Some inflammatory mediators play an important role not only in the pathogenesis of the inflammatory pain, but also in that of neuropathic and visceral pain. We previously showed the antihyperalgesic effect of oATP, the inhibitor of the P2X7 receptors for the pro-nociceptive ATP, in experimental inflammation. Here we show the antihyperalgesic effect of oATP in mouse models of neuropathic and visceral pain, other than in a model of arthritic pain mimicking rheumatoid arthritis in humans. We also show that mice lacking P2X7 receptors (KO) are resistant to hyperalgesic thermal stimuli following the induction of arthritic, neuropathic and visceral pain. Local (injection into the right hind paw) pre-treatment with oATP is able to prevent the successive induction of ATP-dependent hyperalgesia in wild type mice. In addition, KO mice are not insensitive to intraplantar treatment with ATP. Our data suggest that, even if oATP is able to inhibit purinoceptors different from P2X7, the latter are the more important involved in pain transmission.

CP-452,759 and CP-470,947, two covalent [C-14]-labeled diarylsulfonylurea (DASU) inhibitors of the processing and release of IL-1 from human monocytes, have been synthesized. The radiosyntheses utilize [C-14]-labeled phosgene to prepare labeled isocyanates which are coupled with an epoxide sulfonamide to give the [C-14]-DASUs. These tools should allow future studies aimed at purifying and identifying the DASU binding protein involved in the inhibition of the processing and release of IL-1 from human monocytes. Copyright (C) 2002 John Wiley Sons, Ltd.

Glycoproteins containing O-linked N-acetylglucosamine (O-GlcNAc) are present in axons of Aplysia neurons (Gabel et al., 1989) and among transcription factors and other proteins in the nucleus of eukaryotic cells (Jackson and Tjian, 1988). A recently discovered pathway in neurons transports proteins through the axon and then into the nucleus (Ambron et al., 1992). If any of the axonal O-GlcNAc glycoproteins use this pathway, then the axon and the nucleus will have these glycoproteins in common. We addressed this issue by using galactosyltransferase and UDP-3H-galactose to label and identify the glycoproteins in three regions of Aplysia neurons: axoplasm, extruded from nerves; nuclei, isolated by manual dissection of single neurons; and cytoplasm, obtained after removal of nuclei. At least 21 glycoproteins were labeled by this procedure; several, at 200, 180, 83, 76, and 66 kDa, from the nucleus and axoplasm comigrated after SDS- PAGE. Radiolabeled galactosyl-N-acetylglucosaminitol was released from the glycoproteins by base/borohydride, thereby verifying the presence of O-GlcNAc. Comparison of the 83 kDa glycoprotein from the nucleus and axoplasm revealed that both were soluble, had multiple O-GlcNAcs, and were bound to WGA. Thus, the 83 kDa constituent is a good candidate to use the axonal transport/nuclear import pathway.

The role of glucosylated oligosaccharides in the biogenesis of the glycoprotein (G protein) of vesicular stomatitis virus was studied in PhaR2.7, a mouse lymphoma cell line deficient in glucosidase II activity. As expected, the great majority of cell-associated G protein remained glucosylated in PhaR2.7, and the G protein was rapidly deglucosylated in BW5147, the parental cell line. Despite these differences in glucosylation, the rates of G protein trimerization and transport to the cell surface were as rapid and efficient in the PhaR2.7 mutant as in BW5147. Surprisingly, greater than 73% of the oligosaccharides on G proteins recovered from released virions were complex-type units. The efficient processing of the G protein oligosaccharides coincided with the efficient removal of glucose residues from its oligosaccharides. After treatment with deoxynojirimycin, an inhibitor of endoplasmic reticulum (ER) glucosidases I and II, the total percentage of G protein-associated high mannose-type oligosaccharides increased more in the parental cells than in the mutant cells. Furthermore, when the G protein was retained in the ER of PhaR2.7 cells by depletion of the cellular ATP pools with carbonyl cyanide m-chlorophenylhydrazone, its oligosaccharides remained glucosylated. Under identical conditions, BW5147 cells removed the glucose residues from > 90% of the retained G protein's oligosaccharides. Thus, PhaR2.7 cells efficiently remove glucose residues from high mannose-type oligosaccharides of selected proteins using a deoxynojirimycin-insensitive enzyme located in a post-ER compartment. The existence of a second mechanism for the deglucosylation of N-linked oligosaccharides provides evidence for the important role of glucose removal in glycoprotein maturation.

Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum (Einstein, R., and C. A. Gabel. 1989. J. Cell Biol. 109:1037-1046). To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated. This difference in processing indicates that lysosomes themselves exist in a dephosphorylation-competent and -incompetent state. Man 6-P-bearing acid hydrolases endocytosed by the L+ cells in the absence of serum were not distributed uniformly throughout the lysosomal compartment. The change in the dephosphorylation competence of L cells in response to serum suggests, therefore, that these cells contain multiple populations of lysosomes that differ with respect to their content of a mannose 6-phosphatase, and that serum factors affect the distribution of hydrolases between the different compartments.

A 58-kDa Golgi protein (gp58) was previously identified and found to be concentrated in cis Golgi cisternae in several cell types (Saraste, J., Palade, G.E., and Farquhar, M.G. (1987) J. Cell Biol. 105, 2021-2029). In this study the protein was partially purified from rat pancreas and mouse myeloma cells in order to characterize its oligosaccharides. It migrated on sodium dodecyl sulfate-polyacrylamide gels as a 57-58-kDa doublet under reducing conditions or as a single approximately 116-kDa band under nonreducing conditions. Pancreatic gp58 was susceptible to alpha-N-acetylgalactosaminidase digestion and it bound concanavalin A, Helix pomatia, Dolichos biflorus, soybean agglutinin, and Bauhinia purpurea lectins, but not Ricinus communis agglutinin or lectins from Griffonia simplicifolia-1, Arachis hypogaea, and Limulus polyphemus. It bound Ricinus communis agglutinin after galactosylation with GlcNAc galactosyltransferase. These data demonstrate that pancreatic p58 contains immature N-linked moieties with nonreducing terminal GlcNAc residues as well as the initiating GalNAc of O-linked glycoproteins. Myeloma gp58 was sensitive to endo-beta-N-acetylglucosaminidase H, and oligosaccharide analysis of its [3H]glucosamine-labeled glycopeptides indicated that it also contained immature N-linked glycans. Some of the latter consist of high mannose chains (high affinity for concanavalin A, endo-beta-N-acetylglucosaminidase H-sensitive), but the predominant (95%) species are neutral tri- or tetraantennary N-linked chains containing GlcNAc (no binding to concanavalin A). Glycopeptides from biosynthetically labeled myeloma cells did not contain detectable base labile oligosaccharides, indicating that unlike pancreatic p58, myeloma gp58 may not be an O-linked glycoprotein. Neither pancreatic nor myeloma gp58 contained terminally processed oligosaccharides, indicating that gp58 has not been modified by trans-Golgi glycosyltransferases. Thus, the oligosaccharide content of gp58 is consistent with the assumption that this protein is retained in the cis Golgi cisternae during biosynthesis instead of being transported across the Golgi stacks and targeted back to the cis Golgi from the trans side.

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.

Cathepsin L, a lysosomal cysteine protease, is the major excreted protein of transformed mouse NIH 3T3 cells. Previous studies have shown that asparagine-linked oligosaccharides associated with the secreted hydrolase contain mannose 6-phosphate (Man 6-P), the recognition marker for transport of newly synthesized acid hydrolases to lysosomes. To investigate the mechanism by which cathepsin L evades targeting to lysosomes, we determined the structure of the enzyme's oligosaccharides and analyzed its interaction with the cation-independent mannose 6-phosphate (Man 6-PCl) receptor. Oligosaccharides associated with procathepsin L isolated from the medium of [3H]mannose-labeled J774 cells were remarkably homogeneous; all of the radiolabeled structures were high mannose-type units that contained two phosphomonoesters and 7 mannose residues. Both the alpha 1,3- and alpha 1,6-branches of the oligosaccharides were phosphorylated. Oligosaccharides released by endoglycosidase H from [3H]mannose-labeled procathepsin L bound to a Man 6-PCl receptor affinity column. Despite the high affinity binding of these oligosaccharides, the intact glycoprotein was not a good ligand for the Man 6-PCl receptor. Procathepsin L was internalized poorly by Man 6-P receptor-mediated endocytosis and the purified acid protease interacted weakly with a Man 6-PCl affinity column. In contrast, pro-beta-glucuronidase (another acid hydrolase produced by J774 cells) was an excellent ligand for the Man 6-PCl receptor as judged by the endocytosis and affinity chromatographic assays. Phosphorylated oligosaccharides associated with the J774-secreted pro-beta-glucuronidase were heterogeneous and contained both mono- and diphosphorylated species. Tryptic glycopeptides generated from [3H]mannose-labeled procathepsin L, unlike the intact protein, were excellent ligands for the Man 6-PCl receptor. The results indicate that oligosaccharides associated with procathepsin L are processed uniformly to diphosphorylated species that bind with high affinity to the Man 6-PCl receptor. Protein determinants inherent within the intact acid hydrolase, however, inhibit the high affinity binding of these oligosaccharides and, as a result, impair the interaction of procathepsin L with the receptor.

Deficient arylsulfatase-A activity is diagnostic of a neurodegenerative human lysosomal storage disease, metachromatic leukodystrophy. Paradoxically, similar enzyme deficiency also occurs in normal individuals, who are known as being pseudo arylsulfatase-A deficient. We showed previously that this phenotype is associated with a structural gene mutation that produces an exceptionally labile enzyme. We now report on the nature and consequence of this mutation. When the mutant arylsulfatase-A is deglycosylated by endoglycosidase H, only one smaller molecular species was generated, instead of the two from the normal enzyme. This is consistent with the loss of one of the two N-linked oligosaccharide side chains known to be present on the wild-type enzyme. Quantitative analysis of mannose and leucine incorporation showed that the mutant enzyme incorporated two- to tenfold less mannose than the normal enzyme on a molar basis. This deficient glycosylation was specific to arylsulfatase-A. Another lysosomal enzyme not affected in this mutation, beta-hexosaminidase, was glycosylated normally in the mutant cells. The remaining single oligosaccharide side chain released from the mutant arylsulfatase-A by pronase digestion was normally processed to complex and high-mannose forms. However, the high-mannose side chains contained 30% fewer phosphorylated residues than those of the normal enzyme. Nevertheless, this reduced level of phosphorylation did not prevent targeting of the mutant enzyme to the lysosomes, a process normally mediated through phosphorylated mannose residues. In conclusion, pseudo arylsulfatase-A deficiency is a unique human mutation associated with reduced glycosylation and phosphorylation of a lysosomal enzyme with the loss of one of the two carbohydrate side chains. The mutation results in greatly reduced enzyme stability, thus indicating a role for oligosaccharides in maintaining enzyme stability within the degradative environment of the lysosomes. However, the residual catalytic activity or subcellular targeting of the mutant enzyme was not affected. These properties probably account for the benign clinical presentation of pseudo arylsulfatase-A deficiency.

A key step in the sorting of endocytosed ligands from their receptors is dissociation, which is triggered by the acidic pH of endosomes. To determine whether dissociation occurs synchronously for all ligands, we compared in Chinese hamster ovary cells the intracellular dissociation of insulin, which dissociates between pH 6.3 and 7.0, with that of lysosomal hydrolases bearing the mannose 6-phosphate recognition marker (Man-6-P proteins), which dissociate around pH 5.8. Chinese hamster ovary cells were pulsed for 2 min with 125I-insulin, acid-washed to remove surface binding, and chased. During a 40-min period, about 50% of the internalized 125I-insulin was released intact via a retrocytotic pathway. Retrocytosis was not inhibited by monensin, suggesting that the release was not dependent on acidic endosomes. The remaining insulin dissociated from its receptor in an acidification-sensitive manner and was eventually degraded. Dissociation was 70% complete within 5 min of internalization. When cells were similarly incubated with 125I-Man-6-P proteins, about 35% of the internalized radioactivity was released during a 1-h chase, reflecting proteolytic maturation of the Man-6-P proteins. Dissociation of Man-6-P proteins was acidification-dependent (i.e. inhibited by monensin), and was 50% complete after about 11 min. The results indicate that acidification-dependent dissociation of ligands does not occur in a single step and suggest that multiple endocytic compartments are involved in receptor/ligand sorting.

lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines. Partial sequencing of mouse lgp110 allowed oligonucleotide probes to be constructed for the screening of several mouse cDNA libraries. A partial cDNA clone for mouse lgp110 was found and used for additional library screening, generating a cDNA clone covering all of the coding sequence of mature rat lgp110 as well as genomic clones covering most of the mouse gene. These new clones bring to seven the number of lysosomal membrane proteins whose amino acid sequences can be deduced, and two distinct but highly similar groups (designated lgp-A and lgp-B) can now be defined. Sequence comparisons suggest that differences within each group reflect species variations of the same protein and that lgp-A and lgp-B probably diverged from a common ancestor prior to the evolup4f1ary divergence of birds and mammals. Individual cells and individual lysosomes possess both lgp-A and lgp-B, suggesting that these two proteins have different functions. Mouse lgp110 is encoded by at least seven exons; intron positions suggest that the two homologous ectodomains of each lgp arose through gene duplication.

Mouse L-cells that contain the cation-independent (CI) mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor endocytose acid hydrolases and deliver these enzymes to lysosomes. The postendocytic loss of the Man 6-P recognition marker from the cell-associated acid hydrolases was assessed by CI-Man 6-P receptor affinity chromatography. 125I-labeled acid hydrolases internalized by L-cells grown at high density were delivered to lysosomes but were not dephosphorylated. In contrast, the same 125I-labeled hydrolases internalized by L-cells maintained at low density were delivered to lysosomes and were extensively dephosphorylated. The dephosphorylation at low density required 5 h for completion suggesting that the phosphatase responsible for the dephosphorylation is located within the lysosomal compartment. Transition from the high to low density state was rapid and was not inhibited by cycloheximide. Medium substitution experiments indicated that serum factors were necessary to maintain the L-cells in the dephosphorylation-competent (low density) state, and that serum-free conditions led to a dephosphorylation-incompetent (high density) state. Addition of IGF II to cells in serum-free medium allowed acid hydrolases subsequently introduced by endocytosis to be dephosphorylated. The results indicate that the removal of the Man 6-P recognition marker from endocytosed acid hydrolases is regulated by serum factors in the growth medium, including IGF II.

In the accompanying paper (Gabel, Den, and Ambron, in press) it was shown that eight populations of glycopeptides are synthesized by single neurons of Aplysia californica. To see which glycopeptides might mediate interactions with target cells, we first identified glycopeptides that are transported selectively to synapses and growth cones. The giant neuron R2 was injected intrasomatically with 3H-glucosamine. Twenty-four hours later, 3H-glycopeptides in the axon and cell body were isolated and resolved by serial lectin affinity chromatography. Of the eight populations, the biantennary-type glycopeptides (GPbi) and those that bind to WGA (GPwga) were preferentially associated with rapidly transported glycoproteins. In contrast, the glycopeptide that consists of N-acetylglucosamine O-linked to ser/thr was mostly retained in the cell body. GPbi and GPwga were also preferentially transported to growth cones. Analyses of RUQ cells, exposed to 3H-glucosamine in vitro for 36 h showed an enrichment of GPbi and GPwga at the growth cone relative to the cell body. The disposition of the various glycopeptides in growing neurons was also examined using FITC lectins. FITC-coupled WGA, Vicia vellosa, and lentil lectin showed extensive staining of the cell body, but only WGA stained the growth cones. To investigate if GPwga interacts specifically with target cells, these glycopeptides were isolated from the neurons of 180 abdominal ganglia. GPwga, other Aplysia glycopeptides, and glycopeptides prepared from ovalbumin were coupled separately to fluorescent spheres. The spheres were then added to muscle cells isolated from the auricle of the heart, which is innervated by many neurons from the ganglion. While spheres coupled to GPwga bound to the muscle cell surface, the other glycopeptides did not. These results indicate that glycopeptides class GPwga, found among rapidly transported glycoproteins and on the growth cone surface, is able to bind to muscle cells and may therefore play some role in neuron-target interactions.

Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H]mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides. Structural analysis, however, indicated that 60-78% of the 3H-mannose-labeled oligosaccharides contained a single glucose residue and no fewer than eight mannose residues. The 3H-labeled ts045 oligosaccharides were deglucosylated and processed to complex-type units after the infected cells were returned to the permissive temperature. When shifted to the permissive temperature in the presence of a proton ionophore, the G protein oligosaccharides were deglucosylated but remained as high mannose-type units. The glucosylated state was observed, therefore, when the G protein existed in an altered conformation. The ts045 G protein oligosaccharides were deglucosylated in vitro by glucosidase II at both the permissive and nonpermissive temperatures. G protein isolated from ts045-infected cells labeled with [6-3H]galactose in the presence of cycloheximide contained 3H-glucose-labeled monoglucosylated oligosaccharides, indicating that the high mannose oligosaccharides were glucosylated in a posttranslational process. These results suggest that aberrant G proteins are selectively modified by resident ER enzymes to retain monoglucosylated oligosaccharides.

The biosynthetic capabilities of individual neurons of the abdominal ganglion of the marine mollusc Aplysia californica have been analyzed after intrasomatic injection of 3H-monosaccharides. Glycopeptides prepared from the metabolically labeled cells were fractionated using serial lectin affinity and gel filtration chromatography. The fractionation procedure yielded eight populations of glycopeptides, and comparison of two different neurons (R2 and R14) showed that the quantity of the individual species produced is cell-dependent. Structural analysis indicated that the glycoconjugates produced by the Aplysia neuron constitute both O- and N-linked structures as well as an unusual class of oligosaccharide whose linkage to protein is unknown. The O-linked units are small and consist only of N-acetylglucosamine or N-acetylgalactosamine attached to protein. High-mannose-type asparagine-linked units are produced by the neurons, and some of these appear to be processed to biantennary complex-type units that bind to lentil lectin-agarose. Overall, although the Aplysia neurons produce oligosaccharides of a nature similar to that produced by higher eucaryotes, the N- and O-linked structures produced by the neurons do not achieve the complexity of the comparable structures produced by mammalian cells. The results provide a basis for further studies aimed at understanding the role of glycoconjugates in the development of the nervous system.

Phosphorylation of the high mannose-type oligosaccharides attached to newly synthesized acid hydrolases occurs in two sequential steps within the endoplasmic reticulum and the Golgi apparatus, and the products generated at the two sites differ with respect to the location of the phosphorylated mannose residue. To investigate the mechanism of this two-step phosphorylation, biosynthesis of the Man-6-P recognition marker was studied in class E Thy-1- and J774 cells metabolically labeled with [2-3H]mannose. Class E Thy-1- cells produce truncated high mannose oligosaccharides that lack 4 mannose residues from the alpha 1,6-branch of the core beta-linked mannose residue; three of the missing residues are potential phosphorylation sites. Acid hydrolases produced by these mutant cells were phosphorylated on the alpha 1,3-branch of the truncated oligosaccharide even when transport to the Golgi apparatus was inhibited. J774 cells produce normal high mannose oligosaccharides, but they secrete a large percentage of their newly synthesized acid hydrolases. The secreted enzymes contained primarily diphosphorylated units in which a phosphate was positioned to both the alpha 1,3- and alpha 1,6-branches of the core beta-linked mannose. J774 cells treated with deoxymannojirimycin continued to phosphorylate and to secrete acid hydrolases. The secreted hydrolases, however, contained only monophosphorylated oligosaccharides in which the phosphate was restricted to the alpha 1,6-branch. These results indicate that mannose residues within high mannose oligosaccharides impose constraints on the phosphorylation of their composite structures. We conclude that the two-step phosphorylation occurs as a result of a common phosphotransferase at both the pre-Golgi and Golgi locations and a change in the conformation of the oligosaccharides attached to the acid hydrolases through the action of Golgi-associated alpha-mannosidase I.

The biosynthesis of the mannose 6-phosphate recognition marker has been studied in transport-impaired mouse lymphoma cells to determine the subcellular location of the processing enzymes and to characterize the biosynthetic intermediates. Cells were labeled with [2-3H]mannose and chased at a low temperature (15 or 20 degrees C) or at 37 degrees C in the presence of m-chlorocarbonylcyanide phenylhydrazone to disrupt transport of the pulse-labeled molecules within the secretory apparatus. Both treatments inhibited the migration of the pulse-labeled glycoproteins to the Golgi apparatus as measured by the production of complex-type asparagine-linked oligosaccharides. Despite this inhibition in protein transport, acid hydrolases were phosphorylated. Structural analysis of the phosphorylated oligosaccharides indicated that the transport-impaired cells produced a single species of phosphorylated high mannose oligosaccharide; essentially all of the molecules contain a single phosphodiester group that is restricted to the alpha 1,6 branch of the oligosaccharide. The results suggest that synthesis of mannose 6-phosphate-bearing high mannose oligosaccharides occurs in an ordered, compartmentalized posttranslational process. The initial phosphorylation of newly synthesized acid hydrolases occurs at a pre-Golgi site and results in the production of high mannose-type units that contain a single phosphodiester group. In a subsequent compartment, probably within the Golgi apparatus, the monophosphorylated units may be converted to diphosphorylated forms. Finally, at a site distal to the phosphorylation reactions the diesters are hydrolyzed to reveal the mannose 6-phosphate recognition marker.

The 120-kDa lysosomal membrane glycoprotein (lgp120) is an acidic, heavily glycosylated membrane protein enriched in the lysosomal membrane. To determine the basis for its selective transport to and stability in lysosomes, we have investigated the structure of lgp120. By using an oligonucleotide probe corresponding to the amino terminus of rat lgp120, we isolated and characterized cDNA clones containing the entire coding region. The deduced amino acid sequence demonstrates that lgp120 contains a putative signal peptide, 18 sites for N-linked glycosylation, a single membrane-spanning segment, and a short (11 amino acid) cytosolic tail. The sequence suggests a distinct domain organization, with two luminal glycosylated regions separated by a nonglycosylated proline-rich region. Proteolysis in detergent showed that the protein was not intrinsically resistant to exogenous or endogenous proteases. The N-linked oligosaccharides on lgp120, tetraantennary structures with two lactosamine repeats on one of the branches, were not different from those of glycoproteins on the plasma membrane. lgp120 was similar in its domain organization and portions of its amino acid sequence to the avian 100-kDa lysosomal membrane protein LEP100 [Fambrough, D. M., Takeyasu, K., Lippincott-Schwartz, J., Siegel, N. R. & Somerville, D. (1988) J. Cell Biol. 106, 61-67], and to a distinct 110-kDa lysosomal membrane protein (lgp110) that colocalizes with lgp120. The similarities between lysosomal membrane glycoproteins from diverse species, coupled with the fact that at least two distinct lysosomal membrane glycoproteins are expressed in a single species, indicate the existence of a conserved family of glycoproteins enriched in the lysosomal membrane.

The mannose 6-phosphate (Man 6-P) receptor operates to transport both endogenous newly synthesized acid hydrolases and extracellular enzymes to the lysosomal compartment. In a previous study (Gabel, C. A., and S. A. Foster, 1986, J. Cell Biol., 103:1817-1827), we noted that beta-glucuronidase molecules internalized by mouse L-cells via the Man 6-P receptor undergo a proteolytic cleavage and a limited dephosphorylation. In this report, we present evidence that indicates that the postendocytic alterations of the acid hydrolase molecules occur at a site through which the enzymes pass en route to the lysosomal compartment. Mouse L-cells incubated at 20 degrees C with beta-glucuronidase (isolated from mouse macrophage secretions) internalize the enzyme in a process that is inhibited by Man 6-P but unaffected by cycloheximide. As such, the linear accumulation of the ligand observed at 20 degrees C appears to occur through the continued recycling of the cell surface Man 6-P receptor. The subcellular distribution of the internalized ligands was assessed after homogenization of the cells and fractionation of the extracts by density gradient centrifugation. In contrast to the accumulation of the ligand within lysosomes at 37 degrees C, the beta-glucuronidase molecules internalized by the L cells at 20 degrees C accumulate within a population of vesicles that sediment at the same density as endocytic vesicles. Biochemical analysis of the internalized ligands indicates that: (a) the subunit molecular mass of both beta-glucuronidase and beta-galactosidase decrease upon cell association relative to the input form of the enzymes, and (b) the beta-glucuronidase molecules experience a limited dephosphorylation such that high-mannose-type oligosaccharides containing two phosphomonoesters are converted to single phosphomonoester forms. The same two post-endocytic alterations occur after the internalization of beta-glucuronidase by human I-cell disease fibroblasts, despite the low acid hydrolase content of these cells. The results indicate, therefore, that acid hydrolases internalized via the Man 6-P receptor are processed within the endocytic compartment. In that endogenous newly synthesized acid hydrolases display similar alterations during their maturation, the results further suggest that the endosomal compartment is involved in the sorting of ligands transported via both the cell surface and intracellular Man 6-P receptor.