Basis for low affinity binding of a lysosomal cysteine protease to the cation-independent mannose 6-phosphate receptor

In cultured mouse fibroblasts, secretion of the lysosomal cysteine protease, MEP (major excreted protein) is regulated by growth factors and viral transformation. The ability of this protein to be regulated has been attributed to its intrinsic low affinity for the cation-independent mannose 6-phosphate (Man-6-P) receptor (Dong, J., Prence, E. M., and Sahagian, G. G. (1989) J. Biol. Chem. 264, 7377-7383). In this study, the basis for this low affinity was examined. Chromatography on a cation-independent Man-6-P receptor affinity matrix was used to assess relative affinities of Man-6-P-containing oligosaccharides and proteins, and the state of phosphorylation of the oligosaccharides was determined by ion exchange chromatography on QAE-Sephadex. MEP proteins synthesized by normal NIH 3T3 cells or NIH cells transformed with Kirsten sarcoma virus displayed a similar low affinity for the receptor and were found to possess oligosaccharide species with two phosphomonoester moieties. The affinity of these oligosaccharides for the receptor was the same as intact MEP protein and as great as phosphorylated oligosaccharides obtained from lysosomal proteins with the usual high affinity for the receptor. These results indicate that the polypeptide portion of MEP has no effect on binding of the protein to the receptor and that the difference in affinity of MEP and lysosomal proteins with high affinity cannot be attributed to differences in oligosaccharide structure. To investigate this further, we examined the binding characteristics of MEP made by CHO cells. In contrast to mouse MEP, CHO MEP bound to the receptor with high affinity. Partial endoglycosidase H treatment indicated that CHO MEP has two phosphorylated oligosaccharides, whereas the mouse protein has only one. Both oligosaccharides of the CHO cell protein contained two phosphomonoester moieties and displayed an affinity for the receptor that was indistinguishable from that of oligosaccharides of the mouse protein. Conversion of CHO MEP to a one-oligosaccharide species by partial endoglycosidase H treatment produced a protein that displayed low affinity binding similar to that of mouse MEP. A substantial portion of the pool of CHO cell lysosomal protein was also converted to a low affinity ligand by this treatment. Taken together, these results suggest that high affinity binding to the cation-independent receptor involves a divalent interaction with lysosomal proteins that contain two or more phosphorylated oligosaccharides, and that the low affinity of MEP results from an inability to form this multivalent interaction.     

Granzymes A and B are targeted to the lytic granules of lymphocytes by the mannose-6-phosphate receptor

To investigate the question of whether lytic granules share a common biogenesis with lysosomes, cloned cytolytic T cell lines were derived from a patient with I-cell disease. The targeting of two soluble lytic granule components, granzymes A and B, was studied in these cells which lack a functional mannose-6-phosphate (Man-6-P) receptor-mediated pathway to lysosomes. Using antibodies and enzymatic substrates to detect the lytic proteins, I-cells were found to constitutively secrete granzymes A and B in contrast to normal cells in which these proteins were stored for regulated secretion. These results suggest that granzymes A and B are normally targeted to the lytic granules of activated lymphocytes by the Man-6-P receptor. In normal cells, the granzymes bear Man-6-P residues, since the oligosaccharide side chains of granzymes A and B, as well as radioactive phosphate on granzyme A from labeled cells, were removed by endoglycosidase H (Endo H). However, in I-cells, granzymes cannot bear Man-6-P and granzyme B acquires complex glycans, becoming Endo H resistant. Although the levels of granzymes A and B in cytolytic I-cell lymphocytes are < 30% of the normal levels, immunolocalization and cell fractionation of granzyme A demonstrated that this reduced amount is correctly localized in the lytic granules. Therefore, a Man-6-P receptor-independent pathway to the lytic granules must also exist. Cathepsin B colocalizes with granzyme A in both normal and I-cells indicating that lysosomal proteins can also use the Man-6-P receptor-independent pathway in these cells. The complete overlap of these lysosomal and lytic markers implies that the lytic granules perform both lysosomal and secretory roles in cytolytic lymphocytes. The secretory role of lytic granules formed by the Man-6-P receptor-independent pathway is intact as assessed by the ability of I-cell lymphocytes to lyse target cells by regulated secretion.     

Endocytosis of mannose-6-phosphate binding sites by mouse T-lymphoma cells

The endocytosis and intracellular transport of mannose-6-phosphate conjugated to bovine serum albumin (Man-6-P:BSA) by mouse T-lymphoma cells were investigated in detail using several methods of analysis, both morphological and biochemical. Man-6-P:BSA was labeled with fluorescein or 125I and used to locate both surface and intracellular Man-6-P binding sites by light or electron microscopy, respectively. Incubation of cells with either fluorescent- or 125I-labeled Man-6-P:BSA at 0 degree C revealed a uniform distribution of the Man-6-P binding sites over the cell surface. Competition experiments indicate that the Man-6-P:BSA binding sites on the cell surface are the same receptors that can recognize lysosomal hydrolases. After as little as 1 min incubation at 37 degrees C, endocytosis of Man-6-P binding sites was clearly observed to occur through regions of the plasma membrane and via vesicles that also bound anticlathrin antibody. After a 5-15-min incubation of cells at 37 degrees C, the internalized ligand was detected first in the cis region of the Golgi apparatus and then in the Golgi stacks using both autoradiography and immunocytochemistry to visualize the ligand. The appearance of Man-6-P:BSA in the Golgi region after 15-30 min was confirmed by subcellular fractionation, which demonstrated an accumulation of Man-6-P:BSA in light membrane fractions that corresponded with the Golgi fractions. After a 30-min incubation at 37 degrees C, the internalized Man-6-P binding sites were localized primarily in lysosomal structures whose membrane but not lumen co-stained for acid phosphatase. These results demonstrate a temporal participation of clathrin-containing coated vesicles during the initial endocytosis of Man-6-P binding sites and that one step in the Man-6-P:BSA transport pathway between plasma membrane and the lysosomal structure can involve a transit through the Golgi stacks.     

Mechanism for selective secretion of a lysosomal protease by transformed mouse fibroblasts

Studies in recent years have indicated that secretion of certain lysosomal hydrolases can be enhanced under various conditions. One such protein, the major excreted protein (MEP) of Kirsten virus-transformed NIH 3T3 (KNIH) fibroblasts, is a lysosomal cysteine protease whose synthesis and secretion are affected by viral transformation and growth factors. We have been studying the synthesis and transport of MEP in order to understand better the mechanisms responsible for regulation of lysosomal enzyme secretion. Synthesis of MEP in KNIH cells was found to be 25-fold greater than that in untransformed NIH cells, and 94% of the MEP made was secreted. This was in contrast to NIH cells which secreted only 11% of the newly synthesized MEP. The high level of secretion by the transformed cells was relatively specific in that most other lysosomal enzymes were retained. MEP isolated from both NIH and KNIH cells exhibited a low intrinsic affinity for the mannose-6-phosphate receptor which was at least 10-fold lower than that of other lysosomal enzymes. On the basis of these results, we suggest that both the high level of MEP synthesis and the intrinsic low affinity of MEP for the receptor are responsible for the specific increase in MEP secretion by transformed cells.     

Demonstration of lysosomal localization for the mammalian ependymin-related protein using classical approaches combined with a novel density shift method

Most newly synthesized soluble lysosomal proteins are delivered to the lysosome via the mannose 6-phosphate (Man-6-P)-targeting pathway. The presence of the Man-6-P post-translational modification allows these proteins to be affinity-purified on immobilized Man-6-P receptors. This approach has formed the basis for a number of proteomic studies that identified multiple as yet uncharacterized Man-6-P glycoproteins that may represent new lysosomal proteins. Although the presence of Man-6-P is suggestive of lysosomal function, the subcellular localization of such candidates requires experimental verification. Here, we have investigated one such candidate, ependymin-related protein (EPDR). EPDR is a protein of unknown function with some sequence similarity to ependymin, a fish protein thought to play a role in memory consolidation and learning. Using classical subcellular fractionation on rat brain, EPDR co-distributes with lysosomal proteins, but there is significant overlap between lysosomal and mitochondrial markers. For more definitive localization, we have developed a novel approach based upon a selective buoyant density shift of the brain lysosomes in a mutant mouse lacking NPC2, a lysosomal protein involved in lipid transport. EPDR, in parallel with lysosomal markers, shows this density shift in gradient centrifugation experiments comparing mutant and wild type mice. This approach, combined with morphological analyses, demonstrates that EPDR resides in the lysosome. In addition, the lipidosis-induced density shift approach represents a valuable tool for identification and validation of both luminal and membrane lysosomal proteins that should be applicable to high throughput proteomic studies.     

Proteomics analysis of serum from mutant mice reveals lysosomal proteins selectively transported by each of the two mannose 6-phosphate receptors

Most mammalian cells contain two types of mannose 6-phosphate (Man-6-P) receptors (MPRs): the 300 kDa cation-independent (CI) MPR and 46 kDa cation-dependent (CD) MPR. The two MPRs have overlapping function in intracellular targeting of newly synthesized lysosomal proteins, but both are required for efficient targeting. Despite extensive investigation, the relative roles and specialized functions of each MPR in targeting of specific proteins remain questions of fundamental interest. One possibility is that most Man-6-P glycoproteins are transported by both MPRs, but there may be subsets that are preferentially transported by each. To investigate this, we have conducted a proteomics analysis of serum from mice lacking either MPR with the reasoning that lysosomal proteins that are selectively transported by a given MPR should be preferentially secreted into the bloodstream in its absence. We purified and identified Man-6-P glycoproteins and glycopeptides from wild-type, CDMPR-deficient, and CIMPR-deficient mouse serum and found both lysosomal proteins and proteins not currently thought to have lysosomal function. Different mass spectrometric approaches (spectral count analysis of nanospray LC-MS/MS experiments on unlabeled samples and LC-MALDI/TOF/TOF experiments on iTRAQ-labeled samples) revealed a number of proteins that appear specifically elevated in serum from each MPR-deficient mouse. Man-6-P glycoforms of cellular repressor of E1A-stimulated genes 1, tripeptidyl peptidase I, and heparanase were elevated in absence of the CDMPR and Man-6-P glycoforms of alpha-mannosidase B1, cathepsin D, and prosaposin were elevated in the absence of the CIMPR. Results were confirmed by Western blot analyses for select proteins. This study provides a comparison of different quantitative mass spectrometric approaches and provides the first report of proteins whose cellular targeting appears to be MPR-selective under physiological conditions.     

Identification of sites of mannose 6-phosphorylation on lysosomal proteins

Most newly synthesized soluble lysosomal proteins contain mannose 6-phosphate (Man-6-P), a specific carbohydrate modification that is recognized by Man-6-P receptors (MPRs) that direct targeting to the lysosome. A number of proteomic studies have focused on lysosomal proteins, exploiting the fact that Man-6-P-containing forms can be purified by affinity chromatography on immobilized MPRs. These studies have identified many known lysosomal proteins as well as many proteins not previously classified as lysosomal. The latter are of considerable biological interest with potential implications for lysosomal function and as candidates for lysosomal storage diseases of unknown etiology. However, a significant problem in interpreting the biological relevance of such proteins has been in distinguishing true Man-6-P glycoproteins from simple contaminants and from proteins associated with true Man-6-P glycoproteins (e.g. protease inhibitors and lectins). In this report, we describe a mass spectrometric approach to the verification of Man-6-phosphorylation based upon LC-MS of MPR-purified proteolytic glycopeptides. This provided a useful tool in validating novel MPR-purified proteins as true Man-6-P glycoproteins and also allowed identification of low abundance components not observed in the analysis of the total Man-6-P glycoprotein mixture. In addition, this approach allowed the global mapping of 99 Man-6-phosphorylation sites from 44 known lysosomal proteins purified from mouse and human brain. This information is likely to provide useful insights into protein determinants for this modification and may be of significant value in protein engineering approaches designed to optimize protein delivery to the lysosome in therapeutic applications such as gene and enzyme replacement therapies.     

The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis Golgi cisternae

Mannose-6-phosphate (Man-6-P) receptors for lysosomal enzymes were localized by immunocytochemistry in several secretory and adsorptive cell types using monospecific antireceptor antibodies. By immunofluorescence, the receptors were found in the Golgi region of polarized cells. When localized by immunoperoxidase at the electron microscope level, they were detected in Golgi cisternae, coated vesicles, endosomes, and lysosomes of all cell types examined (hepatocytes, exocrine pancreatic and epididymal epithelia). Within the Golgi complex, immunoreactive receptors were restricted in their distribution to one or two cisternae on the cis side of the Golgi stacks. They were not detected in trans Golgi or GERL cisternae. Based on their high concentration of Man-6-P receptors, we propose that the cis Golgi cisternae represent the site where the secretory and lysosomal pathways diverge: lysosomal enzymes bearing the Man-6-P recognition marker bind to Man-6-P receptors in this location and are delivered to endosomes and lysosomes via coated vesicles.     

Mannose-6-phosphate stimulates proliferation of neuronal precursor cells

The mitogenic signal function of mannose-6-phosphate (Man-6-P)/insulin-like growth factor II (IGF-II) receptors was studied in neuronal precursor cells from developing rat brain (E15). About 30% of the cellular Man-6-P/IGF-II receptors were present on the cell surface. Man-6-P and IGF-II stimulated DNA synthesis twofold and their effects were additive. Antibody 3637 to the Man-6-P/IGF-II receptor blocked the response to Man-6-P but not that to IGF-II. Other phosphorylated hexoses were also active. Fructose-1-phosphate was equally potent with Man-6-P, whereas glucose-6-phosphate was 5 times less potent. We conclude that Man-6-P-containing proteins and IGF-II act as mitogens in developing brain by interaction with the Man-6-P/IGF-II receptor and the IGF-I receptor, respectively.     

Processing and lysosomal localization of a glycoprotein whose secretion is transformation stimulated

The major excreted protein (MEP) of transformed mouse fibroblasts is a mannose 6-phosphate-containing glycoprotein whose synthesis and secretion are increased in malignantly transformed 3T3 cells and whose synthesis is increased by treatment of 3T3 cells with tumor promoters or growth factors. When pulse-labeled extracts from Kirsten virus-transformed NIH 3T3 (KNIH) cells were immunoprecipitated using an antibody against secreted MEP, one cellular protein was immunoprecipitated that had the same molecular weight and tryptic peptide map as the secreted protein. Pulse-chase labeling experiments showed that 50-60% of this 39,000-mol-wt form was secreted in transformed cells. Of the 40-50% remaining, approximately 5% was processed into two lower molecular weight forms (29,000 and 20,000) which are sequestered within the cell. Similar processing of these proteins was observed in the nontransformed parent NIH 3T3 (NIH) cells. However, in NIH cells, much less of the synthesized MEP was secreted. Measurements of steady-state levels of these three forms of cellular MEP by Western blot immunolocalization revealed approximately fourfold more MEP in KNIH cells than in NIH cells as well as differences in the relative distribution of MEP forms in transformed and nontransformed cells. Subcellular fractionation of KNIH cells on a Percoll gradient demonstrated a distribution of total MEP similar to that of several lysosomal enzymes. The light lysosomal/Golgi peak from these gradients contained both the precursor 39,000-mol-wt form of MEP and the 20,000-mol-wt form, whereas the heavy lysosomal peak was enriched in the 20,000-mol-wt form. The distribution of MEP forms was found to be similar in NIH cells except that the 29,000-mol-wt form was also seen to be enriched in the heavy lysosomal peak. This biochemical localization of MEP was confirmed by immunolocalization with light and electron microscopy. These data support the hypothesis that MEP is a lysosomal protein that is secreted by transformed 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.     

Identification of residues essential for carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor.

Two distinct mannose 6-phosphate (Man-6-P) receptors (MPRs), the cation-dependent MPR (CD-MPR) and the insulin-like growth factor II/MPR (IGF-II/MPR), recognize a diverse population of Man-6-P-containing ligands. The IGF-II/MPR is a type I transmembrane glycoprotein with a large extracytoplasmic region composed of 15 repeating domains that display sequence identity to each other and to the single extracytoplasmic domain of the CD-MPR. A structure-based sequence alignment of the two distinct Man-6-P-binding sites of the IGF-II/MPR with the CD-MPR implicates several residues of IGF-II/MPR domains 3 and 9 as essential for Man-6-P binding. To test this hypothesis single amino acid substitutions were made in constructs encoding either the N- or the C-terminal Man-6-P-binding sites of the bovine IGF-II/MPR. The mutant IGF-II/MPRs secreted from COS-1 cells were analyzed by pentamannosyl phosphate-agarose affinity chromatography, identifying four residues (Gln-392, Ser-431, Glu-460, and Tyr-465) in domain 3 and four residues (Gln-1292, His-1329, Glu-1354, and Tyr-1360) in domain 9 as essential for Man-6-P recognition. Binding affinity studies using the lysosomal enzyme, beta-glucuronidase, confirmed these results. Together these analyses provide strong evidence that the two Man-6-P-binding sites of the IGF-II/MPR are structurally similar to each other and to the CD-MPR and utilize a similar carbohydrate recognition mechanism.     

Estradiol down-regulates the mannose-6-phosphate/insulin-like growth factor-II receptor gene and induces cathepsin-D in breast cancer cells: a receptor saturation mechanism to increase the secretion of lysosomal proenzymes.

We have studied the regulation by estradiol of the mannose-6-phosphate (Man-6-P)/insulin-like growth factor-II (IGF-II) receptor concentration in different breast cancer cell lines. The mRNA level was assayed by Northern blot using the H5.1 cDNA probe. The protein level was assayed by Western ligand blot, by binding saturation with [125I]procathepsin-D on total membrane preparations, and by immunoprecipitation of 35S-labeled proteins. In three estrogen receptor-positive cell lines (MCF7, T47D, and ZR75-1), estradiol specifically decreased the steady state level of the Man-6-P/IGF-II receptor protein and mRNA. Moreover, in different cell lines and in primary culture of normal mammary cells, the secretion of procathepsin-D was inversely correlated with the level of Man-6-P/IGF-II receptor protein and mRNA. We conclude that estradiol down-regulates the Man-6-P/IGF-II receptor in breast cancer cells. Since two of its ligands, procathepsin-D and IGF-II, are induced by estrogen, we propose that the Man-6-P/IGF-II receptor becomes saturated after estrogen treatment. This model might explain the previously described estrogen-induced secretion of procathepsin-D and other lysosomal proenzymes routed by the same transport system.     

The endosomal concentration of a mannose 6-phosphate receptor is unchanged in the absence of ligand synthesis

The cation-independent mannose-6-phosphate (Man-6-P) receptor is involved in the targeting of newly synthesized lysosomal hydrolases. To investigate the intracellular distribution of this receptor, a conjugate of lactoperoxidase coupled to asialoorosomucoid was used to catalyze its iodination within the endosomes of human hepatoma (HepG2) cells. The 215-kD, cation-independent Man-6-P receptor was iodinated by this procedure as shown by pentamannosyl-6-phosphate-Sepharose affinity chromatography and by immunoprecipitation of labeled cell extracts. The amount of this receptor detected in endosomes was found to be unchanged after inhibition of protein synthesis with cycloheximide. If the Man-6-P receptor accumulates in the Golgi apparatus in the absence of lysosomal hydrolase synthesis, it should have been correspondingly depleted from endosomes after a period of cycloheximide treatment, because these pools of receptor are in rapid equilibrium. Therefore, these data suggest that newly synthesized ligands are not required for the transport of the cation-independent Man-6-P receptor from the Golgi apparatus to endosomes.     

Identification and validation of mannose 6-phosphate glycoproteins in human plasma reveal a wide range of lysosomal and non-lysosomal proteins

Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.     

Identification of a BALB/c-3T3 cell protein modulated by platelet-derived growth factor.

The platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize a protein (pII; Mr, 35,000) that is constitutively synthesized by spontaneously transformed BALB/c-3T3 (ST2-3T3) cells which do not require PDGF for growth. Antisera against a major excreted protein family (MEP) of retrovirus-transformed cells quantitatively precipitated cellular pII. PDGF-stimulated pII has the same molecular weight, a similar charge, and similar antigenic determinants as authentic MEP isolated from ST2-3T3 or retrovirus-transformed cells. MEP represented about 2% of the nonnuclear proteins synthesized by ST2-3T3 cells and 0.3 to 0.6% of the proteins synthesized by PDGF-treated BALB/c-3T3 cells, a three- to sixfold increase over the background. In BALB/c-3T3 cells, less PDGF was required for pII (MEP) synthesis than for DNA synthesis. PDGF induced a selective increase in pII (MEP) within 40 min. Such preferential synthesis was inhibited by brief treatment with actinomycin D, suggesting a requirement for newly formed RNA. The constitutive synthesis of pII (MEP) by ST2-3T3 cells was not inhibited by actinomycin D. Five spontaneously or chemical carcinogen-transformed tumorigenic BALB/c-3T3 cell lines were studied; they neither required PDGF for growth nor responded to it. These cell lines became arrested at confluence with a G1 DNA content. Each of these independently isolated lines synthesized pII (MEP) constitutively. Thus, the synthesis of pII (MEP) may be required, but is not sufficient, for PDGF-modulated DNA synthesis.     

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.     

Lysosomal Hydrolase Mannose 6-Phosphate Uncovering Enzyme Resides in the trans-Golgi Network

A crucial step in lysosomal biogenesis is catalyzed by "uncovering" enzyme (UCE), which removes a covering N-acetylglucosamine from the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal hydrolases. This study shows that UCE resides in the trans-Golgi network (TGN) and cycles between the TGN and plasma membrane. The cytosolic domain of UCE contains two potential endocytosis motifs: (488)YHPL and C-terminal (511)NPFKD. YHPL is shown to be the more potent of the two in retrieval of UCE from the plasma membrane. A green-fluorescent protein-UCE transmembrane-cytosolic domain fusion protein colocalizes with TGN 46, as does endogenous UCE in HeLa cells, showing that the transmembrane and cytosolic domains determine intracellular location. These data imply that the Man-6-P recognition marker is formed in the TGN, the compartment where Man-6-P receptors bind cargo and are packaged into clathrin-coated vesicles.     

Expression of the yeast aspartyl protease, proteinase A. Phosphorylation and binding to the mannose 6-phosphate receptor are altered by addition of cathepsin D sequences

Proteinase A, a yeast aspartyl protease that is highly homologous to the mammalian lysosomal aspartyl protease, cathepsin D, was expressed in Xenopus oocytes and its biosynthesis and post-translational modifications were characterized. While 29-45% of the proteinase A was secreted from oocytes, approximately 37% of the cell-associated proteinase A underwent proteolytic cleavage, characteristic of delivery to a lysosomal organelle. Although proteinase A is not targeted to the yeast vacuole by a mannose 6-phosphate receptor-dependent pathway, 2-5% of the proteinase A molecules expressed in oocytes bound to a Man-6-P receptor column. However, analysis of its [2-3H]mannose-labeled oligosaccharides revealed that 14-23% of these units contain phosphomannosyl residues. A hybrid molecule (H6), in which the propiece and first 12 amino acids of proteinase A were changed to the cathepsin D sequence, was also expressed in oocytes. The binding of H6 to the Man-6-P receptor was approximately 12-fold greater than observed for proteinase A. This increased level of receptor binding could be accounted for by three factors: 1) a small increase in the total amount of phosphorylated oligosaccharides, 2) an increase in the number of oligosaccharides which acquire two phosphomonoesters, and 3) the presence of a greater percentage of oligosaccharides with one phosphomonoester which exhibit high affinity binding to the Man-6-P receptor. These results demonstrate that proteinase A is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. However, this interaction is altered by the addition of cathepsin D sequences, resulting in the generation of a higher affinity ligand for binding to the Man-6-P receptor.