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Subcellular Localization of Mannose 6-Phosphate Glycoproteins in Rat Brain

1999; Elsevier BV; Volume: 274; Issue: 30 Linguagem: Inglês

10.1074/jbc.274.30.21104

1083-351X

Michel Jadot, Lin Li, David E. Sleat, István Sohár, Ming‐Sing Hsu, John E. Pintar, Franz Dubois, Simone Wattiaux‐De Coninck, Robert Wattiaux, Peter Lobel,

Glycosylation and Glycoproteins Research

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification ofN-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 intracellular transport of soluble lysosomal enzymes relies on the post-translational modification ofN-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. Transport of soluble lysosomal precursor proteins to lysosomes relies on the addition of mannose 6-phosphate (Man 6-P) 1The abbreviations used are: Man 6-P, mannose 6-phosphate; MPR, Man 6-P receptors; CI, cation-independent; P, microsomal fraction; M, mitochondrial fraction; L, light mitochondrial fraction; LAP, lysosomal acid phosphatase residues. The nascent polypeptide chain follows the first steps of the secretory pathway, involving the co-translational addition of preformed oligosaccharide chains onto select asparagine residues. A sorting event in the Golgi apparatus allows diversion of the precursor lysosomal proteins from the secretory pathway. This process entails the two-step generation of Man 6-P on N-linked oligosaccharide chains and subsequent binding to Man 6-P receptors (MPRs). The MPR-lysosomal protein complexes exit the Golgi in transport vesicles, which then fuse with an acidic prelysosomal compartment (endosome), where the low pH triggers dissociation of the complexes. The free receptors recycle back to the Golgi, whereas the lysosomal proteins accumulate in lysosomes. In most cell types, the Man 6-P recognition marker is rapidly removed shortly after endosomal/lysosomal targeting. This explains why typically the bulk of the lysosomal proteins are found in their dephosphorylated state (for reviews, see Refs. 1Kornfeld S. J. Clin. Invest. 1986; 77: 1-6Crossref PubMed Scopus (317) Google Scholar, 2von Figura K. Hasilik A. Annu. Rev. Biochem. 1986; 55: 167-193Crossref PubMed Scopus (637) Google Scholar, 3Kornfeld S. Mellman I. Annu. Rev. Cell Biol. 1989; 5: 483-525Crossref PubMed Scopus (1235) Google Scholar, 4Kornfeld S. Annu. Rev. Biochem. 1992; 61: 307-330Crossref PubMed Scopus (933) Google Scholar, 5Gabel C.A. Mechanisms of Intracellular Trafficking and Processing of Proproteins. CRC Press, Inc., Boca Raton, FL1993: 103-130Google Scholar). However, studies using immortalized cells have shown that the extent of dephosphorylation can vary considerably depending on the cell line and culture conditions (6Einstein R. Gabel C.A. J. Cell Biol. 1991; 112: 81-94Crossref PubMed Scopus (24) Google Scholar,7Einstein R. Gabel C.A. J. Cell Biol. 1989; 109: 1037-1046Crossref PubMed Scopus (12) Google Scholar). To investigate the fate of lysosomal enzymes in vivo, Sleatet al. (8Sleat D.E Sohar I. Lackland H. Majercak J. Lobel P. J. Biol. Chem. 1996; 271: 19191-19198Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) analyzed the level of Man 6-P-containing glycoproteins in a series of rat tissues. This study revealed that brain had significantly higher levels of Man 6-P glycoproteins than other tissues analyzed (e.g. 8-fold higher than liver). Importantly, the phosphorylation state of individual brain lysosomal enzymes was greatly elevated (e.g. on average, the amount of total activity for a given lysosomal enzyme represented by the phosphorylated form was 37% in brain compared with 0.7% in liver). This suggests that brain cells are somehow unable to efficiently process lysosomal enzymes to remove the Man 6-P modification. There are at least three possible hypotheses regarding the mechanism for the limited dephosphorylation of brain lysosomal enzymes. First, this could be the consequence of transport of some of the lysosomal precursor proteins to a specialized, metabolically inactive compartment where they are stored for later use. Second, there could be delayed transfer of lysosomal precursor proteins from endosomes to lysosomes. Finally, the lysosomal enzymes could retain the Man 6-P modification after delivery to “classical,” metabolically active lysosomes. Comparing the steady state localization of the phosphorylated and dephosphorylated lysosomal enzymes potentially could distinguish among these three models. The first two would result in distinct subcellular locations, whereas the third would result in identical subcellular locations for the phosphorylated and dephosphorylated species. In this report, we have investigated the subcellular localization of Man 6-P glycoproteins in rat brain using two complementary approaches. In a biochemical approach, we used a wide range of subcellular fractionation methods to compare the relative distributions of Man 6-P glycoproteins and lysosomal marker enzyme activities. In a morphological approach, we used double-label fluorescence and scanning confocal microscopy to compare the relative localization of Man 6-P glycoproteins and representative lysosomal enzymes. Taken together, our results indicate that the bulk of the brain Man 6-P glycoproteins reside in classical neuronal lysosomes, suggesting that these organelles are not able to fully process the Man 6-P signals. Brain and liver were obtained from adult male Wistar rats weighing 250–300 g. After dissection, brain tissue was immersed immediately in ice-cold 0.25 msucrose and homogenized by one passage through a motor-driven Potter apparatus (1500 rpm). Fractionation of subcellular organelles by differential centrifugation was as described by de Duve et al. (9de Duve C. Pressman B.C. Gianetto R. Wattiaux R. Appelmans F. Biochem. J. 1955; 60: 604-617Crossref PubMed Scopus (2567) Google Scholar). Isopycnic centrifugations were carried out according to Beaufay et al. (10Beaufay H. Jacques P. Sellinger O.Z. Berthet J. de Duve C. Biochem. J. 1964; 92: 184-205Crossref PubMed Scopus (199) Google Scholar) using a Spinco model L7–65 Ultracentrifuge and VTi65 rotor. Enzyme measurements were performed using 4-methylumbelliferyl substrates in the presence of 0.1% Triton X-100, incubated at 37 °C, and terminated by the addition of 2 volumes of 0.5 m glycine, 5 mm EDTA, 0.05% Triton X-100, pH 10.5. Reaction conditions were as follows (enzyme, substrate/buffer): β-galactosidase, 1 mm4-methylumbelliferyl-β-galactoside, 80 mm acetate pH 4.0; α-fucosidase, 0.2 mm 4-methylumbelliferyl-α-fucoside, 80 mm citrate, pH 5.0; β-glucosidase, 5 mm4-methylumbelliferyl-β-glucoside, 80 mm citrate, pH 4.5, 6 mm taurocholate. Samples from individual fractions (representing equivalent portions of tissue homogenate) were analyzed by SDS-polyacrylamide gel electrophoresis on 10% reducing gels. Blotting and detection of the Man 6-P glycoproteins using iodinated soluble cation-independent (CI) MPR were as described (11Valenzano K.J. Kallay L.M. Lobel P. Anal. Biochem. 1993; 209: 156-162Crossref PubMed Scopus (42) Google Scholar,12Sleat D.E. Chen T.-L. Raska K. Lobel P. Cancer Res. 1995; 55: 3424-3430PubMed Google Scholar). Total Man 6-P glycoproteins were estimated using a PhosphorImager and ImageQuant 3.3 software (Molecular Dynamics, Sunnyvale, CA) by integrating the total machine counts present in each lane after correcting for background binding to nitrocellulose. Equivalent samples (representing material present in 0.5 mg of tissue homogenate) were submitted to SDS-polyacrylamide gel electrophoresis on 8% non reducing gels. After semi-dry transfer of proteins to PVDF (Immobilon-P Millipore), membranes were probed with a rabbit polyclonal antisera against the CI-MPR kindly provided by Dr. Kurt von Figura (Gottingen, Germany) and the signal revealed using a chemiluminescence system (Boehringer-Mannheim). Sucrose density gradient fractions were analyzed to determine the percentage of total lysosomal enzymes activities represented by phosphorylated forms as described in (8Sleat D.E Sohar I. Lackland H. Majercak J. Lobel P. J. Biol. Chem. 1996; 271: 19191-19198Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Samples (1 ml) were diluted into column buffer (0.15 m NaCl, 0.1% Triton X-100, 25 mm Tris-HCl, pH 7.2) and loaded onto a 1-cm3 bed volume of Affi-Gel 10-immobilized soluble CI-MPR. The column was washed with 6 ml of column buffer, mock-eluted with 2 ml of column buffer containing 10 mm glucose 6-phosphate, and eluted with 4 ml of column buffer containing 10 mm Man 6-P. The starting material, Man 6-P eluate, and the unbound material (pooled run-through, wash, and mock-eluted fractions) were adjusted to the same concentration of glucose 6-phosphate and mannose 6-phosphate before measuring lysosomal enzyme activities as described above. Biotinylated CI-MPR probe was synthesized as described previously (12Sleat D.E. Chen T.-L. Raska K. Lobel P. Cancer Res. 1995; 55: 3424-3430PubMed Google Scholar). Rabbit antisera raised against bovine liver cathepsin D recognizes rodent cathepsin D and has been described previously (13Chen H.J. Remmler J. Delaney J.C. Messner D.J. Lobel P. J. Biol. Chem. 1993; 268: 22338-22346Abstract Full Text PDF PubMed Google Scholar). Rabbit anti rat β-galactosidase and goat anti-rat lysosomal acid phosphatase antisera were generous gifts from Dr. N. F. LaRusso (Mayo Clinic, Rochester, MN) and Dr. M. Himeno (Kyushu University, Fukuoka, Japan), respectively. Sections (10 μm) of Bouin's-fixed paraffin-embedded rat brain were mounted on glass slides and deparaffinized. All incubation and washing steps were conducted in a humidified atmosphere at 4 °C. Specimens were permeabilized with 0.1% saponin in phosphate-buffered saline (137 mm NaCl, 2.7 mm KCl, 10 mmNa2HPO4, 1.76 mmKH2PO4) for 15 min, incubated with blocking buffer (phosphate-buffered saline containing 10 mg/ml bovine serum albumin, 0.2% Tween 20, 0.02% NaN3) for 2 h and incubated with biotinylated CI-MPR (10 μg/ml in blocking buffer) overnight. Specimens were washed 30 min with wash buffer (phosphate-buffered saline containing 0.2% Tween 20) and then incubated for 2 h with blocking buffer containing Cy5-avidin (Amersham Pharmacia Biotech) (1:500 dilution) and primary antisera against lysosomal marker enzymes (1:100 dilution for all). The specimens were washed for 30 min and incubated for 1 h with appropriate fluorescein isothiocyanate-conjugated secondary antibodies diluted into blocking buffer (Life Technologies, Inc., goat anti-rabbit, 1:100 dilution; Sigma rabbit anti-goat, 1:400 dilution). After washing for 30 min, sections were mounted using Vectashield (Vector Laboratory Inc.). Images were obtained using a Zeiss LSM-410 confocal laser-scanning microscope equipped with a 40 × planapochromate objective. We initially used a scheme of differential centrifugation to investigate the subcellular localization of the Man 6-P-containing glycoproteins in rat brain and liver. Equivalent samples were fractionated by SDS-polyacrylamide gel electrophoresis and blotted on nitrocellulose, and Man 6-P glycoproteins were visualized using iodinated soluble CI-MPR probe as described in Valenzano et al. (11Valenzano K.J. Kallay L.M. Lobel P. Anal. Biochem. 1993; 209: 156-162Crossref PubMed Scopus (42) Google Scholar). Fig.1 A shows representative blots obtained from such an experiment. The total amount of specifically bound 125I-CI-MPR probe is 9-fold higher in brain than in liver, consistent with previous results (8Sleat D.E Sohar I. Lackland H. Majercak J. Lobel P. J. Biol. Chem. 1996; 271: 19191-19198Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Total Man 6-P-containing glycoproteins were quantified and compared with lysosomal marker enzymes (Fig. 1 B). In liver, the Man 6-P glycoproteins have a distribution consistent with their being in prelysosomal compartments in transit to lysosomes. In this well characterized tissue, Golgi, plasma membrane, the bulk of endosomes, and fragmented endoplasmic reticulum sediment in the microsomal fraction P (14Amar-Costesec A. Beaufay H. Wibo M. Thinès-Sempoux D. Feytmans E. Robbi M. Berthet J. J. Cell Biol. 1974; 61: 201-212Crossref PubMed Scopus (249) Google Scholar, 15Limet J.N. Quintart J. Schneider Y.-J. Courtoy P.J. Eur. J. Biochem. 1985; 146: 539-548Crossref PubMed Scopus (59) Google Scholar). Classical lysosomes sediment in both the mitochondrial fraction M and the light mitochondrial fraction L (14Amar-Costesec A. Beaufay H. Wibo M. Thinès-Sempoux D. Feytmans E. Robbi M. Berthet J. J. Cell Biol. 1974; 61: 201-212Crossref PubMed Scopus (249) Google Scholar). As expected, the lysosomal markers β-galactosidase, α-fucosidase, and β-glucosidase are predominantly found in M + L (92, 90, and 78%, respectively, of the total activity in M + L + P), whereas most of the Man 6-P-containing glycoproteins are found in P (59% of M + L + P) (Fig. 1 B, right panels). Importantly, this different distribution clearly demonstrates that most of the liver Man 6-P glycoproteins are not in classical lysosomes. In brain, the lysosomal markers β-galactosidase, α-fucosidase, and β-glucosidase are to a large extent found in M + L (76, 68, and 61%, respectively, of the total activity in M + L + P), although the P fraction contains considerably higher activity than in liver. In contrast to liver, the Man 6-P glycoproteins have a distribution very similar to that of the lysosomal marker enzymes (31% of the M + L + P Man 6-P-containing glycoproteins are found in P), consistent with the phosphorylated forms being present in classical lysosomes. Results presented in Fig. 1raise the possibility that the brain Man 6-P glycoproteins are present in lysosomes or in nonlysosomal structures that also sediment in the M + L fraction. To help discriminate between these two possibilities, we analyzed the behavior of the Man 6-P glycoprotein-containing organelles by isopycnic centrifugation in sucrose density gradients (Fig.2). This analysis shows that the distribution of the Man 6-P-containing organelles and the distribution of the lysosomes are very similar. This is true regardless of the source of the organelles (ML, Fig. 2, left panel;P, Fig. 2, right panel) or if the lysosomal marker enzymes are known to be targeted by the Man 6-P-dependent pathway (β-galactosidase, α-fucosidase) or by a Man 6-P-independent mechanism (β-glucosidase). In M + L, the shoulder in the distribution of β-galactosidase and α-fucosidase (fractions 8–9) and the slight shift of β-glucosidase from β-galactosidase and α-fucosidase suggests some heterogeneity in the lysosome population, whereas the Man 6-P glycoproteins seem to be present throughout these organelles. Note that in Fig. 2, left panel, the dip in the Man 6-P glycoprotein profile at fraction 10 is most likely an artifact due to poor electrotransfer. For comparison, liver was also analyzed in parallel (Fig.3). Most of the Man 6-P glycoproteins, found in the microsomal fraction P (see Fig. 1), are present in structures showing an equilibrium density distinct from that observed for lysosomes (Fig. 3, right panel). These organelles most likely represent light endosomes (16Wattiaux R. Jadot M. Misquith S. Dubois F. Wattiaux-De Coninck S. Biochem. Biophys. Res. Commun. 1986; 136: 504-509Crossref PubMed Scopus (5) Google Scholar). The prelysosomal nature of these structures is further supported by the finding that Man 6-P glycoproteins in these lighter fractions have aM r higher than those in the dense fractions, suggesting they are lysosomal enzyme precursors in transit to the lysosome that have not yet been proteolytically processed. One potentially complicating factor in our analysis would be if in brain, lysosomal, and prelysosomal/endosomal compartments have similar physical properties. To test this possibility, we used two different centrifugation-based cell fractionation procedures to compare the distribution of organelles containing the CI-MPR, a marker of prelysosomal compartments (17Griffiths G. Hoflack B. Simons K. Mellman I. Kornfeld S. Cell. 1988; 52: 329-341Abstract Full Text PDF PubMed Scopus (589) Google Scholar), with that of the lysosomal marker β-galactosidase. Following differential centrifugation of brain homogenate, Western blotting revealed that the CI-MPR was present mainly in the P fraction, whereas β-galactosidase was mostly in M + L (Fig. 4 A). Moreover, analysis of the pooled M + L + P fractions by isopycnic centrifugation (Fig.4 B) shows that membranes associated with the CI-MPR have a lower equilibrium density than those containing β-galactosidase activity. Taken together with our previous results (Figs. 1 and 2), this indicates that most of the brain Man 6-P glycoproteins are not located in a prelysosomal/endosomal compartment. These results suggest that the brain Man 6-P glycoproteins are contained in lysosomes. As an additional test of this hypothesis, we used other separation methods in an attempt to separate the Man 6-P glycoprotein-containing organelles and classical lysosomes. One method entailed fractionation of brain M + L and P fractions by isopycnic centrifugation in NycodenzTM media (18Wattiaux R. Wattiaux-De Coninck S. Rickwood D. Iodinated Density Gradient Media, a Practical Approach. IRL Press at Oxford University Press, Oxford1983: 119-137Google Scholar). Consistent with previous results, the distribution of Man 6-P glycoproteins and lysosomal marker enzymes were quite similar (Fig.5). The same conclusion was reached from free flow electrophoresis experiments (data not shown). Finally, we also measured additional lysosomal marker enzymes (α-glucosidase, β-mannosidase, β-glucuronidase, β-hexosaminidase, α-iduronidase, acid phosphatase, sulfatase, and cathepsin B) and obtained essentially the same distributions as those markers reported in Figs. 2, 3, and 5 (data not shown). Taken together, the distributions of Man 6-P glycoproteins and lysosomal marker enzymes reported in Figs. 1, 2, 4, and 5 would be best explained by a steady state co-localization of these molecules in brain lysosomes. A classical approach to assess if a molecule is located in a particular organelle is to compare the effect of membrane-disrupting treatments on the release of the molecule of interest and on known marker proteins. In particular, the osmotic properties of endocytic organelles differ between endosomes and lysosomes. We analyzed the effect of treatments known to damage the lysosomal membrane (suspension in hypotonic media (9de Duve C. Pressman B.C. Gianetto R. Wattiaux R. Appelmans F. Biochem. J. 1955; 60: 604-617Crossref PubMed Scopus (2567) Google Scholar), 37 °C incubation in isotonic glucose (19Jadot M. Wattiaux-De Coninck S. Wattiaux R. Biochem. J. 1989; 262: 981-984Crossref PubMed Scopus (6) Google Scholar), and repeated cycles of freezing and thawing (9de Duve C. Pressman B.C. Gianetto R. Wattiaux R. Appelmans F. Biochem. J. 1955; 60: 604-617Crossref PubMed Scopus (2567) Google Scholar)) on the release of Man 6-P glycoproteins from vesicles present in brain M + L fractions. These treatments were performed in the presence of excess Man 6-P to avoid possible binding of released lysosomal enzymes to MPRs present in other membranes. After sedimentation of membranes by centrifugation, pellets and supernatants were recovered and analyzed for release of Man 6-P glycoproteins and the lysosome marker enzymes α-fucosidase and β-galactosidase. Fig. 6 shows that in all three membrane-disrupting treatments, soluble lysosomal marker enzymes and Man 6-P glycoproteins exhibit very similar release curves, indicating that they are likely to reside within the same organelle. If, as suggested by our previous experiments, the bulk of the brain Man 6-P glycoproteins are located in lysosomes, then the phosphorylated and the dephosphorylated forms of the lysosomal enzymes would have identical distributions. To test this, detergent-solubilized fractions from a brain M + L sucrose density gradient (similar to the one shown in Fig. 2, left panel) were applied to a column of immobilized CI-MPR. Enzyme activities (α-fucosidase and β-galactosidase) were determined on samples of each fraction (total activity), on pooled flow-through and washes, and on Man 6-P eluates. The bound and unbound activities were expressed as the percentage of the total activity present in the gradient. Fig.7 shows that a large proportion of the active α-fucosidase and β-galactosidase are retained on the column (39 and 65% respectively), in accordance with data reported previously (8Sleat D.E Sohar I. Lackland H. Majercak J. Lobel P. J. Biol. Chem. 1996; 271: 19191-19198Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Importantly, the dephosphorylated and phosphorylated forms of each enzyme have similar equilibrium densities, consistent with both forms being located in the same organelle. In addition to subcellular fractionation, we used laser-scanning confocal microscopy to investigate the cellular location of Man 6-P glycoproteins in rat brain. This was compared with the distribution of cathepsin D and β-galactosidase, two lysosomal enzymes targeted by the Man 6-P pathway, and to lysosomal acid phosphatase (LAP), which is synthesized as a membrane protein and is targeted to the lysosome by a Man 6-P-independent pathway. The micrographs shown in Fig. 8 represent 1-μm-thick optical sections of rat cerebral cortex layer 5, an area rich in pyramidal neurons. The Man 6-P glycoproteins (red) are predominantly found in compartments in neuronal cell bodies and some processes. Comparison of the Man 6-P glycoprotein and lysosomal enzyme-labeling (green) indicates that there is some variability both in the relative staining intensity of different neurons and in the pattern of staining. For instance, the β-galactosidase antisera stains neuronal cell bodies, but in addition, there is strong staining of long apical dendritic processes that project toward the surface of the brain (Fig. 8, bottom panels). Also, within a given cell body, there appeared to be minor differences in the relative staining of individual vesicles containing Man 6-P glycoproteins and the different lysosomal markers (most prominent with LAP). Nonetheless, the overall staining patterns showed extensive colocalization (Fig. 8,merged panels). Similar findings were obtained on neurons in numerous areas of the brain, indicating that the majority of neuronal lysosomes contain Man 6-P glycoproteins. In this report, we have investigated the subcellular localization of Man 6-P glycoproteins in rat brain using a number of complementary approaches. The biochemical experiments consistently demonstrate co-migration of Man 6-P glycoproteins and lysosomal marker enzymes in brain subcellular compartments. However, it is possible that even using the wide range fractionation methods employed in this study, distinct compartments with similar physical properties might not be distinguishable. The morphological experiments provide relatively high resolution information but are nonquantitative, and results may be dependent on antigen accessibility considerations. The convergence of these approaches strengthen our conclusion that the bulk of brain Man 6-P glycoproteins reside within neuronal lysosomes. It is reasonable to assume that the low phosphorylation state of lysosomal enzymes in most other tissues is due the short half-life of the Man 6-P marker (1.4 h in mouse BW5147 cells (20Gabel C.A. Goldberg D.E. Kornfeld S. J. Cell Biol. 1982; 95: 536-542Crossref PubMed Scopus (42) Google Scholar)) compared with the long half-lives of lysosomal enzymes (generally days, see Refs.21Skudlarek M.D. Swank R.T. J. Biol. Chem. 1981; 256: 10137-10144Abstract Full Text PDF PubMed Google Scholar, 22Smith K. Ganschow R.E. J. Biol. Chem. 1978; 253: 5437-5442Abstract Full Text PDF PubMed Google Scholar, 23Warburton M.J. Wynn C.H. Biochem. J. 1976; 158: 401-407Crossref PubMed Scopus (11) Google Scholar). Conversely, the high phosphorylation state of lysosomal enzymes in neurons could either be because of a relatively long half-life of the Man 6-P marker or, less likely, to a short half-life of the enzyme itself. A prolonged Man 6-P half-life could result from either the presence of some unusual neuron-specific carbohydrate modification that hinders the action of the dephosphorylating enzyme or from low levels of this enzyme. Further insight into this question will require identification of the endogenous enzyme responsible for removing the Man 6-P modification. This is unlikely to be the classical lysosomal acid phosphatase, as indicated by overexpression studies of LAP in cultured cells (24Bresciani R. Peters C. von Figura K. Eur. J. Cell Biol. 1992; 58: 57-61PubMed Google Scholar) or by our findings that Man 6-P glycoproteins and LAP coexist in the same neuronal compartments.In vitro studies indicate that the Man 6-P modification can be removed by purple acid phosphatase (25Bresciani R. von Figura K. Eur. J. Biochem. 1996; 238: 669-674Crossref PubMed Scopus (29) Google Scholar). If this observation is physiologically relevant, it will be of interest to determine the cellular and intracellular distribution of this enzyme in brain. When considering the biological mechanisms and consequences of dephosphorylation of lysosomal enzymes, it is important to note that different cell lines exhibit marked differences in rates of dephosphorylation of lysosomal enzymes. For instance, Gabel et al. (26Gabel C.A. Goldberg D.E. Kornfeld S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 775-779Crossref PubMed Scopus (139) Google Scholar) find that some CI-MPR-deficient cell lines do not rapidly remove the Man 6-P marker (26Gabel C.A. Goldberg D.E. Kornfeld S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 775-779Crossref PubMed Scopus (139) Google Scholar). This may not be a direct consequence of loss of the CI-MPR, as knock out mice that lack either the CI-MPR or the CD-MPR (27Sohar I. Sleat D. Liu C.-G. Ludwig T. Lobel P. Biochem. J. 1998; 330: 903-908Crossref PubMed Scopus (50) Google Scholar) exhibit little change in overall levels of Man 6-P glycoproteins in various solid tissues (brain, liver, spleen, kidney, and lung) compared with mice that contain both MPRs. 2D.E Sleat, I. Sohar, and P. Lobel, unpublished data. Also, in some CI-MPR-containing cell lines, dephosphorylation is dependent on cell culture conditions such as cell density, serum starvation, and the presence of growth factors such as insulin-like growth factor II (6Einstein R. Gabel C.A. J. Cell Biol. 1991; 112: 81-94Crossref PubMed Scopus (24) Google Scholar,7Einstein R. Gabel C.A. J. Cell Biol. 1989; 109: 1037-1046Crossref PubMed Scopus (12) Google Scholar). Changes in dephosphorylation of lysosomal enzymes may also occurin vivo under certain pathological conditions. For instance, approximately one-third of all human breast carcinomas exhibit elevated levels of Man 6-P glycoproteins compared with normal breast epithelia (12Sleat D.E. Chen T.-L. Raska K. Lobel P. Cancer Res. 1995; 55: 3424-3430PubMed Google Scholar), although the underlying mechanism of this is not known. Our studies on normal brain tissue avoid nonphysiological situations that can occur in cultured cell systems and thus are biologically relevant. However, it is important to note that brain contains ∼10–50-fold more glial cells than neurons, and there are a wide variety of neuronal cell types. This cellular heterogeneity complicates interpretation of the biochemical analysis in two areas. First, we have found that the phosphorylation state of different brain lysosomal enzymes varies considerably (this study and Ref. 8Sleat D.E Sohar I. Lackland H. Majercak J. Lobel P. J. Biol. Chem. 1996; 271: 19191-19198Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It is possible that this reflects heterogeneity within a given cell type. However, given that the relative abundance of different lysosomal enzymes varies in neurons and glia (28Petanceska S. Burke S. Watson S.J. Devi L. Neuroscience. 1994; 59: 729-738Crossref PubMed Scopus (60) Google Scholar), this would also be consistent with a situation where most of the glial-derived hydrolases lacked Man 6-P and the neuron-derived hydrolases contained Man 6-P. Second, the multiple peaks or shoulders in the profiles of the sucrose and Nycodenz density gradients (this study)and in free flow electrophoresis experiments 3M. Jadot, L. Lin, D. E. Sleat, I. Sohar, M.-S. Hsu, J. Pintar, F. Dubois, S. Wattiaux-De Coninck, R. Wattiaux, and P. Lobel, unpublished data. indicate that the brain Man 6-P glycoprotein-containing lysosomes are heterogeneous. This is also consistent with other studies on brain lysosomes (29Fiorilli A. Siniscalco C. Chiarini A. Di Francesco L. Venerando B. Tettamanti G. FEBS Lett. 1991; 282: 235-238Crossref PubMed Scopus (3) Google Scholar, 30Sellinger O.Z. Hiatt R.A. Brain Res. 1968; 7: 191-200Crossref PubMed Scopus (55) Google Scholar). This heterogeneity may occur within a given cell type (possibly reflecting either changes in endogenous proteins and lipids as lysosomes mature or degradative intermediates during digestion of different substrates), or it may be because of the presence of lysosomes derived from different cell types. Thus, it will be important to develop biologically relevant in vitro model systems to clarify these and other mechanistic questions. The increased level of mannose 6-phosphate-containing lysosomal enzymes is not unique to rat. Elevated levels of Man 6-P glycoproteins have been observed in neurons compared with other cell types in humans and mice using biotinylated receptor and histochemistry, and similar results have been observed in bovine and mouse brain by blotting (31O'Brien D.A. Magyar P.L. Sleat D.E. Lobel P. Mol. Biol. Cell. 1994; 5 (abstr.): 221Google Scholar). 4D. E. Sleat, J. Pintar, and P. Lobel, unpublished data. Retention of the Man 6-P modification on neuronal lysosomal enzymes could have important functional consequences. One possibility is that retention of the Man 6-P marker serves a protective function, allowing efficient reuptake of inadvertently secreted hydrolases. This would be particularly important in brain, given that there is extensive vesicular trafficking and exocytosis in neurons, and even a small amount of missorting could result in release of lysosomal enzymes and damage to surrounding tissue. Another intriguing possibility is that the Man 6-P modification is used to tether lysosomal enzymes to surface MPRs, greatly increasing their local concentration and allowing efficient degradation of extracellular material (32Roff C.F. Wozniak R.W. Blenis J. Wang J.L. Exp. Cell Res. 1983; 144: 333-344Crossref PubMed Scopus (16) Google Scholar, 33Brauker J.H. Roff C.F. Wang J.L. Exp. Cell Res. 1986; 164: 115-126Crossref PubMed Scopus (18) Google Scholar). In this manner, the lysosomal enzymes may participate in important processes such as neuronal migration, outgrowth, and synaptic remodeling. We thank Dr. M. Pypaert, for critical reading of the manuscript and M. Savels for the artwork.