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.     

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.     

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.     

The mannose 6-phosphate glycoprotein proteome

Most luminal lysosomal proteins are synthesized as precursors containing mannose 6-phosphate (Man6-P) and a number of recent studies have conducted affinity purification of Man6-P containing proteins as a step toward defining the composition of the lysosome. Approximately 60 known lysosomal proteins have been found in such studies as well as many other Man-6-P glycoproteins, some of which represent new lysosomal proteins. The latter are of considerable interest from cell-biological and biomedical perspectives, but differentiating between them and other proteins remains a significant challenge. The aim of this study was to conduct a global analysis of the mammalian Man6-P glycoproteome, implementing technical and biostatistical methods to aid in the discovery and validation of lysosomal candidates. We purified Man6-P glycoproteins from 17 individual rat tissues. To distinguish nonspecific contaminants (i.e., abundant or "sticky" proteins that are not fully removed during purification) from specifically purified proteins, we conducted a semiquantitative mass spectrometric comparison of protein levels in nonspecific mock eluates versus specific affinity chromatography eluates to identify those proteins that are specifically purified. We identified 60 known lysosomal proteins, representing nearly all that are currently known to contain Man-6-P. We also find 136 other proteins that are specifically purified but which are not known to have lysosomal function. This approach provides a list of candidate lysosomal proteins and also provides insights into the relative distribution of Man6-P glycoproteins.     

The human urine mannose 6-phosphate glycoproteome

Glycoproteins containing the mannose 6-phosphate (Man-6-P) modification represent a class of proteins of considerable biomedical importance. They include over sixty different soluble lysosomal hydrolases and accessory proteins, deficiencies of which result in over forty different known human genetic diseases. In addition, there are patients with lysosomal storage diseases of unknown etiology and lysosomal proteins have been implicated in pathophysiological processes associated with Alzheimer disease, arthritis, and cancer. The aim of this study was to explore urine as a source for the proteomic investigation of lysosomal storage disorders as well as for biomarker studies on the role of Man-6-P containing proteins in other human diseases. To this end, urinary proteins were affinity purified on immobilized Man-6-P receptors, digested with trypsin, and analyzed using nanospray LC/MS/MS. This resulted in identification of 67 proteins, including 48 known lysosomal proteins and 9 proteins that may be lysosomal. The identification of a large proportion of the known set of soluble lysosomal proteins with relatively few contaminants suggests that urine represents a promising substrate for the development of comparative proteomic methods for the investigation of lysosomal disorders and other diseases involving Man-6-P glycoproteins.     

Domain 5 of the cation-independent mannose 6-phosphate receptor preferentially binds phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester)

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46 kDa cation-dependent MPR (CD-MPR) are key components of the lysosomal enzyme targeting system that bind newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and divert them from the secretory pathway. Previous studies have mapped two high-affinity Man-6-P binding sites of the CI-MPR to domains 1-3 and 9 and one low-affinity site to domain 5 within its 15-domain extracytoplasmic region. A structure-based sequence alignment predicts that domain 5 contains the four conserved residues (Gln, Arg, Glu, Tyr) identified as essential for Man-6-P binding by the CD-MPR and domains 1-3 and 9 of the CI-MPR. Here we show by surface plasmon resonance (SPR) analyses of constructs containing single amino acid substitutions that these conserved residues (Gln-644, Arg-687, Glu-709, Tyr-714) are critical for carbohydrate recognition by domain 5. Furthermore, the N-glycosylation site at position 711 of domain 5, which is predicted to be located near the binding pocket, has no influence on the carbohydrate binding affinity. Endogenous ligands for the MPRs that contain solely phosphomonoesters (Man-6-P) or phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester, Man-P-GlcNAc) were generated by treating the lysosomal enzyme acid alpha-glucosidase (GAA) with recombinant GlcNAc-phosphotransferase and uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase). SPR analyses using these modified GAAs demonstrate that, unlike the CD-MPR or domain 9 of the CI-MPR, domain 5 exhibits a 14-18-fold higher affinity for Man-P-GlcNAc than Man-6-P, implicating this region of the receptor in targeting phosphodiester-containing lysosomal enzymes to the lysosome.     

The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins

The lysosome is a membrane delimited cytoplasmic organelle that contains at least 50 hydrolytic enzymes and associated cofactors. The biomedical importance of these enzymes is highlighted by the many lysosomal storage disorders that are associated with mutations in genes encoding lysosomal proteins, and there is also evidence that lysosomal activities may be involved in more widespread human diseases. The aim of this study was to characterize the human brain lysosomal proteome with the goal of establishing a reference map to investigate human diseases of unknown etiology and to gain insights into the cellular function of the lysosome. Proteins containing mannose 6-phosphate (Man6-P), a carbohydrate modification used for targeting resident soluble lysosomal proteins to the lysosome, were affinity-purified using immobilized Man6-P receptor. Fractionation by two-dimensional electrophoresis resolved a complex mixture comprising approximately 800 spots. Constituent proteins in each spot were identified using a combination of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (both peptide mass fingerprinting and tandem mass spectrometry) [corrected] on in-gel tryptic digests and N-terminal sequencing. In a complementary analysis, we also analyzed a tryptic digest of the unfractionated mixture by liquid chromatography MS/MS. In total, 61 different proteins were identified. Seven were likely contaminants associated with true Man6-P glycoproteins. Forty-one were known lysosomal proteins of which 11 have not previously been reported to contain Man6-P. An additional nine proteins were either uncharacterized or proteins not previously reported to have lysosomal function. We found that the human brain Man6-P-containing lysosomal proteome is highly complex and contains more proteins with a much greater number of individual isoforms than found in previous studies of Man6-P glycoproteomes.     

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.     

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.     

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.     

Recognition of Dictyostelium discoideum lysosomal enzymes is conferred by the amino-terminal carbohydrate binding site of the insulin-like growth factor II/mannose 6-phosphate receptor

The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) is a type I glycoprotein that mediates both the intracellular sorting of lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) residues to the lysosome and the bioavailability of IGF-II. The extracytoplasmic region of the IGF-II/MPR contains 15 repeating domains; the two carbohydrate recognition domains (CRDs) have been localized to domains 1-3 and 7-9, and the high-affinity IGF-II binding site maps to domain 11. To characterize the carbohydrate binding properties of the IGF-II/MPR, regions of the receptor encompassing the individual CRDs were produced in a baculovirus expression system. Characterization of the recombinant proteins revealed that the pH optimum for carbohydrate binding is significantly more acidic for the carboxyl-terminal CRD than for the amino-terminal CRD (i.e., pH 6.4-6.5 vs 6.9). Equilibrium binding studies demonstrated that the two CRDs exhibit a similar affinity for Man-6-P. Furthermore, substitution of the conserved arginine residue in domain 3 (R435) or in domain 9 (R1334) with alanine resulted in a similar >1000-fold decrease in the affinity for the lysosomal enzyme, beta-glucuronidase. In contrast, the two CRDs differ dramatically in their ability to recognize the distinctive modifications (i.e., mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes: the amino-terminal CRD binds mannose 6-sulfate and Man-6-P methyl ester with a 14-55-fold higher affinity than the carboxyl-terminal CRD. Taken together, these results demonstrate that the IGF-II/MPR contains two functionally distinct CRDs.     

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.     

Extending the Mannose 6-Phosphate Glycoproteome by High Resolution/Accuracy Mass Spectrometry Analysis of Control and Acid Phosphatase 5-Deficient Mice

In mammals, most newly synthesized lumenal lysosomal proteins are delivered to the lysosome by the mannose 6-phosphate (Man6P) targeting pathway. Man6P -containing proteins can be affinity-purified and characterized using proteomic approaches, and such studies have led to the discovery of new lysosomal proteins and associated human disease genes. One limitation to this approach is that in most cell types the Man6P modification is rapidly removed by acid phosphatase 5 (ACP5) after proteins are targeted to the lysosome, and thus, some lysosomal proteins may escape detection. In this study, we have extended the analysis of the lysosomal proteome using high resolution/accuracy mass spectrometry to identify and quantify proteins in a combined analysis of control and ACP5-deficient mice. To identify Man6P glycoproteins with limited tissue distribution, we analyzed multiple tissues and used statistical approaches to identify proteins that are purified with high specificity. In addition to 68 known Man6P glycoproteins, 165 other murine proteins were identified that may contain Man6P and may thus represent novel lysosomal residents. For four of these lysosomal candidates, (lactoperoxidase, phospholipase D family member 3, ribonuclease 6, and serum amyloid P component), we demonstrate lysosomal residence based on the colocalization of fluorescent fusion proteins with a lysosomal marker.The lysosome is a key site within the cell for the digestion of macromolecules, including proteins, carbohydrates, lipids, and nucleic acids (1), and this catabolic function is enabled by the concerted action of numerous hydrolases that have evolved to function in the acidic environment of the lysosome. Most lysosomal hydrolases and their accessory proteins are soluble and located within the lumen of the lysosome, and to date, ∼70 such proteins have been identified (2, 3). Mutations in the genes encoding more than 30 of these proteins result in lysosomal storage diseases in which unhydrolyzed substrates accumulate within lysosomes, disrupting cellular function and frequently resulting in cell death (32).Most newly synthesized soluble, lumenal lysosomal proteins are directed to the lysosome via the mannose 6-phosphate (Man6P)¹ pathway (4). Here, select N-linked carbohydrates receive the Man6P modification that is recognized by two Man6P receptors (MPRs) that direct the vesicular trafficking of the lysosomal proteins from the trans-Golgi to an acidified prelysosomal compartment. In most cell types, the Man6P modification is rapidly removed by acid phosphatase 5 (ACP5) (5) upon arrival within the lysosome.The presence of Man6P provides a unique opportunity for the study of lysosomal proteins by allowing for the highly specific affinity purification of this class of proteins from complex mixtures using MPRs immobilized on a solid affinity support (6). Man6P-containing lysosomal proteins have been purified from a wide variety of biological sources, and their characterization by proteomic methods has provided valuable insights into the composition and function of the lysosome as well as the roles of lysosomal proteins in human disease (2, 7). In mammals, the tissue distribution of lysosomal proteins can be highly restricted, and thus one approach that has effectively increased proteome coverage has been to analyze multiple individual tissues (3).One limitation to the MPR affinity purification approach is that in most cells and tissues, a relatively small fraction of lysosomal proteins contain Man6P (8) because of natural dephosphorylation by ACP5 (5), with the notable exception of brain (9). As a result, minor lysosomal components or those that are particularly susceptible to dephosphorylation, either in vivo or during the course of purification, may be overlooked in proteomic surveys. In addition, as the proportion of Man6P glycoproteins in the complex mixture of proteins of an initial tissue extract becomes smaller, the proportion of contaminants is likely to increase in the purified preparation. In practical terms, extensive dephosphorylation of lysosomal proteins can make purification from limiting tissue sources in whole organism surveys a difficult or impossible prospect.In this study, we have extended the proteomic analysis of mammalian Man6P glycoproteins with an analysis of multiple tissues from the mouse. We address the problem of dephosphorylation of lysosomal proteins with the inclusion of multiple tissues from ACP5-deficient mice (10), and we apply high resolution/accuracy mass spectrometry to confidently identify components of the mixture. Sixty eight known Man6P glycoproteins (65 lysosomal and 3 nonlysosomal) are identified, which is comparable with earlier studies. However, the modified approach has also resulted in the identification of 165 other murine proteins that are specifically purified by MPR affinity chromatography and that may potentially represent lysosomal proteins. This is the most extensive list obtained to date for candidate mannose 6-phosphorylated proteins, and we demonstrate that four of these proteins localize to the lysosome using morphological methods.     

Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome. The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity ( approximately 1 nm) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9. Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR. A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket. To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris. Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate). Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was approximately 300-fold lower (K(i) = 5.3 mm) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme beta-glucuronidase was also much lower (K(d) = 54 microm) as determined by surface plasmon resonance analysis. Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9.     

Subcellular localization of mannose 6-phosphate glycoproteins in rat brain.

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification of N-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.     

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.     

Mechanisms for high affinity mannose 6-phosphate ligand binding to the insulin-like growth factor II/mannose 6-phosphate receptor

The two mannose 6-phosphate (Man-6-P) binding domains of the insulin-like growth factor II/mannose 6-phosphate receptor (Man-6-P/IGF2R), located in extracytoplasmic repeats 1-3 and 7-9, are capable of binding Man-6-P with low affinity and glycoproteins that contain more than one Man-6-P residue with high affinity. High affinity multivalent ligand binding sites could be formed through two possible mechanisms: the interaction of two Man-6-P binding domains within one Man-6-P/IGF2R molecule or by receptor oligomerization. To discriminate between these mechanisms, truncated FLAG epitope-tagged Man-6-P/IGF2R constructs, containing one or both of the Man-6-P binding domains, were expressed in 293T cells, and characterized for binding of pentamannose phosphate-bovine serum albumin (PMP-BSA), a pseudoglycoprotein bearing multiple Man-6-P residues. A construct containing all 15 repeats of the Man-6-P/IGF2R extracytoplasmic domain bound PMP-BSA with the same affinity as the full-length receptor (K(d) = 0.54 nm) with a curvilinear Scatchard plot. The presence of excess unlabeled PMP-BSA increased the dissociation rate of pre-formed (125)I-PMP-BSA/receptor complexes, suggesting negative cooperativity in multivalent ligand binding and affirming the role of multiple Man-6-P/IGF2R binding domains in forming high affinity binding sites. Truncated receptors containing only one Man-6-P binding domain and mutant receptor constructs, containing an Arg(1325) --> Ala mutation that eliminates binding to the repeats 7-9 binding domain, formed high affinity PMP-BSA binding, but with reduced stoichiometries. Collectively, these observations suggest that alignment of Man-6-P binding domains of separate Man-6-P/IGF2R molecules is responsible for the formation of high affinity Man-6-P binding sites and provide functional evidence for Man-6-P/IGF2R oligomerization.     

Latency-associated peptide of transforming growth factor-β1 is not subject to physiological mannose phosphorylation

Latent TGF-β1 was one of the first non-lysosomal glycoproteins reported to bear mannose 6-phosphate (Man-6-P) residues on its N-glycans. Prior studies have suggested that this sugar modification regulates the activation of latent TGF-β1 by allowing it to bind cell surface-localized Man-6-P receptors. Man-6-P has also been proposed as an anti-scarring therapy based on its ability to directly block the activation of latent TGF-β1. A complete understanding of the physiological relevance of latent TGF-β1 mannose phosphorylation, however, is still lacking. Here we investigate the degree of mannose phosphorylation on secreted latent TGF-β1 and examine its Man-6-P-dependent activation in primary human corneal stromal fibroblasts. Contrary to earlier reports, minimal to no Man-6-P modification was found on secreted and cell-associated latent TGF-β1 produced from multiple primary and transformed cell types. Results showed that the inability to detect Man-6-P residues was not due to masking by the latent TGF-β1-binding protein (LTBP). Moreover, the efficient processing of glycans on latent TGF-β1 to complex type structures was consistent with the lack of mannose phosphorylation during biosynthesis. We further demonstrated that the conversion of corneal stromal fibroblast to myofibroblasts, a well known TGF-β1-dependent process, was not altered by Man-6-P addition when latent forms of this growth factor were present. Collectively, these findings indicate that Man-6-P-dependent effects on latent TGF-β1 activation are not mediated by direct modification of its latency-associated peptide.     

Structural insights into the mechanism of pH-dependent ligand binding and release by the cation-dependent mannose 6-phosphate receptor

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a key component of the lysosomal enzyme targeting system that binds newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and transports them to endosomal compartments. The interaction between the MPRs and its ligands is pH-dependent; the homodimeric CD-MPR binds lysosomal enzymes optimally in the pH environment of the trans Golgi network (pH approximately 6.5) and releases its cargo in acidic endosomal compartments (相似文献