1985). In the present study we have utilized antibodies blocking

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1 The EMBO Journal vol.6 no.9 pp , 1987 Mr mannose 6phosphate specific receptor: its targeting of lysosomal enzymes role in Martin Stein, Jos E.ZijderhandBleekemolen', Hans Geuze', Andrej Hasilik and Kurt von Figura PhysiologischChemisches Institut, Universitiat Munster, Waldeyerstrasse 15, D4400 Munster, FRG, and 'Centre for Electron Microscopy, Medical Faculty, University of Utrecht, Nic. Beetstraat 22, 3511 HG Utrecht, The Netherlands Communicated by B.Dobberstein Antibodies that block the ligand binding site of the cationdependent mannose 6phosphate specific receptor (Mr MIPR) were used to probe the function of the receptor in transport of lysosomal enzymes. Addition of the antibodies to the medium of Morris hepatoma 7777 cells, which express only the Mr MPR, resulted in a decreased intracellular retention and increased secretion of newly synthesized lysosomal enzymes. In fibroblasts and HepG2 cells that express the cationindependent mannose 6phosphate specific receptor (Mr MPR) in addition to the Mr MPR, antibodies against the Mr MPR inhibited the intracellular retention of newly synthesized lysosomal enzymes only when added to the medium together with antibodies against the Mr MPR. Morris hepatoma (M.H.) 7777 did not endocytose lysosomal enzymes, while U937 monocytes, which express both types of MPR, internalized lysosomal enzymes. The uptake was inhibited by antibodies against the Mr MPR, but not by antibodies against the Mr MPR. These observations suggest that Mr MPR mediates transport of endogenous but not endocytosis of exogenous lysosomal enzymes. Internalization of receptor antibodies indicated that the failure to mediate endocytosis of lysosomal enzymes is due to an inability of surface Mr MPR to bind ligands rather than its exclusion from the plasma membrane or from internalization. Key words: mannose 6phosphate specific receptor/lysosomal enzymes/targeting Introduction Targeting of newly synthesized lysosomal enzymes depends in many cell types on mannose 6phosphate residues in lysosomal enzymes and their recognition by mannose 6phosphate specific receptors (MPR) (von Figura and Hasilik, 1986; Komfeld, 1986). A mannose 6phosphate binding protein with a subunit molecular size of has been isolated from bovine liver by Sahagian et al. (1981) and demonstrated in many tissues (reviewed in von Figura and Hasilik, 1986). This receptor is a transmembrane glycoprotein (von Figura et al., 1984; Sahagian and Steer, 1985) that binds its ligands in the absence of divalent cations. Recently, Hoflack and Kornfeld (1985a,b) characterized a second mannose 6phosphate binding protein in membranes from bovine liver and murine macrophages, which requires divalent cations for ligand binding. This second MPR is a glycoprotein with a subunit molecular size of and is immunologically distinct from the Mr MPR. With the aid of antibodies blocking the ligand binding site of the Mr MPR it has been shown IR Press imited, Oxford, England previously that this receptor functions in targeting of newly synthesized lysosomal enzymes as well as in endocytosis of exogenous lysosomal enzymes (von Figura et al., 1984; Gartung et al., 1985). In the present study we have utilized antibodies blocking the ligand binding site of the Mr MPR to examine its role in transport of lysosomal enzymes in cells expressing either both types of MPR or only the Mr MPR. Results Blocking antibodies against the Mr MPR The experimental approach for examining the role of Mr MPR in the transport of lysosomal enzymes depended on the availability of antibodies blocking the binding site of the receptor. We examined the effect of receptor antibodies (Ig fraction of a polyclonal antiserum) on the binding of [125I]pentamannosyl6 phosphate bovine serum albumin (PMPBSA) to membranes of M.H cells and P388D1 cells. Both cells are known to express the M, MPR, but not the Mr MPR (Stein et al., 1987; Hoflack and Kornfeld, 1985a). PMPBSA is a neoglycoprotein with 35 pentamannose 6phosphate residues per polypeptide with high affinity to MPR (Braulke et al., 1987). Membranes prepared from both cell types bound PMPBSA in a mannose 6phosphatedependent manner (Table I). The binding was only partially sensitive to inhibition by EGTA. We assume that the strict cation requirement of binding of lysosomal enzymes to the Mr MPR (Hoflack and Kornfeld, 1985a, b) is compensated for by the high degree of substitution of PMPBSA with pentamannose 6phosphate residues. Treatment of membranes with antimr MPR Ig inhibited the binding of PMPBSA to a similar extent as mannose 6phosphate, while treatment with antimr MPR Ig did not inhibit the binding. The slight increase in ligand binding observed in the presence of antimr MPR Ig was due to nonspecific binding. This increase in binding was neither sensitive to EGTA nor to mannose 6phosphate (not shown). The binding of PMPBSA to membranes of U937 cells, which contain the Mr MPR and the Mr MPR (Stein et al., 1987) was inhibited to 62% of control by antimr MPR Ig (not shown). Mr MPR does not mediate endocytosis of lysosomal enzymes It was previously shown that M.H cells do not internalize lysosomal enzymes (Mainferme et al., 1985). In these earlier experiments endocytosis may have been missed due to the rather high concentrations of divalent cations required for binding of ligands to the Mr MPR. Therefore, uptake of lysosomal enzymes by M.H cells was followed in culture medium supplemented with 5 mm MgCl2. Under these conditions, M.H cells also failed to internalize lysosomal enzymes (not shown). Furthermore, M.H cells in the medium containing 5 mm MgCl2 neither bound nor internalized [1251]PMPBSA. Membranes from M.H cells suspended in culture medium containing 5 mm MgCl2 and 0.5% saponin bound [251I]PMP 2677

2 M.Stein et al. Table I. membranes of M.H cells and P388D, macrophages Effect of antimr MPR Ig on binding of [1251]PMPBSA to Additions Ig ['251]PMPBSA bound (3 mg/ml) (ng/mg protein) M.H P388D1 20 mm MgC12 Control mm MgCl2+10 mm Control mannose 6phosphate 10 mm EGTA Control mm MgCl2 AntiMr MPR mm MgCl2 AntiMr MPR All values are the means of duplicates (maximal deviation of the mean was 9%). For experimental details see Materials and methods. Table H. Binding of ['25I]PMPBSA by U937 monocytes Addition ['251]PMPBSA (ng/mg cell protein) Cell surface bounda mm Man6P AntiMr MPR Ig, 10 Ag AntiMr MPR Ig, 10 jg AntiMr MPR antimr MPR Ig, 10 ltg each Internalizeda athe values are the means of duplicates. For experimental details see Materials and methods. BSA in amounts comparable with membranes suspended in Hepes buffer supplemented with 20 mm MgCl2 (Table 1). This finding excluded the possibility that the failure of M.H cells to bind (and to internalize) PMPBSA was due to conditions unsuitable for ligand binding. Furthermore, incubation of the M.H cells for 15 min at 0 C with 5 mm mannose 6phosphate prior to the binding assay did not result in subsequent binding of PMPBSA. This indicated that the lack of the binding of PMP BSA was not due to an interference of endogenous ligands. U937 monocytes, which contain both types of MPR, bound and internalized [1251]PMPBSA (Table H). In the presence of 5 mm mannose 6phosphate, binding was reduced to 20% and uptake to 3% of control. Antibodies against the Mr MPR decreased the binding to 51 % and the uptake to 26% of control, while antibodies against the Mr MPR had no effect on binding and uptake. From the inability of M.H cells to bind and internalize mannose 6phosphatecontaining ligands and from the failure of blocking antibodies against the Mr MPR to inhibit binding and uptake of the ligands by U937 monocytes we concluded that the Mr MPR is not involved in endocytosis of ligands. Receptormediated uptake of antimr MPR Ig A small fraction of Mr MPR is located at the cell surface. In U937 monocytes the Mr MPR at the cell surface accounts for 3% of the total receptor (Stein et al., 1987). Preliminary immunocytochemical data suggest that in M.H cells 10% of the Mr MPR is located at the cell surface (H.Geuze, unpublished). We examined the ability of fibroblasts to internalize antibodies against the Mr MPR. Fibroblasts were incubated for 4 h at 0 and 370C with '25Ilabelled antimr MPR Ig. The experiments were performed in 2678 Table Im. Uptake of [1251]antiMr MPR Ig by fibroblasts Cellassociated [1251]antiMr 0 C A 5.4 (3.4) B 3.8 (2.7) AB C A 14.9 (4.1) B 4.4 (2.6) AB MPR Ig (ng/mg cell protein) Cells were incubated for 4 h at 0 or 37 C in medium containing 250 ng/ml iodinated antibody and 20% preimmune serum (A) or 20% antimr MPR serum (B). The amount of cellassociated radioactivity was determined. The numbers in parentheses refer to the radioactivity that could be released from the cells by incubation with pronase for 1 h at 0 C. Specific association is defined as the difference between A and B. All values are the means of duplicates. *1 *~; I A ;, r" Cctitlpsin D : I' PS 1'ii K *CIet i 'v 1 ' j ; Cl!i I' 4 ),* S ;vi.o a& s~~~~~~~~~~~~" 9 _ e )C ;; , I4 f5 Fig. 1. Effect of antimr MPR serum on sorting of cathepsin C and cathepsin D in M.H cells. M.H. cells were labelled with [35S]methionine in the presence of up to 10% antimr MPR serum (a46). The final concentration of serum was adjusted to 15% with control rabbit serum. Cathepsins D and C were immunoprecipitated from extracts of cells and medium. Precursor (P) and mature (M) forms of cathepsin D are indicated at the left margin, that of cathepsin C (4) at the right margin. Mature cathepsin C is represented by at least 14 polypeptides with Mr values ranging from to (Mainferme et ai., 1985). The numbers below the lanes give the percentage of intracellular retained 35Slabelled cathepsins D and C. the presence of an excess of unlabelled preimmune serum or antimr MPR serum. Specific association of the 125ilabelled antibodies to the cells was defined as that inhibitable by the antimr MPR serum. About 6 times more radioactivity associated with the cells at 37 C than at 0 C (Table III). A similar amount of radioactivity was released from the cells that had been incubated at 0 and 370C with the '251labelled antibodies during a 1h incubation at 0 C with pronase. The pronase

3 Role of Mr MPR in targeting of lysosomal enzymes C C.; 1) 3;a f: Ca a Cellsz Medi jr Cells Cells a _ F i robic s'ts + ; le w*... dm VW ft Aum low",* + Oa qf,; _ kj " 0w_ Oe. 9" _ 1.s dome 40 do A.~VA _m _m _''imp mw ma.n 4m ~, w r..l nm3 Fig. 2. Effect of antisera against the Mr MPR and the Mr MPR on targeting of cathepsin D, (3hexosaminidase and arylsulfatase A in fibroblast and HepG2 cells. Fibroblasts and HepG2 cells were labelled with [35S]methionine in the presence of 10% antimr MPR serum (a46) and/or 10% antimr MPR serum (a215). The final concentration of the serum was adjusted to 20% with control rabbit serum. The lysosomal enzymes were sequentially immunoprecipitated from extracts of cells and medium. The precursor (P), intermediate (I) and mature (M) forms of cathepsin D, the precursor (pa, p,b) and mature forms (ma, m13) of the a and 13chain of hexosaminidase and arylsulfatase A (arrow) are indicated. The secreted lysosomal enzyme precursors are shown only for cathepsin D. The numbers below the lanes give the percentage of intracellularly retained 35Slabelled lysosomal enzyme. releasable fraction is assumed to derive from the cell surface. In cells incubated at 0 C with '25Ilabelled antibodies it accounted for about twothirds of cellassociated radioactivity. We conclude from these results that the higher association of radioactivity at 370C results from uptake of the antibodies mediated by the cell surface Mr MPR. Role of Mr MPR in targeting of endogenous lysosomal enzymes M.H cells were metabolically labelled in the presence of preimmune serum or antiserum against the Mr MPR. Extracts of cells and media were analysed for labelled cathepsin C and cathepsin D (Figure 1). In cells that had been incubated in the presence of receptor antiserum, the fraction of intracellular retained cathepsin C and cathepsin D was lower (50 and 78%) than in the control cells (86 and 93%). The secreted enzymes were represented exclusively by the precursor forms, while the cellassociated enzymes were represented largely by the mature (lysosomal) forms of cathepsin C and cathepsin D. The inhibitory effect of the receptor antiserum on intracellular retention of newly synthesized lysosomal enzyme precursors was reproducible, although subject to variation. Occasionally only 30% or less of the newly synthesized cathepsin C and cathepsin D was retained intracellularly. The inhibitory effect of the receptor antiserum on the targeting of newly synthesized cathepsin C and cathepsin D indicates that the Mr MPR mediates transport of newly synthesized lysosomal enzymes to lysosomes. The effect of the receptor antiserum is tentatively explained by the assumptions that (i) binding of the antibodies to receptors at the cell surface functionally inactivates the receptors and (ii) internal receptors are subject to the inactivation due to an equilibrium of internal and cell surface receptors. The functional inactivation of the receptors could be due to the blocking of the ligand binding site (see above) and/or the sequestration of antibodytagged receptors into a pool excluded from targeting of newly synthesized lysosomal enzymes. The effect of the antireceptor serum on intracellular retention of newly synthesized lysosomal enzymes was also examined in human skin fibroblasts and HepG2 cells. These cells express both the Mr MPR and the Mr MPR (Stein et al., 1987). When fibroblasts and HepG2 cells were metabolically labelled in the presence of 10% of antimr MPR serum, the retention of newly synthesized flhexosaminidase and arylsulfatase A was unaffected (Figure 2, lanes 1 and 2). Only in fibroblasts a small, but reproducible increase in secretion was noted for cathepsin D (Figure 2, lanes 1 and 2). Incubation of fibroblasts and HepG2 cells in the presence of 10% of antimr MPR serum significantly decreased the intracellular retention of cathepsin D, 3hexosaminidase and arylsulfatase A (Figure 2, lane 3) as has been reported earlier for fibroblasts (von Figura et al., 1984; Gartung et al., 1985). Increasing the concentration of the antireceptor sera to 20% did not further decrease the retention of the newly synthesized lysosomal enzymes. When fibroblasts and HepG2 cells were incubated in the presence of a combination of the antisera against the Mr MPR and Mr MPR, the retention of the three lysosomal enzymes was significantly lower than in the presence of antimr MPR antiserum alone (Figure 2 lane 4). Discussion The targeting of newly synthesized lysosomal enzymes to lysosomes was inhibited when M.H cells were exposed to antiserum against the Mr MPR. This indicated that the Mr MPR participates in transport of endogenous lysosomal enzymes. We assume that blocking antibodies bind to receptors at the cell surface and that a deficiency of functionally active receptors at the sorting site results from equilibrium of antibodytagged cell surface receptors with internal receptors. In contrast to the antimr MPR serum the antimr MPR serum was ineffective in inhibiting the targeting of lysosomal enzymes in cells that express both the Mr MPR and Mr MPR. In these cells the antimr MPR serum exerted an inhibitory effect on targeting of newly synthesized lysosomal enzymes only in combination with anti 2679

4 M.Stein et al. R A R1 B R1 C Fig. 3. Models for targeting of lysosomal enzymes () by two receptors (RI, ). Binding of the lysosomal enzyme to RI and (models A and B) as well as binding to RI, dissociation from RI and binding to (model C) is assumed to occur in the secretory pathway. Dissociation from the RI and (models A and B) or (model C) is assumed to occur after segregation from the secretory route. Mr MPR serum. This suggests that the Mr MPR contributes to targeting only when the Mr MPR is blocked or that antibodymediated inactivation of Mr MPR can be compensated for by the Mr MPR. Receptordependent targeting is assumed to depend on the binding of the newly synthesized lysosomal enzymes to the receptors within the secretory route, segregation of the MPigand complexes into specific vesicles, delivery of the ligands to elements of the endocytic route and recycling of the receptors to the binding site (von Figura and Hasilik, 1986). Several models are conceivable to explain the function of the two types of MPR within the sorting compartment of a cell. Three models representing extreme views are schematically depicted in Figure 3 (the receptors are designated RI and, the lysosomal enzyme precursors ). In model A the ligand binds either to or. If ligands bind at the same intracellular site to either receptor, compensation of the inactivation of one receptor depends on the transport capacity of the other receptor. If binding to RI and occurs at different sites, only the receptor located more distal along the secretory route could compensate for a functional loss of the other receptor. In the latter case compensation of the inactivation of the Mr MPR in fibroblasts and HepG2 cells by the Mr MPR would therefore imply that the Mr MPR is located proximal to the Mr MPR. According to model B the two receptors are located at different sites and RI, the receptor located more proximally along the secretory route, is responsible for the transport of the bulk of ligands. This model predicts that functional inactivation of has only a marginal effect on targeting of ligands and that functional inactivation of RI may be compensated for by. If this model holds true, the Mr MPR would correspond to proximal receptor RI and the Mr MPR to the distal receptor, since inactivation of the Mr MPR had only a minor effect on targeting. In model C the two receptors operate in sequence in transporting the same ligands. Inactivation of either of the two receptors should produce the same effect. Model C is not compatible with the observation that inactivation of the Mr MPR inhibited targeting only when the Mr MPR was inactivated simultaneously. The available data are compatible with models A and B. Knowledge of the subcellular distribution of the Mr MPR and the Mr MPR among the internal membranes of one cell type may help in understanding how the two types of MPR function in the targeting of newly synthesized lysosomal enzymes. Although present at the cell surface, the Mr MPR did not mediate endocytosis of mannose 6phosphatecontaining ligands such as lysosomal enzymes or the neoglycoprotein PMP BSA. The inability to mediate endocytosis distinguishes the M, MPR from the Mr MPR, which mediates endocytosis of exogenous lysosomal enzymes (Gartung et al., 1985). The failure of the cells to internalize ligands via the M, MPR correlated with the inability of the cell surface Mr MPR to bind ligands. Our results indicate that the failure to bind ligands to the cell surface receptors is not due to the composition of the incubation medium or to interference of the endogenous ligands. Several observations suggested that the receptors at the surface are in equilibrium with internal receptors, e.g. the inhibitory effect of receptor antibodies on the targeting of endogenous lysosomal enzymes is supposed to depend on the exchange of cell surface receptors with internal receptors. Furthermore, antibodies bound to cell surface Mr MPR are subject to internalization. It is unclear why Mr MPR at the cell surface do not bind ligands. It is conceivable that exposure of the receptors at the cell surface alters the conformation or subunit arrangement [the Mr MPR occurs in membranes mostly as a dimer, Stein et al. (1987)], in a manner that is not compatible with ligand binding. In summary, the results presented in this study provide evidence for a function of the Mr MPR in targeting of endogenous lysosomal enzymes. In cells that simultaneously express the Mr MPR and Mr MPR, both receptors are involved in targeting of endogenous lysosomal enzymes. Materials and methods Materials 1251labelled PMPBSA sp. act c.p.m./mg protein was kindly provided by Dr T.Braulke of this institute. The ['2I]PMPBSA has a Kd of x 109 M for the Mr MPR (Braulke et al., 1987). Antibodies The antibodies against rat cathepsin C were kindly provided by Dr F.Mainferme, University of Namur (Mainferme et al., 1985) and the antibodies against rat cathepsin D by Dr Baccino, University of Torino. The antisera and affinitypurified antibodies, human Mr MPR (von Figura et al., 1984), human (hexosaminidase (Hasilik and Neufeld, 1980), human cathepsin D (Gieselmann et al., 1983) and human arylsulfatase A (Waheed et al., 1982) were those described. The antiserum against the Mr MPR purified from human liver according to Hoflack and Komfeld (1985a) was raised in rabbits. The antiserum and affinitypurified immunoglobulins were monospecific for the Mr MPR as shown in immunoblots and by immunoprecipitation of the receptor from extracts of metabolically labelled cells (Stein et al., 1987). Cell culture and metabolic labelling Fibroblasts, U937 monocytes, HepG2 cells (all of human origin), rat M.H cells were grown and metabolically labelled as described (Stein et al., 1987), murine P388D, macrophages according to Hoflack and Kornfeld (1985a). The labelling period with [35S]methionine (sp. act TBcq/mmol) was 12 h. Where indicated the fetal calf serum in the labelling medium was replaced by rabbit serum (control serum or antiserum against the Mr MPR and Mr MPR). The rabbit sera were heat inactivated (56 C for 30 min) and dialysed overnight against serumfree medium (Gorham and Waymouth, 1965, modified, as formulated in the catalogue of GIBCO) or against RPMI 1640 if used for labelling of U937 monocytes. Endocytosis of labelled lysosomal enzymes Radioactive secretions were prepared from human skin fibroblasts incubated in the presence of [35S]methionine and 10 mm NH4Cl as described (von Figura et al., 1983). Recipient M.H cells were incubated in medium supplemented with the radioactive secretions and 5 mm MgCl2 for 24 h and analysed for internalized flhexosaminidase and arylsulfatase A (von Figura et al., 1983). 2680

5 Role of Mr MPR in targeting of lysosomal enzymes Immunoprecipitation Extracts of cells and medium of fibroblasts, HepG2 cells, U937 monocytes (Gieselmann et al., 1983) and M.H cells (Mainferme et al., 1985) were prepared and subjected to sequential immunoprecipitation of the lysosomal enzymes as described. The immunoprecipitates were solubilized in the presence of SDS and dithiothreitol (except for rat cathepsin C, which was solubilized with SDS only), separated by electrophoresis in % polyacrylamide gels (aemmli, 1970) and visualized by fluorography (askey and Mills, 1975). Bands visible in fluorograms were quantified by densitometry. Binding and uptake of iodinated antimr MPR Ig The affinitypurified receptor antibodies were iodinated using Na125I (17 Ci/mg I, Amersham) and lodogen (Pierce Chemical Co., Rockford), accroding to Parker and Strominger (1983) to a specific activity of 4000 c.p.m./ng protein. Fibroblasts were incubated for 4 h at 0 C (placed on ice water) or at 37 C in the respective culture medium containing 250 ng/ml of the iodinated antibody. The fetal calf serum of the culture medium was replaced by 20% heatinactivated (56 C for 30 min) rabbit serum (preimmune or antimr MPR serum). After incubation the cells were washed five times with icecold Hank's balanced salt solution and incubated for 1 h at 0 C with 0.1% pronase (Calbiochem). The radioactivity solubilized with pronase from cells incubated with the ligand at 37 C represents receptor antibodies associated with the cell surface, while the radioactivity resistant to pronase represents by and large the internalized receptor antibodies. Binding and uptake of [1251]PMPBSA by cells Cells were incubated for 2 h at 37 C in medium supplemented with 10% heatinactivated fetal calf serum, 5 mm MgCl2 and 108 M [1251]PMPBSA ( c.p.m./ml). As indicated the medium was supplemented with 5 mm mannose 6phosphate, 5 mm glucose 6phosphate or affinitypurified antibodies against the Mr MPR or Mr MPR. Cell surfaceassociated [125I]PMP BSA was released by incubation with Hank's balanced salt solution adjusted to ph 3.0 for 15 min. This incubation was repeated once. The radioactivity remaining with the cells was resistant to solubilization with trypsin and represented the faction of internalized [1251]PMPBSA. Binding of [1251]PMPBSA by membranes A mixture of cell surface and internal membranes from M.H cells and P388D, macrophages was prepared and incubated with ligand following the procedure of Hoflack and Kornfeld (1985b). The membranes, 300 ug protein, were incubated for 90 min on ice in 0.15 mi of 50 mm Hepes ph 7.8, containing 0.15 M NaCl, 0.5% saponin, 0.17 tiypsin inhibitor units aprotinin, 5 mm sodium,bglycerophosphate, the additions (MgCl2, mannose 6phosphate, EGTA) and 3 mg/ml Ig as indicated. The Ig were purified with the aid of protein ASepharose 4B (Pharmacia). Then [125I]PMPBSA (150 ng) was added. After incubation for 90 min on ice, the membranes were collected by centrifugation for 20 min at g and washed twice with Hepes buffer prior to determination of radioactivity. Other methods Protein was detenmined according to Peterson (1977) using bovine serum albumin as standard. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. References Braulke,T., Gartung,C., Hasilik,A. and von Figura,K. (1987) J. Cell Biol., 104, Gartung,C., Braulke,T., Hasilik,A. and von Figura,K. (1985) EMBO J., 4, Gieselmann,V., Pohlmann,R., Hasilik,A. and von Figura,K. (1983) J. Cell Biol., 97, 15. Gorham,.W. and Waymouth,G. (1965) Proc. Soc. Exp. Biol. Med., 119, Hasilik,A. and Neufeld,E.F. (1980) J. Biol. Chem., 255, Hoflack,B. and Kornfeld,S. (1985a) J. Biol. Chem., 260, Hoflack,B. and Kornfeld,S. (1985b) Proc. Natl. Acad. Sci. USA, 82, Kornfeld,S. (1986) J. Clin. Invest., 77, 16. aemmli,u.k. (1970) Nature, 227, askey,r.a. and Mills,A.D. (1975) Eur. J. Biochem., 56, Mainferme,F., Wattiaux,R. and von Figura,K. (1985) Eur. J. Biochem., 153, Parker,K.C. and Strominger,J.. (1983) Biochemistry, 22, Peterson,G.. (1977) Anal. Biochem., 83, Sahagian,G.G. and Steer,C.J. (1985) J. Biol. Chem., 260, Sahagian,G.G., Distler,J. and Jourdian,G.W. (1981) Proc. Natl. Acad. Sci. USA, 78, Stein,M., Braulke,T., Krentler,C., Hasilik,A. and von Figura,K. (1987) Biol. Chem. HoppeSeyler, 36, von Figura,K. and Hasilik,A. (1986) Annu. Rev. Biochem., 55, von Figura,K., Steckel,F. and Hasilik,A. (1983) Proc. Natl. Acad. Sci. USA, 80, von Figura,K., Gieselmann,V. and Hasilik,A. (1984) EMBO J., 3, von Figura,K., Gieselmann,V. and Hasilik,A. (1986) Biochem. J., 225, Waheed,A., Hasilik,A. and von Figura,K. (1982) Hoppe Seyler's Z. Physiol. Chem., 363, Received on March 11, 1987; revised on May 15,