Purification and characterization of human lysosomal protective protein expressed in stably transformed Chinese hamster ovary cells.

Chinese hamster ovary cells were transfected with a recombinant DNA containing the entire coding sequence of human lysosomal protective protein cDNA under the control of mouse metallothionein I promoter. Neomycin and methotrexate-resistant stably transformed cell lines expressing this protein were isolated. Immunoprecipitation of the product with antiserum against human placental protective protein-beta-galactosidase complex revealed a 52-kDa protective protein precursor, which was then processed to mature form, a heterodimer of 32- and 20-kDa polypeptides. The precursor secreted in the culture medium was taken up by the mannose 6-phosphate receptor system and restored acid carboxypeptidase, beta-galactosidase, and neuraminidase activities in galactosialidosis fibroblasts. The expressed protein showed a granular pattern in intracellular distribution, was fractionated at the density of lysosomes, and had serine esterase activities; acid carboxypeptidase at pH 5.6, esterase at pH 7.0, and carboxyl-terminal deamidase at pH 7.0. They were inhibited simultaneously by phenylmethylsulfonyl fluoride, N-benzyloxycarbonyl-L-phenylalanine chloromethyl ketone, or iodoacetamide. The acid carboxypeptidase activity of the purified monomeric mature protective protein was labile in vitro under the acidic condition. Saposins (sphingolipid activator proteins) stabilized the activity at micromolar level concentrations.

galactosidase to form a high molecular weight complex also involving neuraminidase (5)(6)(7). The precursor protein secreted from normal fibroblasts treated with ammonium chloride is taken up by galactosialidosis fibroblasts via the mannose 6-phosphate receptor system and restores p-galactosidase and neuraminidase activities (1,2).
A cDNA encoding this protein was cloned by Galjart et al. (8), and the deduced amino acid sequence was found to share homology to yeast carboxypeptidase Y and KEXl gene product (8,9) as well as plant carboxypeptidase (10). In fact, acid carboxypeptidase activity, with optimal pH at 5.5, is deficient in fibroblasts and lymphoblastoid cells from galactosialidosis patients (11,12). On the other hand, an enzyme with deamidase, esterase, acid carboxy peptidase, and cathespin A-like activities was purified from human platelets, which modified the carboxyl termini of tachykinins (13) and endothelin 1 (14). The sequences of the N-terminal25 amino acids in each of its two polypeptide chains were identical with those deduced for two polypeptides of the mature form protective protein (13). We demonstrated simultaneous deficiency of their activities in galactosialidosis fibroblasts (15). A significant increase of cathepsin A-like activity was confirmed in COS-1 cells transiently expressing the cDNA (16).
In this study, we established a stably transformed Chinese hamster ovary (CHO)' cell line overexpressing human protective protein and multifunctional serine esterase (acid carboxypeptidase, esterase, and deamidase) activities. The gene product was then purified and characterized. Its activity was stablized by saposins (sphingolipid activator proteins).
Cell Culture-Skin fibroblasts were obtained from patients with late onset galactosialidosis. The diagnosis of the disease was established by clinical manifestations and enzyme assays (3). CHO cells and skin fibroblasts were routinely maintained in Ham's F-10 medium supplemented with 10% fetal calf serum (FCS). For eznyme purification, the transformed cells (5 X lo7) were cultured for a week in a roller bottle (Falcon 303.5, Becton Dickinson Labware, NJ; surface area 1750 cm2) with FCS-free GIT medium (Wako Pure Chemicals, Osaka, Japan, 300 mlhottle) in the presence of 4 p~ MTX.
Enzyme Assays-8-Galactosidase, neuraminidase, and p-hexosaminidase activities were assayed fluorometrically (17). Serine esterase activities of protective protein were measured as described previously (15) using Z-Phe-Leu for acid carboxypeptidase, benzoyltyrosine ethyl ester for esterase, and DME-NH2 for deamidase as substrates. Protein determination was performed by the method of Bradford (18) using bovine serum albumin as standard.
For characterization of acid carboxypeptidase activity, some protease inhibitors or active site reagents were added to the assay mixture in 20 mM sodium acetate (pH 5.6). They did not interfere with determination of liberated L-leucine.
pMSXND (19) (20) was subcloned into the XhoI site of pMSXND, which also contained the genes for both G418 resistance and mouse dihydrofolate reductase, and designated as pMSXND(PP11). Transfection of CHO Cells and Selection of Stably Transformed Cells-The procedures were performed as described by Kyle et al. (19) with minor modifications. CHO cells (1-2 X lo5 cells) were seeded on 60-mm dishes 18 h prior to the addition of DNA. Transfection was conducted using a lipofection reagent (Bethesda Research Laboratories) (21) containing 20 pg of pMSXND or pMSXND(PP11). The lipofection reagent-plasmid DNA complex was left on the cells in the FCS-free medium for 15 h, and then an equal volume of the medium containing 20% FCS was added. After 48 h in culture, the cells on each 60-mm dish were trypsinized and split 1:5 into two 100-mm dishes, and a selection medium (Ham's F-10 with 10% FCS and 400 pg/ml G418) was added. After 18 days, G418resistant colonies were isolated and further cultured in the first amplification medium (Ham's F-10 with 10% dialyzed FCS and 0.2 p~ MTX). The MTX concentration was increased four times every 4 days up to 4 p~ and maintained. The selected cell lines were analyzed for expression of human protective protein.
Indirect Immunofluorescence Staining-The transformed CHO cells were seeded on a tissue culture chamber/slide (Lab-Tec; Nunc, Naperville, IL). After a 24-h culture, they were fixed with cold 20% methanol and immunostained by a two-step incubation method (22): first with a 1:lOOO dilution of 8-galactosidase complex antiserum (4) and then with a 1:250 dilution of goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (Seikagaku Kogyo, Tokyo, Japan). Subcellular Fractionation of Protective Protein-Confluent cells on a 125-mm dish were harvested by trypsinization, suspended in 300 p1 of 0.25 M sucrose, and homogenized by sonication for 5 s twice. The homogenate was centrifuged at 5000 X g for 10 min at 4 "C. The resultant postmitochondrial fraction was loaded on 8 ml of 25% Percoll (Pharmacia LKB Biotechnology, Uppsala, Sweden) in 0.25 M sucrose. Acid carboxypeptidase, @-galactosidase, and P-hexosaminidase activities were assayed after centrifugation (30,000 rpm for 40 min; Beckman 50Ti rotor) and fractionation. A density marker bead kit (Pharmacia) was utilized for calibration of the density gradients.
Immunoprecipitation of Protective Protein-Subconfluent transformed CHO cells were incubated for 1 h in methionine/cysteine-free Dulbecco's minimum essential medium and labeled for 16 h with The cell extract was then precleared by incubation with rabbit anti-human placental 8-hexosaminidase serum and with 40 pl of 3% (w/v) protein A-Sepharose CL-4B (Pharmacia) and centrifuged. The supernatant was incubated with an antiserum against human placental high molecular weight complex of 8-galactosidase associated with protective protein (@-galactosidase complex antiserum) (4) overnight at 4 "C with shaking.
The immune complex was precipitated by the addition of 40 pl of 3% (w/v) protein A-Sepharose CL-4B, and the pellet was washed three times with washing buffer (25 mM Tris-HC1 (pH 7.5) containing 0.5 M NaCl, 0.5% Triton X-100, 0.1% SDS, and 1 mM EDTA). The antigen-antibody complex was eluted from the protein A-Sepharose pellet by boiling in the sample buffer for 3 min and was analyzed in SDS-polyacrylamide gel electrophoresis (PAGE) using 10-20% gradient gel (Daiichi Pure Chemicals, Tokyo, Japan) according to the method of Laemmli (23). The gel was subsequently impregnated with EN3HANCE (Amersham, Buckinghamshire, UK), and the autoradiograph of dried gel was obtained on x-ray film at -80 "C.
Preparation of Protective Protein Precursor-The stably transformed CHO cells were seeded on 125-mm dishes in subconfluency and cultured for 24 h. They were washed with PBS twice and further cultured in 25 ml of FCS-free Ham's F-10 containing 10 mM ammonium chloride. After a 3-day incubation, the culture medium was harvested. Ammonium chloride was removed by dilution with 25 volumes of F-10 and concentration with Centriprep 30 (Amicon Corp., Danvers, MA). The concentrate (0.5 ml) was treated at 25 "C for 1 h with 0.2 mM 8-Gal MNT to eliminate endogenous 8-galactosidase activity (24). The inhibitor was removed by dilution with 200 volumes of PBS and concentration with Centriprep 30. The concentrate was stored at -80 "C as a preparation of protective protein precursor.
Uptake of Protective Protein Precursor in Galactosialidosis Fibroblasts-The preparation described above was thawed and sterilized by passing through a 0.22-nm membrane filter (Mirex GV; Millipore, Tokyo, Japan). Galactosialidosis fibroblasts were seeded on 35-mm dishes in subconfluency and cultured in 2 ml of regular F-10. After a 24-h incubation, the culture medium was replaced with fresh medium, and 0.25 volume of the sterile preparation of protective protein precursor was added. Some experiments were performed in the presence of Man-6-P. After 3 days, the cells were washed with PBS three times, harvested, and homogenized in 0.2 ml of distilled water by sonication for 5 s. Enzyme activities and protein concentration in the homogenate were measured as described above.
Immunotitration of Protective Protein-The transformed cells (1-2 X lo9) harvested from 20 roller bottle cultures were homogenized in distilled water containing 0.25 mM leupeptin and 1 mM EDTA by sonication for 10 s twice and then centrifuged at 100,000 X g for 1 h at 4 "C. Each 25 pl of the resultant supernatant (cell extract) was incubated with various dosages of serum IgG in 20 mM sodium phosphate (pH 6.0) (final volume, 50 pl) and left for 3 h at 4 "C. The IgG was prepared from the @-galactosidase complex antiserum or from normal rabbit serum by ammonium sulfate precipitation (60% saturation) and %fold Concentration. Subsequently, 20 pl of 3% (w/ v) protein A-Sepharose was added. After incubation for 1 h at 4 "C with shaking, the mixture was centrifuged at 10,000 X g for 5 min. 8-Galactosidase and acid carboxypeptidase activities in the supernatant were measured as described above.
Purification of Mature Protective Protein-One-ninth volume of 0.25 M imidazole HC1 buffer (pH 7.4) was added to the cell extract prepared as described above, and then the suspension (12 ml) was loaded (120 ml/h) on a chromatofocusing column (Pharmacia PBE94, 0.9 X 30 cm) preequilibrated with 25 mM imidazole HCl buffer (pH 7.4) at 4 "C. The column was washed with 2.5 volumes of the buffer, and the enzyme was eluted (12 ml/h) with Polybuffer 74 (Pharmacia) that had been diluted 1% with water (pH 4.0). Mainly acid carboxypeptidase activity was monitored for protective protein, but deamidase and esterase activities were also assayed. The fractions with enzyme activities were pooled and stored at -80 "C. The combined fraction was thawed, and the buffer composition was changed three times by concentration and dilution with 30 volumes of 20 mM sodium phosphate buffer (pH 7.2) in Centriprep 30 (Amicon).
Finally, the fraction was concentrated to 1 ml, and 1 ml of 4 M ammonium sulfate was added. The suspension was loaded (15 ml/h) at room temperature on a TSK gel Ether-5PW column (0.75 X 7.5 cm; Tosoh, Tokyo, Japan) preequilibrated with 20 mM sodium phosphate (pH 6.3) containing 2 M ammonium sulfate and washed with the same buffer. After the absorbance of the effluent at 280 nm was decreased to the base line, protein was eluted with a pH gradient (6.3-8.0)/ammonium sulfate gradient (2.0-0 M ) made hy the starting buffer and 20 mM sodium phosphate buffer (pH 8.0). The fractions with enzyme activities were pooled, and 0.01 volume of 10% CHAPS was added to each fraction.
The buffer composition was then changed to 20 mM sodium acetate buffer (pH 4.8) containing 2 mM CHAPS with Centriprep 30. The fraction was concentrated to 1 ml and applied to a Mono S column (HR5/5, Pharmacia) preequilihrated with the same huffer and eluted with a sodium chloride gradient (0-0.3 M ) at room temperature. The fractions with enzyme activities were pooled and stored at -80 "C.
Purity of the preparation was monitored hy SDS-PAGE on a 10-20% gradient gel. Proteins were visualized with a silver stain kit (Wako Pure Chemicals) or by immunostaining. SDS-PAGE gel was electroblotted to a nitrocellulose memhrnne (HA 85, Schleicher and Schuell) (25) and prohed with a 1:500 dilution of the (f-galactosidase complex antiserum. Detection of immunoreactive hands was performed with a blotting detection kit for rahhit antibodies (RI'N 23, Amersham).
Gel Fi'iltration-The molecular mass of purified protective protein was determined hy gel filtration according to the method of Siege1 and Monty (26) on a TSK gel G3000SWsl. column (0.78 X 30 cm) preequilihrated and eluted with 20 mM sodium acetate buffer, pH 5.6, containing 0.1 M NaCl and 2 mM CHAPS at a flow rate of 0 5 ml/ min. The protein preparation was applied in a volume of 25 p l , and 0.5-ml fractions were collected.
Characterization of Acid Carboxypeptidase Actiuity-The stahility of acid Carboxypeptidase activity in the purified mature protective protein was examined in the Ether-5PW preparation of 37 "C, either at pH 4.4 in 40 mM sodium acetate or at pH 7.2 in 40 mM sodium phosphate (total volume, 50 pl; activity, 30 nmol/h for each experiment). In some experiments, saposin, control proteins (lysozyme, hovine serum albumin), or laboratory detergent was added. Prosaposin was purified from human milk (27), and saposins A, R. C, and D were purified from human Caucher disense spleen (28.29).  Fig. 1, lanes I, 5, 7, and 9). They were not detected in mock transfected cells (lanes 3 and 4 ) . After a chase of 6 h, their intensities decreased (lanes 2, 6, 8, and IO). Immunoreactive bands were not detected in the culture medium (data not shown).

Expression of Protective Protein in Transformed
Intracellular Distribution of Expressed Protective Protein-Indirect immunofluorescence staining was performed with the @-galactosidase complex antiserum for subcellular localization of expressed protective protein. A granular pattern was observed (Fig. 2). Mock transfected cells reacted poorly with the antiserum (data not shown).
Restoration of Enzyme Activities in Galactosialidosis Fibroblasts by Protective Protein Precursor-@-Galactosidase and neuraminidase activities were restored in galactosialidosis fibroblasts after the preparation of protective protein precursor from the transformed CHO cells was added in the culture medium (Table I). Acid carboxypeptidase activity was also increased. These changes were not observed when Man-6-P was added in the culture medium. We concluded that functionally active protective protein was expressed in the cells and secreted in the culture medium in the presence of ammonium chloride.
Expression of Serine Esterase Activities-Serine esterase activities were increased in the stably transformed cells; acid Carboxypeptidase 7-fold, esterase %fold, and deamidase 1.4fold, as compared with mock transfected cells (Fig.   3). T h e basal levels of the endogenous activities of parental CHO cells were different among the three enzyme activities, and therefore, the calculated amounts of increase were variable. Immunotitration of Acid Carhoxypcptidasr. Acticity-Both /$galactosidase and acid carboxypept idase activities in the transformed cells were dose dependently immunoprecipitated together, by the /?-galactosidase complex antiserum, hy 50 and 7055, respectively, with 10 p1 of the antiserum (Fig. 4). Neither of them was precipitated hy control senlm. This result indicated the presence of acid carhox?lpeptidase activity in the /jgalactosidase-protective protein complex. Endogenous CHO /j-galactosidase was less efficiently precipitated prohahly clue t o low cross-immunoreactivity with human antihodv. Subccllular Ilistrihution of Acid Carboxypeptidase Activity-Half of the acid carbox-peptidase activity in the postmitochondrial fraction was found in the heavy fraction (density, 1.06 g/ml) and another half in the light fraction (densitv, 1.04 g/ml) (Fig. 5 ) . /j-Hexosaminidase and /3-galactosidase were recovered a s lysosomal marker enzymes in the light fraction. This result indicates that half of the expressed protein was localized in the lysosome; the remainder staved in heavy density granules and WAS not associated with the IJ-galactosidase molecule.
Effects of Protcnse Inhibitors on Acid Cnrhr~xyprptidasc Ac- Restoration of @-galactosidase, neuraminidase, and acid carboxypeptidase activities in galactosialidosis fibroblasts after addition of protective protein precursor The precursor fraction prepared as described under "Experimental Procedures" was added to the culture medium of galactosialidosis fibroblasts. After 3 days, cells were harvested, and enzyme activities were measured. Substrates are 4-methylumbelliferyl derivatives for neuraminidase, @-galactosidase, and @-hexosaminidase and Z-Phe-Leu for acid carboxypeptidase. Enzyme activity is expressed as nmol/h/mg of protein (acid carboxypeptidase, neuraminidase, @-galactosidase) or as pmol/h/mg of protein (@-hexosaminidase) (mean f S.D., four experiments).

FIG. 4. Immunotitration of &galactosidase and acid carboxypeptidase activities in transformed CHO cells.
Increasing amounts of IgG fractions prepared from human protective protein-@galactosidase complex antiserum (0) or control normal serum (0) were added to the cell extract. The immune complex was removed by the addition of protein A-Sepharose, and the remaining P-galactosidase (panel A ) and acid carboxypeptidase (panel B ) activities were measured in the supernatant. 10 mM). Low molecular weight protease inhibitors of microbial origin (30), such as leupeptin, pepstatin A, or phosphoramidon, did not affect the activity at pH 5.6 ( Table 11).
Purification of Mature Protective Protein Monitored by Serine Esterase Activities- Table I11    galactosidase activity in the starting material and the second peak 24.3%.
The first peak with relatively high specific activity of acid carboxypeptidase and low P-galactosidase activity was concentrated and was applied to hydrophobic chromatography and then to Mono S. Initially, the enzyme activity disappeared almost completely after the second step (Ether-5PW); it was later found that the enzyme was unstable and lost its activity simply by standing a t 4 "C for several hours. Detergents were found to maintain the enzyme activity. We therefore added 2 mM CHAPS for subsequent procedures. The recovery of the enzyme activity was 876, and the degree of purification was about 230-fold. Esterase and deamidase activities were copurified with acid carboxypeptidase activity.
The resultant fraction contained two polypeptides of 32 and 20 kDa in SDS-PAGE under the reducing condition (Fig.  6A). They were immunostained by the @-galactosidase complex antiserum (Fig. 6R). The 20-kDa polypeptide was more intensely stained because of a higher content of IgG against it in the antiserum. +Galactosidase or neuroaminidase activity was hardly detected in this preparation. The molecular mass of purified enzyme was estimated to he approximately 52.5 kDa by gel filtration at pH 5.6 ( Fig. 7 ) . It wvas prohahly a monomeric mature protective protein. The value did not change significantly when estimated at pH 6.8 (data not shown). Stability of Acid Carboxypeptidase Acfioity-The purified protective protein lost its acid carhox-ypeptidase activity in the eluate of hydrophohic chromatographv after incuhation at 37 "C for 1 h, either at pH 4.4 or at pH 7.2 (Fig. 8). This inactivation was almost completely protected by 2 mM CHAPS under the acidic condition but not at neutral pH. Other detergents also showed the same effect as CHAPS: Triton X-100 at 0.02-0.2 mM and n-octvl glucoside a t 2-10 mM (data not shown).
We then surveyed the effects of saposins as natural detergents of lipid substrates or as acid hydrolase activators. As shown in Fig. 9, each molecular species of saposins A-D showed stahilizing effects on the enzvme activity under the acidic condition. No effect was ohserved at neutral pH. The half-maximal effective dose was 3.6 pg/ml (360 n M ) for saposin A, 1.4 bg/ml (140 nM) for saposin R, 4.9 pg/ml (490 nM) for saposin c , and 5.4 pg/ml (540 n M ) for saposin D. Saposin R was slightly more effective than the others. Their precursor prosaposin as well as lysozyme did not have remarkahle effects on the enzyme activity. Bovine serum alhumin also showed the same effect, hut the half-maximal dose was much higher as compared with those for saposins.

DISCUSSION
We established a stably transformed CHO cell line permanently expressing human protective protein cDNA in this study. Immunoprecipitation analysis indicated that the gene product was synthesized as a 52-kDa precursor, which was processed to a mature heterodimer of 32-and 20-kDa polypeptides. Immunofluorescence staining of the transformed cells showed a granular pattern characteristic of lysosomes. The precursor form of this protein, secreted in the culture medium in the presence of alkalizing agent, was taken up via the Man-6-P receptor pathway and restored enzyme activities in galactosialidosis fibroblasts. These transformed cells expressed serine esterase activities assayed as acid carboxypeptidase, esterase, and deamidase. About half of the acid carboxypeptidase activity was recovered in the same fraction as other lysosomal enzymes by subcellular fractionation. This result indicates that the expressed protective protein was transported to lysosomes.
We reported that galactosialidosis cells are deficient in serine esterase activities as well as @-galactosidase and neuraminidase activities (12, 15). In this study, we further demonstrated that the mature form of protective protein was multifunctional serine esterase activities in a single molecule on the basis of the following observations: 1) simultaneous increase of protective effects and enzyme activities; 2) immunoprecipitation of acid carboxypeptidase activity by an antiserum against protective protein-@-galactosidase complex; 3) the same sensitivity to three enzyme inhibitors acting on different sites of the protein (11,12), PMSF as serine reagent, ZPCK as histidine reagent, and iodoacetamide as a cysteine reagent; 4) the presence of neutral esterase and deamidase activities in a purified protective protein preparation; 5) the purified protein was recovered as a heterodimeric form composed of two smaller polypeptides (32 and 20 kDa) that were detected by an antiserum against the protective protein-@galactosidase complex.
Protective protein interacts with @-galactosidase and neuraminidase in the lysosome to form a high molecular weight aggregate (5-7), and an inactive form of neuraminidase protein was activated by association with @-galactosidase and protective protein (31). In our present study, overexpressed human protective protein in transformed CHO cells was purified as a monomeric mature form dissociated from the complex with @-galactosidase and neuraminidase. The monomeric enzyme was labile at acidic pH. Jackman et al. (13) reported that purified human platelet deamidase, probably identical with mature protective protein, has a homodimer structure in gel filtration at pH 6.0. These results suggest that serine esterase activity of protective protein is also stabilized by self-dimerization or by aggregate formation with other proteins in lysosomes. The interaction may be physiologically important for mutual stabilization of the three functional proteins in the cell.
Recently, Zhou et al. (32) demonstrated that the protective protein precursor overexpressed in COS cells exists as a homodimer a t neutral pH. They also identified a point mutation in the protective protein gene from a patient with late infantile galactosialidosis, causing a substitution of Phe-412 with Val in the gene product. The expressed mutant precursor protein was partially retained in the endoplasmic reticulum and did not form the homodimeric structure. The dimerization process might be a condition for the proper targeting from the endoplasmic reticulum to endosome or prelysosome and for stable conformation of the protein (32). It is possible that a specific mutation of protective protein gene causing a defect in aggregate formation leads to inactivation of serine esterase functions, even if the enzyme activities are not lost by the mutation itself, in some cases of galactosialidosis.
In this study, the enzymatic instability of dissociated mature protective protein was restored by experimental detergents or saposins in uitro. Saposins are heat-stable proteins of low molecular mass (8-13 kDa) necessary for hydrolysis of sphingolipids by lysosomal hydrolases (33). They are produced by a specific proteolytic processing of a 70-kDa precursor protein (prosaposin) consisting of four structurally similar domains (34)(35)(36). Each of them (saposins A-D) is approximately 800 amino acids long, with six similarly spaced cysteine residues, a glycosylation site, and specifically located proline residues. Saposin B binds and solubilizes sphingolipid substrates (sulfatide, ganglioside GMl, and globotriaosylceramide) (37)(38)(39). Saposin C associates with glucosylceramidase or galactosylceramidase and activates the enzyme (28,40,41).

Protective Protein Gene Expression in CHO Cells
Saposin D has been reported to act as a sphingomyelinase activator (29). The modes of intracellular processing are different among various tissues, and their intermediate forms of proteolytic conversion have been detected (28).
This is the first demonstration of the stabilizing effect of saposins on protective protein with serine esterase activities. The effect seems to be caused by physicochemical interaction occurring under a hydrophobic environment, as experimental detergents have the same effect at higher concentrations. The physiological significance of this result is not known at present. Among the four activators for this study, saposin B showed a relatively high stabilizing effect toward acid carboxypeptidase activity. Its relation to the multienzymic complex, probably induced also by hydrophobic interaction in uiuo, should be considered.
Recently, we purified human fi-galactosidase precursor overexpressed in Spodopteru frugiperdu cells infected with recombinant baculovirus that also has strong hydrophobicity (42,43). Further analysis of intermolecular assembly between protective and P-galactosidase proteins will provide us with a clue to the molecular mechanism of multienzymic complex formation in the lysosome.