β-Glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are β-glucuronidase (β-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that β-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas β-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, β-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that β-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed. This study was funded by the University of the Basque Country through a grant to Consolidated Research Groups. U.I. is a recipient of a pre-doctoral fellowship from the Basque Government.     

Long - lasting synchrony of the division of enteric bacteria

Recent finding of α-N-acetylglucosamine(1)phospho(6)mannose diesters in lysosomal enzymes suggested that formation of mannose 6-phosphate residues involves transfer of N-acetylglucosamine 1-phosphate to mannose. Using dephosphorylated β-hexosaminidase as acceptor and [β-³²P]UDP-N-acetylglucosamine as donor for the phosphate group, phosphorylation of β-hexosaminidase by microsomes from rat liver, human placenta and human skin fibroblasts was achieved. The reaction was not affected by tunicamycin. Acid hydrolysis released mannose 6-[³²P]phosphate from the phosphorylated β-hexosaminidase. Our results suggest that lysosomal enzymes are phosphorylated by transfer of N-acetylglucosamine 1-phosphate from UDP-N-acetylglucosamine. The transferase activity was deficient in fibroblasts from patients affected with l-cell disease. This deficiency is proposed to be the primary enzyme defect in l-cell disease.     

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.     

Studies of the synthesis,structure and function of the phosphorylated oligosaccharides of lysosomal enzymes

Newly synthesized lysosomal enzymes were found to contain N-acetylglucosamine residues in phosphodiester linkage to the 6 position of the mannose residues on high-mannose type oligosaccharides. The formation of these structures was shown to be catalyzed by a specific N-acetylglucosaminylphosphotransferase enzyme, that utilises UDP-N-acetylglucosamine as a donor. The phosphorylation reaction can take place on any of four or five positions on the high-mannose oligosaccharide. Subsequently an α-N-acetylglucosaminylphosphodiesterase removes the outer blocking N-acetylglucosamine residues to generate the mature phosphomannsoyl recognition signal. This signal is responsible for the targetting of newly synthesized lysosomal enzymes to lysosomes. The human syndromes of I-cell disease (Mucolipidosis II) and pseudo-Hurler polydystrophy (Mucolipidosis III) were shown to be caused by deficiency of the first enzyme in the pathway, the UDP-N-acetylglucosamine: Glycoprotein N-acetylglucosaminylphosphotransferase.     

An Inhibition and Kinetic Study of Acid Phosphatase in Paramecium caudatum and Paramecium tetraurelia1

ABSTRACT. Inhibition, inactivation, pH, and kinetic studies using both homogenates and purified lysosomal fractions of Paramecium caudalum and of P. tetraurelia were carried out to examine the lysosomal acid phosphatase (AcPase) and its relationship to p-nitrophenylphosphatase (pNPPase), glucose-6-phosphatase (G6Pase), and 5′-nucleotidase (AMPase). The results generally support the idea that Paramecium cells contain a distinct lysosomal AcPase with a broad substrate specificity. The hydrolysis of glucose-6-phosphate (G6P) and adenosine 5′-monophosphate (AMP) was shown to be due to this enzyme, suggesting that true G6Pase and AMPase may be lacking in these two species; however, some hydrolysis of AMP at pH 7.5 catalyzed by an unknown soluble enzyme distinct from alkaline phosphatase and Na⁺-K⁺-ATPase was observed. Since the hydrolysis of p-nitrophenylphosphate (pNPP) at acid pH was also shown to be due to AcPase alone, pNPPase could be used as a rapid assay for Paramecium AcPase. At an alkaline pH, however, this activity was catalyzed by an alkaline phosphatase located in the cytosol fraction. P. caudatum AcPase was shown to have kinetic properties similar to those of purified rat liver and human prostatic AcPase and to have relative substrate affinities in the order of G6P < β-glycerophosphate < pNPP < AMP. These different substrate affinities might account for the observed differences in the inhibition of the four lysosomal activities by NaF, L(+)-tartrate, and molybdate, all of which inhibited the hydrolysis of G6P, β-glycerophosphate, and pNPP competitively, but which exhibited a noncompetitive inhibition of a mixed type with the hydrolysis of AMP.     

Isolation and characterization of phosphorylated oligosaccharides from alpha-N-acetylglucosaminidase that are recognized by cell-surface receptors.

Adsorptive endocytosis of lysosomal enzymes by fibroblasts and hepatocytes involves binding to cell surface receptors that recognize on lysosomal enzymes a phosphorylated carbohydrate, most likely a mannose 6-phosphate residue [Kaplan et al. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 2026-2030; Ullrich et al. (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 1591-1598]. Loss of alpha-N-acetylglucosaminidase endocytosis after treatment with endoglucosaminidase H indicated that the recognition site of alpha-N-acetylglucosaminidase is located on N-glycosidically linked oligosaccharides of the high mannose type. Acidic oligosaccharides with an average molecular weight of 2200 were liberated from alpha-N-acetylglucosaminidase by endoglucosaminidase H. These oligosaccharides were susceptible to degradation by alkaline phosphatase, alpha-mannosidase and beta-N-acetylglucosaminidase. At the non-reducing terminal these oligosaccharides bear phosphorylated mannose and/or N-acetylglucosamine residues.     

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.     

Identification of a rat liver alpha-N-acetylglucosaminyl phosphodiesterase capable of removing "blocking" alpha-N-acetylglucosamine residues from phosphorylated high mannose oligosaccharides of lysosomal enzymes

We recently reported that the high mannose-type oligosaccharides of the biosynthetic intermediates of beta-glucuronidase contain phosphate groups in diester linkage between mannose residues and outer alpha-linked N-acetylglucosamine residues (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 255, 6633-6639). We now describe an alpha-N-acetylglucosaminyl phosphodiesterase from rat liver that is capable of removing the N-acetyl-glucosamine residues, leaving phosphomonoester groups on the high mannose oligosaccharide units. This activity is greatly enriched in smooth membrane preparations. It can be distinguished from a lysosomal alpha-N-acetylglucosaminidase by several criteria, including subcellular localization and differential inhibition by amino sugars. In addition, human fibroblasts with mutations which lead to a deficiency of the lysosomal activity have normal levels of the alpha-N-acetylglucosaminyl phosphodiesterase. This enzyme may be involved in the "unmasking" of the phosphomannosyl recognition marker on newly synthesized acid hydrolases which could then direct the targeting of these enzymes to lysosomes.     

Convergence of the endocytic and lysosomal pathways in soybean protoplasts

Ultrastructural analysis of endocytosis of cationized ferritin (CF) has been combined with ultrastructural localization of acid phosphatases (AcPase) in soybean (Glycine max (L.) Merr.) protoplasts. While CF is an electron-dense marker of organelles of the endocytic pathway, ultrastructural histochemistry of AcPase identifies the organelles involved in the synthesis, transport, and storage of lytic-compartment enzymes, i.e. the lysosomal pathway. Acid phosphatases have been localized using both lead- and cerium-precipitation techniques. Protoplasts have been exposed to CF for 5 min, 30 min, or 3 h and processed for AcPase localization. At 5 min, smooth vesicles contain both CF and AcPase. By 30 min, Golgi cisternae and multivesicular bodies contain both labels. By 3 h, vacuoles become labelled with both CF and AcPase. The large central vacuoles contain intraluminal membranes which are associated with both AcPase and CF. These observations extend the analogy between plant vacuoles and animal lysosomes and demonstrate the points at which the endocytic pathway of plants converges with the lysosomal pathway.Abbreviations AcPase acid phosphatase - CF cationized ferritin - ER endoplasmic reticulum - MVB multivesicular body - PCR partially coated reticulum - PM plasma membrane     

Carbohydrate mediated recognition of lysosomal enzymes by cell surface receptors

The recognition of lysosomal enzymes by various carbohydrate specific cell surface receptors is reviewed. In particular the biosynthesis of mannose 6-phosphate residues in lysosomal enzymes and their role for targeting of lysosomal enzymes to lysosomes are discussed.     

The binding requirements of monkey brain lysosomal enzymes to their immobilised receptor protein

The lysosomal enzyme binding protein (receptor protein) isolated from monkey brain was immobilised on Sepharose 4B and used to study the binding of brain lysosomal enzymes. The immobilised protein could bind \-D-glucosaminidase, α-D-mannosidase, α-L-fucosidase and2-D-glucuronidase. The bound enzymes could be eluted either at an acid pH of 4.5 or by mannose 6-phosphate but not by a number of other sugars tested. Binding could be abolished by prior treatment of the lysosomal enzymes with sodium periodate. Alkaline phosphatase treatment of the enzymes did not prevent the binding of the lysosomal enzymes to the column but decreased their affinity, as seen by a shift in their elution profile, when a gradient elution with mannose 6-phosphate was employed. These results suggested that an ‘uncovered’ phosphate on the carbohydrate moiety of the enzymes was not essential for binding but can enhance the binding affinity.     

Processing of the phosphorylated recognition marker in lysosomal enzymes. Characterization and partial purification of a microsomal alpha-N-acetylglucosaminyl phosphodiesterase

N-Acetylglucosamine(1)phospho(6)mannose groups recently identified in lysosomal enzymes were proposed to be precursors of the recognition markers terminating with mannose 6-phosphate (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 225, 6633-6639; Hasilik, A., Klein, U., Waheed, A., Strecker, G., and von Figura, K. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7074-7078). To study the presumptive enzyme removing N-acetylglucosamine from the diester, an assay was developed using a radioactive oligosaccharide containing diester groups of the above structure. An alpha-N-acetylglucosaminyl phosphodiesterase cleaving this substrate in vitro was found in human placenta and in rat liver. The enzyme was solubilized from the microsomal fraction of human placenta and more than 800-fold purified with 75% yield. It is distinct from the lysosomal alpha-N-acetylglucosaminidase by the criteria of immunological cross-reactivity, substrate specificity, and heat stability. The partially purified enzyme cleaves alpha-N-acetylglucosamine phosphodiester bonds in oligosaccharides from lysosomal enzymes, in lysosomal enzymes, and in UDP-N-acetylglucosamine. We propose that the microsomal alpha-N-acetylglucosaminyl phosphodiesterase is involved in the processing of the phosphorylated recognition marker in lysosomal enzymes.     

Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? evidence from I-cell disease

We have examined frozen liver tissue for N-acetylglucosamine-l-phosphotransferase, an enzyme required for the formation of the mannose 6-phosphate recognition marker of lysosomal enzymes. Using [β³²P]-UDPGlcNAc and placental β-hexosaminidase B as N-acetylglucosamine l-phosphate donor and acceptor, respectively, we were unable to find activity of the transferase in 100,000 × g membranes prepared from livers of patients with I-cell disease, whereas activity was readily observed in membranes from control livers stored under the same conditions. Yet the activity of several lysosomal enzymes (β-N-acetylglucosaminidase, β-glucuronidase, α-mannosidase and α-$\text{L}$-iduronidase) was comparable in liver tissue of I-cell patients and controls, and only β-galactosidase activity showed a marked reduction. These results suggest that in contrast to cultured skin fibroblasts, liver may be able to introduce into lysosomes acid hydrolases that lack the mannose 6-phosphate recognition marker.     

Binding of cathepsin D to the mannose receptor on rat peritoneal macrophages

Adherent cultures of rat peritoneal macrophages secrete lysozyme and the lysosomal marker enzymes beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase; the levels of secreted lysosomal cathepsin D, however, were found to be insignificant. Incubation of the cells at 4 degrees C for 15 min with yeast mannan or with 50 mM mannose, methyl alpha-glucopyranoside, or N-acetylglucosamine caused the concentration of cathepsin D in the culture medium to increase 30-40-fold; mannose-6-phosphate had no effect. 125I-labeled cathepsin D was prepared and the binding constant to the macrophage cell surface was determined to be KD = 27 nM. The data suggest that cathepsin D binds to the mannose receptor of macrophages and that binding to this receptor is not in equilibrium with the bulk medium.     

Density gradient separation of two populations of lysosomes from rat parotid acinar cells

Exocrine acinar cells possess two cytochemically distinct populations of secondary lysosomes. One population is Golgi associated and has demonstrable acid phosphatase (AcPase) activity, whereas the second is basally located and lacks AcPase activity but has trimetaphosphatase (TMPase) activity. The basal lysosomes are tubular in shape and rapidly label with horseradish peroxidase (HRP) after intravenous injection. In the present study using isolated rat parotid acinar cells, the two lysosomal populations were separated by cell fractionation on Percoll density gradients and were analyzed biochemically and by EM cytochemistry. On 35% Percoll gradients, two peaks of AcPase and beta-hexosaminidase, both lysosomal marker enzymes, and succinic dehydrogenase, an enzyme marker for mitochondria, could be resolved. The major peaks of beta-hexosaminidase and succinic dehydrogenase and the minor peak of AcPase corresponded with the dense lysosome fraction. The major peak of AcPase and the minor peaks for beta-hexosaminidase and succinic dehydrogenase coincided with the light membrane fraction. Galactosyl transferase (a marker enzyme for Golgi saccules) and 5'-nucleotidase (a plasma membrane marker) were also associated with this fraction. By electron microscopy, the light membrane fraction was seen to contain tubular elements, multivesicular bodies (MVB), Golgi saccules, GERL, immature secretory granules, and some mitochondria. Electron microscopic cytochemical examination showed that these tubular structures were lysosomes. The dense lysosome fraction contained lysosomes positive for both AcPase and TMPase. After continuous incubation of isolated acinar cells with HRP, reaction product was rapidly localized to the light membrane fraction (greater than 2 min), where it was found in vesicles and tubular lysosomes. By 10 min it was present in MVB and tubular lysosomes, but by 60 min no HRP reaction product had appeared in the dense lysosomes. These results demonstrate that the tubular lysosomes are separable from dense lysosomes, typical secondary lysosomes, and are involved in the initial stages of endocytosis.     

Endocytosis of lysosomal enzymes by non-parenchymal rat liver cells. Comparative study of lysosomal-enzyme uptake by hepatocytes and non-parenchymal liver cells

Cultured non-parenchymal rat liver cells internalize human urine alpha-N-acetylglucosaminidase, human skin beta-N-acetylglucosaminidase and pig kidney alpha-mannosidase. Different heat-stabilities of endocytosed and endogenous alpha-mannosidase activity provided indirect evidence that the increase in intracellular activity resulted from uptake. The high efficiency and the saturation kinetics of uptake indicated that these enzymes become internalized by adsorptive endocytosis. Competition experiments with glycoproteins bearing known carbohydrates at their non-reducing terminals, with mannans, methyl glycosides and monosaccharides, established that the uptake of these three lysosomal enzymes is mediated by the binding to cell-surface receptors that recognize mannose and N-acetylglucosamine residues. The decreased uptake after treatment of these enzymes with either beta-N-acetylglucosaminidase or alpha-mannosidase was in accordance with the results of the inhibition experiments. Removal of oligosaccharides of the high-mannose type by treatment with endoglucosaminidase H inhibited uptake almost completely, suggesting that the sugars recognized by cell-surface receptors of non-parenchymal liver cells are located in the outer core of these oligosaccharides. A comparison of the uptake of these three lysosomal enzymes by parenchymal and non-parenchymal rat liver cells indicates that infused alpha-N-acetylglucosaminidase is taken up preferentially by hepatocytes, whereas alpha-mannosidase and beta-N-acetylglucosaminidase are localized predominantly in non-parenchymal rat liver cells.     

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.     

Purification and characterization of rat liver alpha-N-acetylglucosaminyl phosphodiesterase

In an earlier report we described the identification of an alpha-N-acetylglucosaminyl phosphodiesterase that is capable of cleaving the outer phosphodiester-linked alpha-N-acetylglucosamine residues present on the high mannose oligosaccharides of newly synthesized lysosomal enzymes (Varki, A., and Kornfeld, S. (1980) J. Biol. Chem. 255, 8398-8401). We have now purified this enzyme 1800-fold with a 24% yield from rat liver, using subcellular fractionation, differential extraction with Triton X-10, DEAE-cellulose chromatography, heparin-Sepharose chromatography, concanavalin A-Sepharose affinity chromatography, and gel filtration on Sephacryl S-300. The purified preparation is free of lysosomal alpha-N-acetylglucosaminidase. The enzyme exhibited a single form on both the ion exchange and gel filtration steps. It has a broad pH optimum between 6.0-8.0 and is unaffected by divalent cations or reducing agents. The enzyme cleaves alpha-N-acetylglucosamine residues from five different locations on the high mannose oligosaccharide. In the case of molecules with one phosphodiester, the rate of cleavage is not affected by the size of the underlying oligosaccharide or the presence or absence of an asparagine-linked peptide. Molecules with two phosphodiesters are cleaved in a nonrandom manner. The enzyme has no activity toward p-nitrophenyl-alpha-N-acetylglucosamine but is capable of cleaving phosphodiester-linked N-acetylglucosamine in molecules such as UDP-N-acetylglucosamine, indicating that it can only hydrolyze N-acetylglucosamine residues that are alpha-linked to a phosphate group.     

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

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