccharides by MOPC 315 plasmacytoma cells. Decreased synthesis by a nonsecretory variant

MOPC 315 is a BALB/c plasmacytoma which secretes a trinitrophenol-binding IgA lambda 2 paraprotein. We have investigated the incorporation of [3H]mannose into lipid-linked oligosaccharide precursors in wild-type MOPC 315/J and variant nonsecretory 315/P cells. In pulse labeling experiments, no differences could be detected in the ability of the two cell types to incorporate [3H]mannose into lipid-linked oligosaccharides containing 5 or less mannose residues. In contrast, quantitation of the incorporation of [3H]mannose into larger lipid-linked oligosaccharides and proteins revealed a 49 and 40% decrease, respectively, in the 315/P cells compared to wild-type cells. Further characterization of the lipid-linked structures documented a marked decrease in glucosylated oligosaccharides isolated from 315/P cells. When membranes from the two cell lines were analyzed for their ability to transfer [3H]glucose from UDP-[3H]glucose to [3H]glucosylphosphoryldolichol, an apparent deficiency was noted in the 315/P preparations. However, if assay conditions were adjusted to include AMP in the reaction mixtures, no differences in the in vitro synthesis of [3H]glucosylphosphoryldolichol or [3H]glucose-labeled oligosaccharide-lipid could be detected. In these reactions AMP was found to prevent hydrolysis of UDP-[3H]glucose by inhibiting nucleotide pyrophosphatase (EC 3.6.1.9), the specific activity of which was determined to be more than 100 times greater in variant 315/P compared to wild-type MOPC 315/J cells. This large difference in specific activity was not accompanied by similar differences in the activity of several other enzymes analyzed. A decrease in whole cell UDP-glucose pool size was not detected in 315/P cells. Therefore, if nucleotide pyrophosphatase is important for the control of substrates for glycosylation, it must regulate nucleotide sugar levels at a site other than the cytoplasm of cells, perhaps at the location of synthesis of the larger lipid-linked oligosaccharides.     

Lysosomal enzyme oligosaccharide phosphorylation in mouse lymphoma cells: specificity and kinetics of binding to the mannose 6-phosphate receptor in vivo

Phosphomannosyl residues on lysosomal enzymes serve as an essential component of the recognition marker necessary for binding to the mannose 6-phosphate (Man 6-P) receptor and translocation to lysosomes. The high mannose-type oligosaccharide units of lysosomal enzymes are phosphorylated by the following mechanism: N-acetylglucosamine 1-phosphate is transferred to the 6 position of a mannose residue to form a phosphodiester; then N- acetylglucosamine is removed to expose a phosphomonoester. We examined the kinetics of this phosphorylation pathway in the murine lymphoma BW5147.3 cell line to determine the state of oligosaccharide phosphorylation at the time the newly synthesized lysosomal enzymes bind to the receptor. Cells were labeled with [2-(3)H]mannose for 20 min and then chased for various times up to 4 h. The binding of newly synthesized glycoproteins to the Man 6-P receptor was followed by eluting the bound ligand with Man 6-P. Receptor-bound material was first detected at 30 min of chase and reached a maximum at 60 min of chase, at which time approximately 10 percent of the total phosphorylated oligosaccharides were associated with the receptor. During longer chase times, the total quantity of cellular phosphorylated oligosaccharides decreased with a half-time of 1.4 h, suggesting that the lysosomal enzymes had reached their destination and had been dephosphorylated. The structures of the phosphorylated aligosaccharides of the eluted ligand were then determined and compared with the phosphorylated oligosaccharides of molecules which were not bond to the receptor. The major phosphorylated oligosaccharide species present in the nonreceptor-bound material contained a single phosphosphodiester at all time examined. In contrast, receptor-bound oligosaccharides were greatly enriched in species possessing one and two phosphomonoesters. These results indicate that binding of newly synthesized lysosomal enzymes to the Man 6-P receptor occurs only after removal of the covering N- acetylglucosamine residues.     

Renin, a secretory glycoprotein, acquires phosphomannosyl residues

Renin is an aspartyl protease which is highly homologous to the lysosomal aspartyl protease cathepsin D. During its biosynthesis, cathepsin D acquires phosphomannosyl residues that enable it to bind to the mannose 6-phosphate (Man-6-P) receptor and to be targeted to lysosomes. The phosphorylation of lysosomal enzymes by UDP- GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase (phosphotransferase) occurs by recognition of a protein domain that is thought to be present only on lysosomal enzymes. In order to determine whether renin, being structurally similar to cathepsin D, also acquires phosphomannosyl residues, human renin was expressed from cloned DNA in Xenopus oocytes and a mouse L cell line and its biosynthesis and posttranslational modifications were characterized. In Xenopus oocytes, the majority of the renin remained intracellular and underwent a proteolytic cleavage which removed the propiece. Most of the renin synthesized by oocytes was able to bind to a Man-6-P receptor affinity column (53%, 57%, and 90%, in different experiments), indicating the presence of phosphomannosyl residues. In the L cells, the majority of the renin was secreted but 5-6% of the renin molecules contained phosphomannosyl residues as demonstrated by binding of [35S]methionine- labeled renin to the Man-6-P receptor as well as direct analysis of [2- 3H]mannose-labeled oligosaccharides. Although the level of renin phosphorylation differed greatly between the two cell types examined, these results demonstrate that renin is recognized by the phosphotransferase and suggest that renin contains at least part of the lysosomal protein recognition domain.     

beta-Glucuronidase is transported slowly to lysosomes in BW5147 mouse lymphoma cells: evidence that the prelysosomal enzyme is not restricted to the endoplasmic reticulum

The post-translational processing of beta-glucuronidase in BW5147 mouse lymphoma cells is slow relative to other newly synthesized lysosomal enzymes. To characterize this slow maturation the acid hydrolase was immunoprecipitated from cells pulse-labeled with [2-3H]mannose. Radiolabeled beta-glucuronidase migrated as the precursor form of the enzyme for up to 4 h of chase, whereas another acid hydrolase, beta-galactosidase, was processed completely to its mature form within this same time period. Both beta-glucuronidase and beta-galactosidase obtained high levels of mannose 6-phosphate (Man 6-P) within 60 min of their biosynthesis. The Man 6-P content of beta-galactosidase declined rapidly during a subsequent chase while that of beta-glucuronidase remained high during the first 4 h of chase and then slowly declined. 3H-Labeled phosphorylated high mannose-type oligosaccharides isolated from beta-glucuronidase after 1 h of chase were composed primarily of species with one or two phosphodiester groups, but oligosaccharides with one and two phosphomonoesters became the predominant phosphorylated species with longer chase times. The phosphorylated oligosaccharides attached to other newly synthesized acid hydrolases, on the other hand, contained primarily phosphodiester species at all chase times. When BW5147 cells were pulsed with [3H]mannose and chased in the presence of monensin to disrupt transport, the number of phosphorylated oligosaccharides recovered from beta-glucuronidase was comparable to the quantity recovered from the enzyme produced by non-drug-treated cells. The number of phosphorylated units recovered from all other newly synthesized acid hydrolases, however, was greater in the presence of the ionophore than in its absence. Nondenaturing gel electrophoresis studies indicated that beta-glucuronidase existed in two forms at steady state within BW5147 cells and, as such, was similar to liver beta-glucuronidase in which a large percentage of the enzyme was present as a complex bound to egasyn. These data suggest that newly synthesized beta-glucuronidase produced by BW5147 cells complexes with an egasyn-like protein within the endoplasmic reticulum. This interaction retards the enzyme's migration through the secretory apparatus but does not prevent its access to Golgi-associated processing enzymes.     

Differential sorting of lysosomal enzymes in mannose 6-phosphate receptor-deficient fibroblasts.

In higher eukaryotes, the transport of soluble lysosomal enzymes involves the recognition of their mannose 6-phosphate signal by two receptors: the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). It is not known why these two different proteins are present in most cell types. To investigate their relative function in lysosomal enzyme targeting, we created cell lines that lack either or both MPRs. This was accomplished by mating CD-MPR-deficient mice with Thp mice that carry a CI-MPR deleted allele. Fibroblasts prepared from embryos that lack the two receptors exhibit a massive missorting of multiple lysosomal enzymes and accumulate undigested material in their endocytic compartments. Fibroblasts that lack the CI-MPR, like those lacking the CD-MPR, exhibit a milder phenotype and are only partially impaired in sorting. This demonstrates that both receptors are required for efficient intracellular targeting of lysosomal enzymes. More importantly, comparison of the phosphorylated proteins secreted by the different cell types indicates that the two receptors may interact in vivo with different subgroups of hydrolases. This observation may provide a rational explanation for the existence of two distinct mannose 6-phosphate binding proteins in mammalian cells.     

A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function.

The determinants on the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) required for lysosomal enzyme sorting have been analyzed. Mouse L cells deficient in the mannose 6-phosphate/insulin-like growth factor-II receptor were transfected with normal bovine CD-MPR cDNA or cDNAs containing mutations in the 67-amino acid cytoplasmic tail and assayed for their ability to target the lysosomal enzyme cathepsin D to lysosomes. Cells expressing the wild-type bovine CD-MPR sorted 67 +/- 2% of newly synthesized cathepsin D compared with the base-line value of 47 +/- 1%. The presence of mannose 6-phosphate in the medium did not affect the efficiency of cathepsin D sorting, indicating that the routing of the ligand-receptor complex is completely intracellular. Mutant receptors with the carboxyl-terminal His-Leu-Leu-Pro-Met67 residues deleted or replaced with alanines sorted cathepsin D below the base-line value. A mutant receptor with the outermost Pro-Met residues replaced with alanines sorted cathepsin D better than the wild-type receptor, indicating that the essential residues for sorting are the His-Leu-Leu sequence. Disruption of a putative casein kinase II phosphorylation site at Ser57 had no detectable effect on sorting. The mutant receptor with the five-amino acid deletion was able to bind to a phosphopentamannose affinity column, proving that its ligand binding site was grossly intact. Resialylation experiments showed that this mutant receptor recycled from the cell surface to the Golgi at a rate similar to the normal CD-MPR, indicating that the defect in sorting is at the level of the Golgi.     

The novel Drosophila lysosomal enzyme receptor protein mediates lysosomal sorting in mammalian cells and binds mammalian and Drosophila GGA adaptors

Biogenesis of lysosomes depends in mammalian cells on the specific recognition and targeting of mannose 6-phosphate-containing lysosomal enzymes by two mannose 6-phosphate receptors (MPR46, MPR300), key components of the extensively studied receptor-mediated lysosomal sorting system in complex metazoans. In contrast, the biogenesis of lysosomes is poorly investigated in the less complex metazoan Drosophila melanogaster. We identified the novel type I transmembrane protein lysosomal enzyme receptor protein (LERP) with partial homology to the mammalian MPR300 encoded by Drosophila gene CG31072. LERP contains 5 lumenal repeats that share homology to the 15 lumenal repeats found in all identified MPR300. Four of the repeats display the P-lectin type pattern of conserved cysteine residues. However, the arginine residues identified to be essential for mannose 6-phosphate binding are not conserved. The recombinant LERP protein was expressed in mammalian cells and displayed an intracellular localization pattern similar to the mammalian MPR300. The LERP cytoplasmic domain shows highly conserved interactions with Drosophila and mammalian GGA adaptors known to mediate Golgi-endosome traffic of MPRs and other transmembrane cargo. Moreover, LERP rescues missorting of soluble lysosomal enzymes in MPR-deficient cells, giving strong evidence for a function that is equivalent to the mammalian counterpart. However, unlike the mammalian MPRs, LERP did not bind to the multimeric mannose 6-phosphate ligand phosphomannan. Thus ligand recognition by LERP does not depend on mannose 6-phosphate but may depend on a common feature present in mammalian lysosomal enzymes. Our data establish a potential important role for LERP in biogenesis of Drosophila lysosomes and suggest a GGA function also in the receptor-mediated lysosomal transport system in the fruit fly.     

Fibroblast chemotaxis and prolidase activity modulation by insulin-like growth factor II and mannose 6-phosphate

Chemotactic locomotion of fibroblasts requires extensive degradation of extracellular matrix components. The degradation is provided by a variety of proteases, including lysosomal enzymes. The process is regulated by cytokines. The present study shows that mannose 6-phosphate and insulin-like growth factor II (IGF-II) enhance fibroblast chemotaxis toward platelet-derived growth factor (PDGF). It is suggested that lysosomal enzymes (bearing mannose 6-phosphate molecules) are involved in chemotactic activity of the cells. The suggestion is supported by the observation that a-mannosidase and cathepsin D inhibitor - pepstatin are very potent inhibitors of fibroblast chemotaxis. Simultaneously, mannose 6-phosphate stimulates extracellular collagen degradation. The final step in collagen degradation is catalyzed by the cytosolic enzyme - prolidase. It has been found that mannose 6-phosphate stimulates also fibroblast prolidase activity with a concomitant increase in lysosomal enzymes activity. The present study demonstrates that the prolidase activity in fibroblasts may reflect the chemotactic activity of the cells and suggests that the mechanism of cell locomotion may involve lysosomal enzyme targeting, probably through IGF-II/mannose 6-phosphate receptor.     

Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo.     

In vitro mutagenesis of potential N-glycosylation sites of arylsulfatase A. Effects on glycosylation, phosphorylation, and intracellular sorting.

The correct intracellular sorting of lysosomal enzymes such as arylsulfatase A depends on the presence of mannose 6-phosphate residues on high mannose type oligosaccharides. The arylsulfatase A cDNA contains three potential N-glycosylation sites, two of which are utilized. We have mutated one or two of the N-glycosylation sites and analyzed the glycosylation, phosphorylation, and intracellular sorting of the mutant arylsulfatase A polypeptides. The results show that each of the three glycosylation sites (I, II, and III) can be glycosylated, but glycosylation at sites I and II is mutually exclusive. In mutants with one oligosaccharide side chain at positions I, II, or III all side chains can acquire mannose 6-phosphate residues irrespective of their location. This demonstrates spatial flexibility of the phosphotransferase, which specifically recognizes lysosomal enzymes and initiates the addition of mannose 6-phosphate residues on oligosaccharide side chains. However, these mutants have different intracellular sorting efficiencies and seem to use different (mannose 6-phosphate receptor-dependent and -independent) sorting pathways.     

Strategies for carbohydrate recognition by the mannose 6-phosphate receptors

The two members of the P-type lectin family, the 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) and the 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR), are ubiquitously expressed throughout the animal kingdom and are distinguished from all other lectins by their ability to recognize phosphorylated mannose residues. The best-characterized function of the MPRs is their ability to direct the delivery of approximately 60 different newly synthesized soluble lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) on their N-linked oligosaccharides to the lysosome. In addition to its intracellular role in lysosome biogenesis, the CI-MPR, but not the CD-MPR, participates in a number of other biological processes by interacting with various molecules at the cell surface. The list of extracellular ligands recognized by this multifunctional receptor has grown to include a diverse spectrum of Man-6-P-containing proteins as well as several non-Man-6-P-containing ligands. Recent structural studies have given us a clearer view of how these two receptors use related, but yet distinct, approaches in the recognition of phosphomannosyl residues.     

Mouse lymphoma cell lines resistant to pea lectin are defective in fucose metabolism

Two mutants of the BW5147 mouse lymphoma cell line have been selected for their resistance to the toxic effects of pea lectin. These cell lines, termed PLR1.3 and PHAR1.8 PLR7.2, have a decreased number of high affinity pea lectin-binding sites (Trowbridge, I.S., Hyman, R., Ferson, T., and Mazauskas, C. (1978) Eur. J. Immunol. 8, 716-723). Intact cell labeling experiments using [2-3H]mannose indicated that PLR1.3 cells have a block in the conversion of GDP-[3H]mannose to GDP-[3H]fucose whereas PHAR1.8 PLR7.2 cells appear to be blocked in the transfer of fucose from GDP-[3H]fucose to glycoprotein acceptors. In vitro experiments with extracts of PLR1.3 cells confirmed the failure to convert GDP-mannose to GDP-fucose and indicated that the defect is in GDP-mannose 4,6-dehydratase (EC 4.2.1.47), the first enzyme in the conversion of GDP-mannose to GDP-fucose. The block in the PLR1.3 cells could be bypassed by growing the cells in the presence of fucose, demonstrating that an alternate pathway for the production of GDP-fucose presumably via fucose 1-phosphate is functional in this line. PLR1.3 cells grown in 10 mM fucose showed normal high affinity pea lectin binding. PHRA1.8 PLR7.2 cells synthesize GDP-fucose and have normal or increased levels of GDP-fucose:glycoprotein fucosyltransferase when assayed in vitro. The fucosyltransferases of this clone can utilize its own glycoproteins as fucose acceptors in in vitro assays. These findings indicate that this cell line fails to carry out the fucosyltransferase reaction in vivo despite the fact that it possesses the appropriate nucleotide sugar, glycoprotein acceptors, and fucosyltransferase. The finding of decreased glycoprotein fucose in two independent isolates of pea lectin-resistant cell lines and the restoration of high affinity pea lectin binding to PLR1.3 cells following fucose feeding strongly implicates fucose as a major determinant of pea lectin binding.     

Targeted disruption of the mouse cation-dependent mannose 6-phosphate receptor results in partial missorting of multiple lysosomal enzymes.

In mammalian cells two mannose 6-phosphate receptors (MPRs) are involved in lysosomal enzyme transport. To understand the precise function of the cation-dependent mannose 6-phosphate receptor (CD-MPR), one allele of the corresponding gene has been disrupted in mouse embryonic stem cells and homozygous mice lacking this receptor have been generated. The homozygous mice appear normal, suggesting that other targeting mechanisms can partially compensate for the loss of the CD-MPR in vivo. However, homozygous receptor-deficient cells and animals clearly exhibit defects in targeting of multiple lysosomal enzymes when compared with wild-types. Increased levels of phosphorylated lysosomal enzymes were present in body fluids of homozygous animals. In thymocytes from homozygous mice or in primary cultures of fibroblasts from homozygous embryos, there is a marked increase in the amount of phosphorylated lysosomal enzymes that are secreted into the extracellular medium. The cultured fibroblasts have decreased intracellular levels of multiple lysosomal enzymes and accumulate macromolecules within their endosomal/lysosomal system. Taken together, these results clearly indicate that the CD-MPR is required for efficient intracellular targeting of multiple lysosomal enzymes.     

Effects of brefeldin A on the endocytic route. Redistribution of mannose 6-phosphate/insulin-like growth factor II receptors to the cell surface.

The effect of brefeldin A (BFA) on the trafficking of the mannose 6-phosphate/insulin-like growth factor II receptor within the endocytic route was analyzed. Treatment with BFA induced a redistribution of the receptor to the cell surface and increased both the binding and internalization of ligands 2-4-fold. The effect of BFA was dose- and time-dependent and reversible. Determinations of transport rates showed that BFA increases the internalization rate and the externalization rate of the receptor. This implies that the higher surface concentration is due to higher concentrations of receptor at the intracellular sites from where they recycle to the cell surface. The effect of BFA was additive to the redistribution induced by insulin-like growth factors I and II and was observed in all human and rodent cell lines analyzed. BFA increased also the cell surface expression of the Mr 46,000 mannose 6-phosphate receptor but not of the transferrin receptor. The results indicate that BFA interferes with the transport of mannose 6-phosphate receptors and affects the endocytosis of lysosomal enzymes by increasing the number of receptors available for recycling to the cell surface.     

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.