Glycoprotein phosphorylation in simple eucaryotic organisms. Identification of UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase activity and analysis of substrate specificity

UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase activity has been identified in both Acanthamoeba castellani and Dictyostelium discoideum. Each of these activities exhibits a different in vitro specificity toward various purified glycoproteins. The N-acetylglucosaminyl-phosphotransferase of A. castellani is very similar to the mammalian enzyme in that it phosphorylates the lysosomal enzymes cathepsin D and uteroferrin much more efficiently than nonlysosomal glycoproteins and appears to recognize a determinant on the protein portion of these good acceptors. In contrast the D. discoideum enzyme cannot utilize cathepsin D as a good substrate and, although it phosphorylates uteroferrin efficiently, it does not recognize the protein portion of this acceptor. The oligosaccharide of uteroferrin appears to assume a different conformation than the oligosaccharides of other glycoproteins and glycopeptides, as evidenced by its enhanced sensitivity to mannosidase digestion. This conformation, presumably induced by some interaction with the underlying protein, may be responsible for the specific phosphorylation of uteroferrin by the N-acetylglucosaminylphosphotransferase of D. discoideum.     

Lysosomal enzyme phosphorylation. Recognition of a protein-dependent determinant allows specific phosphorylation of oligosaccharides present on lysosomal enzymes

We have investigated the basis for the specific recognition of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. This enzyme is responsible for the selective phosphorylation of mannose residues on lysosomal enzymes. Two mammalian lysosomal enzymes, cathepsin D and uteroferrin, and two nonlysosomal glycoproteins were treated with endo-beta-N-acetylglucosaminidase H to remove those high mannose oligosaccharide units which are accessible on the native protein. These proteins were then tested as inhibitors of three different glycosyltransferases. The endo H-treated lysosomal enzymes were shown to be specific inhibitors of the phosphorylation of intact lysosomal enzymes. Proteolytic fragments of cathepsin D, including the entire light chain and heavy chain, did not retain the ability to be recognized by the N-acetylglucosaminylphosphotransferase. These findings indicate that the intact protein portion of lysosomal enzymes contains a specific recognition determinant which leads to high-affinity binding to the N-acetylglucosaminylphosphotransferase. The expression of this determinant appears to be dependent on the conformation of the protein.     

Purification of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii and identification of a subunit of the enzyme.

UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) from the soil amoeba Acanthamoeba castellanii has been purified over 100,000-fold by means of wheat germ agglutinin-Sepharose affinity chromatography, DEAE-cellulose chromatography, concanavalin A-Sepharose affinity chromatography, orange A-agarose dye chromatography, and gel filtration on Superose 6. The most purified enzyme has an estimated specific activity of at least 5 mumol of GlcNAc-phosphate transferred/min/mg of protein using alpha-methylmannoside as acceptor. The molecular weight of the native enzyme is approximately 250,000, as determined by gel filtration and glycerol gradients in H2O and D2O. A protein with an apparent M(r) of 97,000 in small scale preparations and its putative proteolytic fragment of 43,000 in large scale preparations co-purifies with the enzyme activity. This protein is covalently modified with GlcNAc-[32P]phosphate when the enzyme preparation is incubated with [beta-32P]UDP-GlcNAc in the absence of an acceptor substrate. The labeling of the 97(43)-kDa protein requires active enzyme and is completely inhibited by the addition of the acceptor substrate alpha-methylmannoside. The GlcNAc-[32P]phosphate transferred to the protein is not bound to serine, threonine, tyrosine, or mannose residues. The 97(43)-kDa protein with covalently bound GlcNAc-P does not serve as a kinetically competent enzyme-substrate intermediate. However, preincubation of GlcNAc-phosphotransferase with UDP-GlcNAc does result in a decrease in the Vmax of the enzyme in subsequent assays. Taken together, these data are consistent with the 97(43)-kDa protein being a subunit of GlcNAc-phosphotransferase.     

The alpha- and beta-subunits of the human UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase [corrected] are encoded by a single cDNA

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an alpha2beta2gamma2-subunit structure was proposed. Although cDNA encoding the gamma-subunit has been described, cDNAs for the alpha- and beta-subunits have not. Using partial amino acid sequences from the bovine alpha- and beta-subunits, we have isolated a human cDNA that encodes both the alpha- and beta-subunits. Both subunits contain a single predicted membrane-spanning domain. The alpha- and beta-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the alpha/beta-subunits-precursor cDNA with or without the gamma-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the alpha-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.     

Role of N-linked oligosaccharide flexibility in mannose phosphorylation of lysosomal enzyme cathepsin L

Mannose phosphorylation of N-linked oligosaccharides by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is a key step in the targeting of lysosomal enzymes in mammalian cells and tissues. The selectivity of this process is determined by lysine-based phosphorylation signals shared by lysosomal enzymes of diverse structure and function. By introducing new glycosylation sites at several locations on the surface of mouse procathepsin L and modeling oligosaccharide conformations for sites that are phosphorylated, it was shown that the inherent flexibility of N-linked oligosaccharides can account for the specificity of the transferase for oligosaccharides at different locations on the protein. By using this approach, the physical relationship between the lysine-based signal and the site of phosphorylation of mannose residues was determined. The analysis also revealed the existence of additional independent lysine-based phosphorylation signals on procathepsin L, which account for the low level of phosphorylation observed when the primary Lys-54/Lys-99 signal is ablated. Mutagenesis of residues that surround Lys-54 and Lys-99 and demonstration of mannose phosphorylation of a glycosylated derivative of green fluorescent protein provide strong evidence that the cathepsin L phosphorylation signal is a simple structure composed of as few as two well placed lysine residues.     

Glucose-1-phosphotransferase and N-acetylglucosamine-1-phosphotransferase have distinct acceptor specificities

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha Glc-1-P from UDP-Glc to endoglycosidase H-sensitive oligosaccharides on acceptor glycoproteins. We have previously demonstrated that Glc-phosphotransferase was specific for UDP-Glc as its nucleotide sugar substrate and thus appeared to be distinct from UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase), an enzyme specific for lysosomally destined acceptor glycoproteins. Here, sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs of endogenous acceptor glycoproteins in embryonic chick neural retina homogenates labeled by the presence of [beta-32P]UDP-Glc were shown to be distinct from those labeled by [beta-32P]UDP-GlcNAc, indicating that the two enzymatic activities recognize different populations of endogenous glycoproteins. To further probe the acceptor specificities of these enzymes, three glycoproteins known to be exogenous acceptors for GlcNAc-phosphotransferase were included in assays for Glc-phosphotransferase from retinal homogenates. Cathepsin D and beta-N-acetylhexosaminidase had no significant effects on phosphoglucose incorporation. Uteroferrin, an acid phosphatase, had a pronounced inhibitory effect on incorporation from UDP-Glc, and subsequent experiments suggested that phosphorylation of the Glc-phosphotransferase or another protein may be necessary for maximal activity to be seen. Also, I-cells, which have previously been shown to possess no GlcNAc-phosphotransferase activity, and control human fibroblasts were assayed for both Glc-phosphotransferase and GlcNAc-phosphotransferase. GlcNAc-phosphotransferase activity was observed only in control cells, whereas Glc-phosphotransferase was observed in both I-cells and controls at similar specific activities.     

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.     

Mucolipidosis II (I-cell disease) and mucolipidosis IIIA (classical pseudo-hurler polydystrophy) are caused by mutations in the GlcNAc-phosphotransferase alpha / beta -subunits precursor gene

Mucolipidosis II (MLII; I-cell disease) and mucolipidosis IIIA (MLIIIA; classical pseudo-Hurler polydystrophy) are diseases in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent or reduced, respectively. In the absence of mannose phosphorylation, trafficking of lysosomal hydrolases to the lysosome is impaired. In these diseases, mistargeted lysosomal hydrolases are secreted into the blood, resulting in lysosomal deficiency of many hydrolases and a storage-disease phenotype. To determine whether these diseases are caused by mutations in the GlcNAc-phosphotransferase alpha / beta -subunits precursor gene (GNPTAB), we sequenced GNPTAB exons and flanking intronic sequences and measured GlcNAc-phosphotransferase activity in patient fibroblasts. We identified 15 different mutations in GNPTAB from 18 pedigrees with MLII or MLIIIA and demonstrated that these two diseases are allelic. Mutations in both alleles were identified in each case, which demonstrated that GNPTAB mutations are the cause of both diseases. Some pedigrees had identical mutations. One frameshift mutation (truncation at amino acid 1171) predominated and was found in both MLII and MLIIIA. This mutation was found in combination with severe mutations (i.e., mutations preventing the generation of active enzyme) in MLII and with mild mutations (i.e., mutations allowing the generation of active enzyme) in MLIIIA. Some cases of MLII and MLIIIA were the result of mutations that cause aberrant splicing. Substitutions were inside the invariant splice-site sequence in MLII and were outside it in MLIIIA. When the mutations were analyzed along with GlcNAc-phosphotransferase activity, it was possible to confidently distinguish these two clinically related but distinct diseases. We propose criteria for distinguishing these two disorders by a combination of mutation detection and GlcNAc-phosphotransferase activity determination.     

Phosphorylation of the oligosaccharide of uteroferrin by UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum requires alpha 1,2-linked mannose residues

We have investigated the oligosaccharide requirements of the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum. Uteroferrin, an acid hydrolase, was phosphorylated by the three N-acetylglucosaminylphosphotransferases, and the phosphorylated oligosaccharides were isolated and analyzed by ion suppression high performance liquid chromatography. In all three cases, the phosphorylated species contained 6 or more mannose residues. Phosphorylation of the Man5GlcNAc2 oligosaccharide could not be detected even though this was the major species on the native uteroferrin. The Man5GlcNAc2 oligosaccharides lack alpha 1,2-linked mannose residues, whereas the larger oligosaccharides contain 1 or more mannose residues in this linkage. Treatment of intact uteroferrin with an alpha 1,2-specific mannosidase-generated molecules whose oligosaccharides consisted almost entirely of species with 5 mannose residues. The N-acetylglucosaminylphosphotransferases could no longer phosphorylate such molecules. These data indicate that at least 1 alpha 1,2-linked mannose residue must be present on uteroferrin's oligosaccharide for phosphorylation to occur.     

Lysosomal storage causes cellular dysfunction in mucolipidosis II skin fibroblasts

Mucolipidosis II (ML-II) is a fatal inherited metabolic disease caused by deficiency of GlcNAc-phosphotransferase, which plays a role in generating the mannose 6-phosphate recognition marker on lysosomal enzymes. In ML-II, many lysosomal acid hydrolases are mistargeted out of cells, and lysosomes become filled with undigested substrates, which explains inclusion cell disease as an alternative name for this disease. In this study, we revealed various cellular phenotypes in ML-II skin fibroblasts. We quantitated phospholipid and cholesterol within cells and showed ~2-fold accumulation in ML-II as compared with normal cells. Lysosomal pH of ML-II cells was higher than that of normal cells (5.29 ± 0.08 versus 4.79 ± 0.10, p < 0.001). The proliferated lysosomes in ML-II cells were accumulated ~3-fold in amount as compared with normal cells. Intracellular logistics including endocytosis and mannose 6-phosphate receptor recycling were impaired in ML-II cells. To confirm whether these ML-II cellular phenotypes derive from deficient lysosomal acid hydrolases within lysosomes, we performed supplementation of lysosomal enzymes using a partially purified total enzyme mixture, which was derived from the conditioned culture medium of normal skin fibroblasts after NH(4)Cl treatment. This supplementation corrected all of the previously described ML-II phenotypes. In addition, the autophagic and mitochondrial impairment that we have previously reported improved, and inclusion bodies disappeared on electron micrography following total lysosomal enzyme supplementation. Our results indicate that various cellular phenotypes in ML-II are caused by the deficiency of many lysosomal enzymes and massive accumulation of undigested substrates.     

Inhibition by lysosomal enzymes and mannose-6-phosphate of the phosphorylation of the lysosomal enzyme binding receptor protein from monkey brain

The lysosomal enzymes beta-glucuronidase and alpha-L-fucosidase and mannose-6-phosphate inhibited the phosphorylation of the lysosomal enzyme binding receptor protein prepared from monkey brain. Inhibition of both serine and tyrosine phosphorylation was observed. A non-lysosomal glycoprotein enzyme butyrylcholinesterase, mannose or glucose did not inhibit phosphorylation. Tyrosine phosphorylation of histone by the receptor protein was also inhibited by the lysosomal enzymes and mannose-6-phosphate.     

The role of extra-hepatic tissues in the receptor-mediated plasma clearance of glycoproteins terminated by mannose or N-acetylglucosamine.

The mannose- and N-acetylglucosamine-specific pathway for the clearance of mammalian glycoproteins has been characterized by using 125I-labelled neoglycoproteins, glycosidase-treated orosomucoid and lysosomal glycosidases (beta-glucuronidase and beta-N-acetylglucosaminidase) as probes. There are two components to this pathway in vivo; one liver-dependent and the other extrahepatic or liver-independent. Cells that mediate clearance by the latter component of the pathway are present in spleen, bone and in elements of the reticuloendothelial system, but not in the kidney. Glycoproteins that possess terminal mannose, glucose or N-acetylglucosamine residues, including various lysosomal enzymes, are rapidly cleared from plasma via this pathway. Glucose-terminated glycoproteins are recognized by two pathways in the intact animal; the hepatic galactose-specific pathway and the mannose/N-acetylglycosamine-specific pathway, which is present in liver and in peripheral tissues. Following removal of the liver by surgical evisceration, glucose-terminated glycoproteins are cleared whereas glycoproteins bearing galactose are not cleared. Uptake of 125I-labelled neoglycoproteins and agalacto-orosomucoid by isolated alveolar macrophages closely mimics clearance in vivo by the mannose/N-acetylglucosamine pathway. Neoglycoproteins terminated by mannose, glucose or N-acetylglucosamine all compete with 125I-labelled agalacto-orosomucoid for uptake by receptor-mediated pinocytosis. The extent of substitution of the neoglycoproteins is a critical determinant of their inhibitory potency. It is proposed that mononuclear phagocytes are in important component of the clearance pathway in vivo. The mannose/N-acetylglucosamine pathway may be important in the regulation of extracellular levels of various glycosylated macromolecules, including lysosomal hydrolases.     

Identification of the minimal lysosomal enzyme recognition domain in cathepsin D

Specific recognition of lysosomal hydrolases by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues, is governed by a common protein determinant. Previously, we generated a lysosomal enzyme recognition domain in the secretory protein glycopepsinogen by substituting in two regions (lysine 203 and amino acids 265-293 of the beta loop) from cathepsin D, a highly related lysosomal protease. Here we show that substitution of just two lysines (Lys-203 and Lys-267) stimulates mannose phosphorylation 116-fold. Substitution of additional residues in the beta loop, particularly lysines, increased phosphorylation 4-fold further, approaching the level obtained with intact cathepsin D. All the phosphorylation occurred at the carboxyl lobe glycan, indicating that additional elements are required for phosphorylation of the amino lobe glycan. These data support the proposal that as few as two lysines in the correct orientation to each other and to the glycan can serve as the minimal elements of the lysosomal enzyme recognition domain. However, our findings show that the spacing between lysines is flexible and other residues contribute to the recognition marker.     

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.     

Mannose 6-phosphate-independent targeting of lysosomal enzymes in I- cell disease B lymphoblasts

B lymphocytes from patients with I-cell disease (ICD) maintain normal cellular levels of lysosomal enzymes despite a deficiency of the enzyme UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1- phosphotransferase. We find that an ICD B lymphoblastoid cell line targets about 45% of the lysosomal protease cathepsin D to dense lysosomes. This targeting occurs in the absence of detectable mannose 6- phosphate residues on the cathepsin D and is not observed in ICD fibroblasts. The secretory protein pepsinogen, which is closely related to cathepsin D in both amino acid sequence and three-dimensional structure, is mostly excluded from dense lysosomes, indicating that the lymphoblast targeting pathway is specific. Carbohydrate residues are not required for lysosomal targeting, since a non-glycosylated mutant cathepsin D is sorted with comparable efficiency to the wild type protein. Analysis of a number of cathepsin D/pepsinogen chimeric proteins indicates that an extensive polypeptide determinant in the cathepsin D carboxyl lobe can confer efficient lysosomal sorting when introduced into the pepsinogen sequence. This determinant overlaps but is not identical to the recognition marker for phosphotransferase. These results indicate that a specific protein recognition event underlies Man-6-P-independent lysosomal sorting in ICD lymphoblasts.     

Murine UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase lacking the gamma-subunit retains substantial activity toward acid hydrolases

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on acid hydrolases. The transferase exists as an alpha(2)beta(2)gamma(2) hexameric complex with the alpha- and beta-subunits derived from a single precursor molecule. The catalytic function of the transferase is attributed to the alpha- and beta-subunits, whereas the gamma-subunit is believed to be involved in the recognition of a conformation-dependent protein determinant common to acid hydrolases. Using knock-out mice with mutations in either the alpha/beta gene or the gamma gene, we show that disruption of the alpha/beta gene completely abolishes phosphorylation of high mannose oligosaccharides on acid hydrolases whereas knock-out of the gamma gene results in only a partial loss of phosphorylation. These findings demonstrate that the alpha/beta-subunits, in addition to their catalytic function, have some ability to recognize acid hydrolases as specific substrates. This process is enhanced by the gamma-subunit.     

Purification and biochemical characterization of a lysosomal α-fucosidase from the deuterostomia Asterias rubens

In vertebrates, mannose 6-phosphate receptors [MPR300 (Mr 300 kDa) and MPR46 (Mr 46 kDa)] are highly conserved transmembrane glycoproteins that mediate transport of lysosomal enzymes to lysosomes. Our studies have revealed the appearance of these putative receptors in invertebrates such as the molluscs and deuterostomes. Starfish tissue extracts contain several lysosomal enzyme activities and here we describe the affinity purification of α-fucosidase. The purified enzyme is a glycoprotein that exhibited a molecular mass of ∼56 kDa in SDS-PAGE under reducing conditions. It has also cross-reacted with an antiserum to the mollusc enzyme suggesting antigenic similarities among the two invertebrate enzymes. LC–MS/MS analysis of the proteolytic peptides of the purified enzyme in combination with de novo sequencing allowed us to do partial amino acid sequence determination of the enzyme. These data suggest that this invertebrate enzyme is homologous to the known mammalian enzyme. The purified enzyme exhibited a mannose 6-phosphate dependent interaction with the immobilized starfish MPR300 protein. Our results demonstrate that the lysosomal enzyme targeting pathway is conserved even among the invertebrates.     

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

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

Lysosomal acid phosphatase is not involved in the dephosphorylation of mannose 6-phosphate containing lysosomal proteins.

The mannose 6-phosphate (Man6P) residues that are necessary for the targeting of newly synthesized lysosomal proteins are dephosphorylated after delivery of lysosomal proteins to lysosomes. To examine the role of lysosomal acid phosphatase (LAP) for the dephosphorylation of Man6P residues in lysosomal proteins, the phosphorylation of endogenous lysosomal proteins and of internalized arylsulfatase A was analyzed in mouse L-cells that overexpress human LAP. Non-transfected L-cells dephosphorylate endogenous lysosomal proteins slowly (half time approximately 13 h) as well as internalized arylsulfatase A. A more than 100-fold overexpression of LAP in these cells did not affect the dephosphorylation rate. Control experiments showed that the internalized arylsulfatase A and overexpressed LAP partially colocalize and that under in vitro conditions purified LAP does not dephosphorylate arylsulfatase A. Taken together, these results indicate that LAP is not the mannose 6-phosphatase that dephosphorylates lysosomal proteins after their delivery to lysosomes.     

Lysosomal enzyme phosphorylation. II. Protein recognition determinants in either lobe of procathepsin D are sufficient for phosphorylation of both the amino and carboxyl lobe oligosaccharides.

Cathepsin D is a bilobed lysosomal aspartyl protease that contains one Asn-linked oligosaccharide/lobe. Each lobe also contains protein determinants that serve as recognition domains for binding of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the first enzyme in the biosynthesis of the mannose 6-phosphate residues on lysosomal enzymes. In this study we examined whether the location of the protein recognition domain influences the relative phosphorylation of the amino and carboxyl lobe oligosaccharides. To do this, chimeric proteins containing either amino or carboxyl lobe sequences of cathepsin D substituted into a glycosylated form of the homologous secretory protein pepsinogen were expressed in Xenopus oocytes. The amino and carboxyl lobe oligosaccharides were then isolated from the various chimeric proteins and independently analyzed for their mannose 6-phosphate content. This analysis has shown that a phosphotransferase recognition domain located on either lobe of a cathepsin D/glycopepsinogen chimeric molecule is sufficient to allow phosphorylation of oligosaccharides on both lobes. However, phosphorylation of the oligosaccharide on the lobe containing the recognition domain is favored. We also found that the majority of the carboxyl lobe oligosaccharides of cathepsin D acquire two phosphates, whereas the amino lobe oligosaccharides only acquire one phosphate.