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Binding of monoclonal antibody to cathepsin M located on the external surface of rabbit lysosomes
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 233, No. 1, August 15, pp. 26’7-2’71, 1984
Binding of Monoclonal Antibody to Cathepsin M Located on the External Surface of Rabbit Lysosomes S. PONTREMOLI,
Institute
of Biological
E. MELLONI, G. DAMIANI, M. MICHETTI, B. SPARATORE, AND B. L. HORECKER*v’ Chemistry, University of Genoa, Italy and *Roehe Institute Roche Research Center, Nut&, New Jersey 07110 Received March
F. SALAMINO,
of Molecular
Biology,
12, 1984
A monoclonal antibody raised against rabbit liver cathepsin M binds to intact rabbit liver lysosomes. The binding is specific and is abolished by treating the lysosomes with trypsin, which has previously been shown to digest the membrane-bound cathepsin M [S. Pontremoli, E. Melloni, M. Michetti, F. Salamino, B. Sparatore, and B. L. Horecker (1982) B&hem. Biophys. Res. Commun, 106,903-9091. Rabbit liver lysosomes are adsorbed onto Sepharose 4B coupled to anti-cathepsin M, but not to Sepharose 4B itself or to Sepharose coupled to a nonspecific antibody. The results confirm the location of membrane-bound cathepsin M on the outer surface of the lysosomal membrane.
Several lysosomal proteinases that catalyze limited proteolysis of cytosolic proteins have been shown to be partly associated with the lysosomal membrane fraction (1, 2). The activities of these membrane-bound proteinases are expressed at neutral pH by undisrupted lysosomes (for a review see (3)), which suggests that they are located on the outer surface of the lysosomes. More direct evidence for this surface orientation was obtained by studying the effects of treatment of the intact lysosomes with soluble or immobilized trypsin. The membrane-bound activities, but not the same activities present within the lysosomes, were inactivated by this treatment (4). In the present report we describe experiments with a monoclonal antibody raised against cathepsin M that confirm the presence of this enzyme on the outer lysosomal surface. EXPERIMENTAL
rified from rabbit liver as described. Sepharose 4B was purchased from Pharmacia Fine Chemicals, and Ultrogel AcA34 from LKB. Trypsin, NADH, fructose 1,6-bisphosphate, bovine serum albumin, sodium dodecyl sulfate (SDS),2 and glycyl phenylalanine-2naphthylamide were from Sigma Chemical Company. The monoclonal antibody (PBM 6,4) directed against glycoproteins IIb and IIIa of human platelets (referred to here as “nonspecific antibody”) and the same antibody labeled with i*C were prepared as described (7). Mice were immunized by intraperitoneal injections at 7-day intervals with 50 pg of purified cathepsin M in complete Freund’s adjuvant. Monoclonal anti-cathepsin M was obtained by fusing spleen cells from the immunized mouse, collected 4 days after the fourth injection, with P3X63Ag8Ul myeloma cells as described (8,9). Fourteen days after fusion, each culture medium was tested for anti-cathepsin M activity by a solid-phase binding assay (8,ll) using 50 pl(25 rg)aliquots of purified cathepsin M in 10 mM sodium phosphate buffer, pH 7.2, containing0.15 M NaCl (PBS) distributed in 72 wells of a flexible polyvinyl chloride microtitration plate (Microtiter Plates, No. l-220-29). After incubation for 24 h at 25°C. the wells were
PROCEDURES
Materials. Cathepsin M (2), the endogenous cytosolic cathepsin M inhibitor (5), and aldolase (6) were pu-
i To whom correspondence
’ Abbreviations used: PBS, 10 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl; SDS, sodium dodecyl sulfate; IgG, immunoglobulin G; Hepes, 4-(2hydroxyethyl)-l-piperazineethanesulfonic acid.
should be addressed. 267
0003-9861/84 $3.00 Copyright All rights
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
268
PONTREMOLI
FIG. 1. Specificity of monoclonal anti-cathepsin M antibody. A solution (1.0 ml) containing washed lysosomal membranes (2 mg protein) solubilized in 0.1 ml 1 M NaCl, 20 mM sodium phosphate buffer, pH 7.2, 0.15 M NaCl (PBS) was treated with 0.1 ml (settled gel volume) Sepharose-coupled nonspecific antibody (monoclonal antibody directed against glycoproteins IIb and IIIa of human platelets). The suspension was rotated end-over-end for 30 min at 5°C. The gel was then removed by centrifugation, and the supernatant solution was treated with 0.1 ml Sepharose-coupled DNAse to remove actin-like substances. After removing the gel by centrifugation, the supernatant solution was treated with 0.05 ml Sepharose-coupled monoclonal anti-cathepsin M antibody and suspended in 0.05 ml PBS. The suspension was gently stirred for 1 h at 5”C, and the gel was collected and washed three times with l-ml portions of 0.1 M Tris-HCl, pH 8.0, containing 0.5 M NaCl, followed by three washings with PBS. The washed gel was suspended in 100 ~1 10% SDS and heated for 1 min at 100°C. Undissolved material was removed by centrifugation, and 50 ~1 of the supernatant solution was used for radioiodination (see Materials and Methods). Solutions (50 ~1) containing 5 Fg purified cathepsin M or 50 pg total lysosomal membrane proteins after solubilization with 1 M NaCl were also labeled with ‘%I as described for the labeling of the antibodies. Aliquots of each sample containing 25,000 cpm were suspended in 0.1 M sodium phosphate, pH 7.0, containing 2% SDS and 2% mercaptoethanol, and analyzed on 10% polyacrylamide slab-gels containing 0.1% SDS. After electrophoresis for 4 h in 0.1 M sodium phosphate buffer,
ET AL. washed two times with PBS. Each well then received 200 ~1 of 5% bovine serum albumin dissolved in the washing buffer, and was incubated for 2 h at 25°C. The albumin solution was removed and the wells were washed extensively with PBS. Aliquots (70 ~1) of each culture supernatant were then added to the treated wells and left for 4 h at 25°C. After washing five times with PBS, 70 pl (0.48 pg, 12 X lo6 cpm/mg) of ‘%Ilabeled rabbit anti-mouse IgG antiserum was added to each well. After 2 h at 25°C the wells were washed five times and cut apart, and the radioactivity in each was determined. A culture medium was considered positive when the bound radioactivity exceeded by at least fourfold the quantity detected in a control containing medium from P3X63Ag8Ul cells. Four of the wells were found to be positive for anti-cathepsin activity. One (CM 7), showing the highest activity, was selected and repeatedly cloned in soft agar as described by Coffino et al. (11). Subclone CM 7 was injected intraperitoneally into BALB/c mice pretreated with Pristane (2,6,10,14-tetramethylpentadecane). The ascitic fluids (45 ml) were collected, and the proteins were precipitated with 80% saturated ammonium sulfate. The precipitates were suspended in PBS and chromatographed on an Ultrogel AcA 34 column (2.5 X 120 cm) previously equilibrated with the same buffer. The fractions containing anti-cathepsin M were concentrated to 4.0 ml by ultrafiltration on an Amicon UMlO membrane. The antibody was shown to be of the IgG class by its molecular weight (160,000) and by Ouchterlony analysis with anti-w and anti-X antisera. For the preparation of “Clabeled anti-cathepsin M, CM 7 cells were incubated in culture medium containing [i4C]leucine (350 Ci/ mol) [‘“C]valine (200 Ci/mol), and [Wlthreonine (250 Ci/mol) (12). After 6 h the culture medium was collected, and the labeled antibody was recovered as described above for the unlabeled anti-cathepsin M. Methods. The monoclonal antibodies (CM 7.2 and PBM 6.4) were coupled to BrCN-activated Sepharose 4B by the procedure described by Pontremoli et al. (4). Proteins were labeled by iodination using chloramine T according to Greenwood et al (13). Gel electrophoresis in SDS was carried out in 10% polyaerylamide gels (14). Assays for cathepsin M (2) and cathepsin C (15) were as described. The heavy particle fraction was prepared from rabbit liver, and the membrane fraction recovered after freezing and thawing of the heavy particle fraction was solubilized with 1 M NaCl as described (15). Treatment of the heavy particle fraction with trypsin
pH 7.0, the protein bands were revealed by autoradiography. Lane 1, ‘%I-labeled lysosomal membrane proteins; Lane 2, ‘?-labeled proteins recovered from anti-cathepsin M-Sepharose; lane 3, ‘%I-labeled cathepsin M.
CATHEPSIN
M MONOCLONAL
ANTIBODY
BINDING
269
was as described (4), except that the incubation mixtures contained 10 IIIM sodium borate, pH 7.4, instead of Hepes buffer, pH 7.2. RESULTS
Characterization of the anti-cathepsin M mono&ma1 antibody. Binding of cathepsin M was demonstrated with the Sepharosecoupled antibody. A suspension of matrixbound antibody containing 40 pug protein was capable of binding 50 units (10 pg) of purified soluble cathepsin M, or the same number of units of cathepsin M from a preparation of solubilized lysosomal membranes. Fructose-1,6-bisphosphatase-converting enzyme activity (15) in the same preparation was not removed by the immobilized antibody (data not shown). Specificity of binding was confirmed in a similar experiment in which the mixture of proteins eluted from the lysosomal membranes was labeled by iodination (Fig. 1). In addition to other proteins, this mixture contains cathepsin M (M, = 30,000) (2) and fructose-1,6-bisphosphatase converting-enzyme (M,. = 70,000) (15). When the adsorbed proteins were recovered from the gel and analyzed by SDS-polyacrylamide slab-gel electrophoresis, only a single band was detected (Fig. 1, lane 2), which migrated at the same position as purified iodinated cathepsin M (Fig. 1, lane 3). No bands were detected corresponding to the converting enzyme or to other proteins present in the membrane eluate (cf. Fig. 1, lane 1). Binding of the mono&ma1 antibody to intact lysosomes. This was demonstrated by incubating the 14C-labeled anti-cathepsin M with intact lysosomes in isotonic sucrose solution (Fig. 2). Binding was specific for the anti-cathepsin M antibody, and was not observed to a significant extent with the radioactive monoclonal antibody directed against membrane glycoproteins IIb and IIIa of human platelets. Binding of anti-cathepsin M was not affected by the presence of the nonspecific antibody. Trypsin-treated lysosomes showed little binding of anti-cathepsin M. We have previously reported (4) that membrane-bound cathepsin M was inactivated by this treatment.
Heavy
particle
fraction
(pl)
FIG. 2. Binding of %-labeled anti-cathepsin M to intact lysosomes. The indicated volumes of heavy particle suspension (1.98 mg protein/ml) were diluted with 1 ml 0.25 M sucrose containing 10 mM sodium acetate, pH 6.5, 1 mg/ml of bovine serum albumin, and 4500 cpm of %-labeled monoclonal anti-cathepsin M alone (filled circles), or together with 25 c(g unlabeled nonspecific monoclonal antibody (open circles). Other reaction mixtures contained trypsin-treated lysosomes and W-labeled anti-cathepsin M (filled triangles), or untreated lysosomes and 5000 cpm nonspecific antibody, labeled as described for the monoclonal anti-cathepsin M antibody (open triangles). The suspensions were rotated end-over-end for 30 min at 5°C and the lysosomes were recovered by centrifugation at 80009 for 10 min. The pellets were washed with 2 ml of isotonic sucrose containing 10 mM sodium acetate, pH 6.5, and 1 mg/ml of bovine serum albumin, and the washed pellets were suspended in 0.5 ml formic acid; 0.2-ml aliquots were added to 10 ml of Pica-Fluor 30 (Packard) for determination of radioactivity.
The binding of anti-cathepsin M to intact lysosomes was also measured with unlabeled antibody. The presence of the antibody on the surface of the lysosomes was detected with lz51-labeled anti-mouse IgG (Fig. 3). No binding of anti-mouse IgG was observed unless the lysosomes were first allowed to bind the specific anti-cathepsin M antibody. Binding of lysosomes to immobilized mono&ma1 anti-cathepsin M. A suspension of anti-cathepsin M coupled to Sepharose 4B showed significant binding of intact rabbit liver lysosomes (Table I). This binding was evaluated by measuring the cathepsin C activity associated with the antibody-coupled resin. No binding was observed to Sepharose 4B itself or to
PONTREMOLI
270
Heavy
particle
fraction
(~111
FIG. 3. Binding of anti-cathepsin M antibody to intact lysosomes determined with ‘?-labeled antimouse IgG. The reaction mixtures containing the indicated volumes of the heavy particle suspension and 25 pg unlabeled anti-cathepsin M antibody were incubated, and the lysosomes were recovered as described in Fig. 2. The washed pellets were suspended in 2 ml isotonic sucrose containing 10 mM sodium acetate, pH 6.5, and 0.1 ml of a solution containing 2 X 10’ cpm ‘?-labeled rabbit anti-mouse IgG was added. The suspensions were stirred at 5°C for 30 min, and the particles were recovered by centrifugation and counted (see Fig. 2, filled circles). Control experiments were carried out with no anti-cathepsin M (open circles) or with anti-cathepsin M replaced by the nonspecific monoclonal antibody (see Fig. 1, open triangles).
Sepharose 4B coupled to a nonspecific monoclonal antibody. Lysosomes were also specifically adsorbed to Falcon tissue-culture plates that had been coated with monoclonal anti-cathepsin M (Table II). Little or no binding was observed when the dishes were treated with PBS alone, or with PBS containing either nonspecific monoclonal antibody against glycoproteins IIb and IIIa of human platelets or Cohn Fraction II human IgG. DISCUSSION
The results reported here further support the surface location of lysosomal cathepsin M by demonstrating the binding of 14C-labeled monoclonal antibody to intact lysosomes. That this is binding, and not uptake and internalization of the antibody, is confirmed by the reciprocal binding of lysosomes to immobilized mono-
ET AL.
clonal antibody and of labeled anti-mouse IgG to lysosomes that had been allowed to adsorb unlabeled monoclonal anti-cathepsin M. Lysosomal proteinases have generally been considered to function in the degradation of proteins, particularly extracellular proteins carried into cells as pinocytic vesicles. Their role in the degradation of native cytosolic proteins is less well understood. The finding that some lysosomal enzymes are located on the outer lysosomal surface and in this form are active at neutral pH suggests that they may play a role in regulating the activities of certain cytosolic enzymes. The activity of fructosel,&bisphosphate aldolase is decreased in livers of fasted rabbits (16), apparently due to modification in vivo by limited proteolysis that is similar to that observed when liver aldolase is incubated with purified
TABLE BINDING
I
OF LYSOSOMES TO ANTI-CATHEPSIN M-SEPHAROSE~
Cathepsin
Resin Sepharose 4B Anti-cathepsin M coupled to Sepharose 4B Nonspecific antibody coupled to Sepharose 4B
Units 0
0.11
0.005
C activity Percentage of total 0
21
0.7
’ Aliquots of Sepharose 4B (0.5 ml settled volume), of Sepharose 4B coupled to anti-cathepsin M, or to nonspecific antibody, as indicated, were washed with 0.25 M sucrose containing 10 mM sodium borate, pH 7.4. The resins were then suspended in 2 ml of the same buffered sucrose solution containing 1 mg/ml bovine serum albumin and 100 ~1 of the heavy particle fraction (0.99 mg/ml of protein). The mixtures were rotated end-over-end for 30 min at 5°C and the resins were collected by free sedimentation and washed six times with the same buffered isotonic sucrose mixture. The washed gels were suspended in 0.5 ml 0.5% Triton X-100 and, after 10 min at room temperature, the supernatant solutions were assayed for cathepsin C activity.
CATHEPSIN TABLE
M MONOCLONAL
ANTIBODY
II
BINDING OF INTACT LYSOSOMESTO PLASTIC DISHES COATED WITH MONOCLONAL ANTI-CATHEPSIN M” Solution added to the plastic dishes
Cathepsin activity
2.
C 3.
PBS Anti-cathepsin M monoclonal antibody Nonspecific monoclonal antibodyb Human IgG’
0.0 0.060 0.001 0.001
4.
” Falcon 3046 6-well tissue culture plates were coated with l-ml aliquots of the following solutions: 20 mM PBS, anti-cathepsin M monoclonal antibody (1 mg/ ml in PBS), nonspecific monoclonal antibody directed against glycoproteins IIb and IIIa of human platelets (1 mg/ml in PBS), or human IgG (1 mg/ml in PBS). After 24 h at 25°C the wells were washed with PBS, and 0.1 ml of a suspension of heavy particles (0.99 mg/ml of protein) diluted to 1 ml in 0.25 M sucrose containing 10 mM sodium acetate, pH 6.5, and 1 mg/ ml bovine serum albumin was added to each well. The mixtures were incubated at 5°C for 30 min, with gentle stirring every 5 min to maintain the heavy particles in suspension. The particles were then removed, and the wells were washed five times with 2ml portions of 0.25 M sucrose containing 10 mM sodium acetate, pH 6.5, and 1 mg/ml bovine serum albumin. Two-tenths milliliter of 0.2% Triton X-100 was then added to each well. After incubation at 25°C for 5 min with gentle stirring, the solutions were collected, and aliquots (0.1 ml) were assayed for cathepsin C activity. b Nonspecific monoclonal antibody prepared against glycoproteins IIb and IIIa of human platelets. ‘Human IgG was the Cohn Fraction II human IgG from Sigma Chemical Company.
cathepsin M in vitro (17). It remains, however, to be determined whether this modification is indeed due to the action of the surface-oriented lysosomal proteinases.
6.
7.
8.
9.
10.
11.
12. 13. 14.
15.
ACKNOWLEDGMENT The Institute of Biological Chemistry of the versity of Genoa acknowledges support from the ian Consiglio Nazionale delle Ricerche Progetti alizzato Ingegneria Genetica e Basi Moleculari Malattie Ereditarie.
5.
UniItalFindelle
REFERENCES 1. PONTREMOLI, S., ACCORSI, A., MELLONI, E., SCHIAVO, E., DE FLORA, A., AND HORECKER,
16.
17.
BINDING
271
B. L. (1974) Arch. Biochem. Biophys. 164, 716721. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE, B., MICHETTI, M., AND HORECKER, B. L. (1982) Arch. Biochem. Biophys. 214,376385. PONTREMOLI, S., MELLONI, E., AND HORECKER, B. L. (1984) in Lysosomes in Biology and Pathology (Dingle, J. T., Dean, R. I., and Sly, W. A., eds.), Vol. 7, in press. PONTREMOLI, S., MELLONI, E., MICHETTI, M., SALAMINO, F., SPARATORE, B., AND HORECKER, B. L. (1982) B&hem. Biophys. Res. Commun. 106, 903-909. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE, B., MICHETTI, M., AND HORECKER, B. L. (1983) Proc. Nut1 Acad. Sci. USA 80,12611264. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE,B., MICHEITI, M., SINGH, V. N., AND HORECKER, B. L. (1979) Arch Biochem Bicrphys. 197, 356-363. DAMIANI, G., ZOCCHI, E., FABBI, M., BARGELLESI, A., AND PATRONE, F. (1983) Exp. Hematol. 11, 169-171. ZOLLINGER, W. D., DALRYMPLE, J. M., AND ARTENSTEIN, M. S. (1976) J ZmmunoL 117, 17881798. CORTE, G., MORETTA, L., DAMIANI, G., MINGARI, M. C., AND BARGELLESI, A. (1981) Eur. .I Zmmunol 11, 162-164. DAMIANI, G., FRASCIO, M., BENATTI, U., MORELLI, A., ZOCCHI, M. F., BARGELLESI, A., PONTREMOLI, S., AND DE FLORA, A. (1980) FEBS Z&t. 119, 169-173. COFFINO, P., BAUMAL, R., LASKOV, R., AND SCHARFF, M. D. (1979) J. Cell PhysioL 79,429440. DAMIANI, G., COSULICH, E., AND BARGELLESI, A. (1979) Exp. Cell Res. 118,295-303. GREENWOOD, F. C., HUNTER, W. M., AND GLOVER, J. S. (1963) Biochem. J. 89, 114-123. MAIZEL, J. V., JR. (1971) in Methods in Virology (Maramorosch, K., and Koprowski, H., eds.), Vol. 5, pp. 179-246, Academic Press, New York. MELLONI, E., PONTREMOLI, S., SALAMINO, F., SPARATORE, B., MICHETTI, M., AND HORECKER, B. L. (1981) Arch. Biochem. Biophys. 208, 175183. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE, B., MICHETTI, M., AND HORECKER, B. L. (1979) Proc. NatL Acad. Sci. USA 76,63236325. PONTREMOLI, S., MELLONI, E., MICHETTI, M., SALAMINO, F., SPARATORE, B., AND HORECKER, B. L. (1982) Proc. NatL Acd Sci. USA 79,51945196.
Binding of Monoclonal Antibody to Cathepsin M Located on the External Surface of Rabbit Lysosomes S. PONTREMOLI,
Institute
of Biological
E. MELLONI, G. DAMIANI, M. MICHETTI, B. SPARATORE, AND B. L. HORECKER*v’ Chemistry, University of Genoa, Italy and *Roehe Institute Roche Research Center, Nut&, New Jersey 07110 Received March
F. SALAMINO,
of Molecular
Biology,
12, 1984
A monoclonal antibody raised against rabbit liver cathepsin M binds to intact rabbit liver lysosomes. The binding is specific and is abolished by treating the lysosomes with trypsin, which has previously been shown to digest the membrane-bound cathepsin M [S. Pontremoli, E. Melloni, M. Michetti, F. Salamino, B. Sparatore, and B. L. Horecker (1982) B&hem. Biophys. Res. Commun, 106,903-9091. Rabbit liver lysosomes are adsorbed onto Sepharose 4B coupled to anti-cathepsin M, but not to Sepharose 4B itself or to Sepharose coupled to a nonspecific antibody. The results confirm the location of membrane-bound cathepsin M on the outer surface of the lysosomal membrane.
Several lysosomal proteinases that catalyze limited proteolysis of cytosolic proteins have been shown to be partly associated with the lysosomal membrane fraction (1, 2). The activities of these membrane-bound proteinases are expressed at neutral pH by undisrupted lysosomes (for a review see (3)), which suggests that they are located on the outer surface of the lysosomes. More direct evidence for this surface orientation was obtained by studying the effects of treatment of the intact lysosomes with soluble or immobilized trypsin. The membrane-bound activities, but not the same activities present within the lysosomes, were inactivated by this treatment (4). In the present report we describe experiments with a monoclonal antibody raised against cathepsin M that confirm the presence of this enzyme on the outer lysosomal surface. EXPERIMENTAL
rified from rabbit liver as described. Sepharose 4B was purchased from Pharmacia Fine Chemicals, and Ultrogel AcA34 from LKB. Trypsin, NADH, fructose 1,6-bisphosphate, bovine serum albumin, sodium dodecyl sulfate (SDS),2 and glycyl phenylalanine-2naphthylamide were from Sigma Chemical Company. The monoclonal antibody (PBM 6,4) directed against glycoproteins IIb and IIIa of human platelets (referred to here as “nonspecific antibody”) and the same antibody labeled with i*C were prepared as described (7). Mice were immunized by intraperitoneal injections at 7-day intervals with 50 pg of purified cathepsin M in complete Freund’s adjuvant. Monoclonal anti-cathepsin M was obtained by fusing spleen cells from the immunized mouse, collected 4 days after the fourth injection, with P3X63Ag8Ul myeloma cells as described (8,9). Fourteen days after fusion, each culture medium was tested for anti-cathepsin M activity by a solid-phase binding assay (8,ll) using 50 pl(25 rg)aliquots of purified cathepsin M in 10 mM sodium phosphate buffer, pH 7.2, containing0.15 M NaCl (PBS) distributed in 72 wells of a flexible polyvinyl chloride microtitration plate (Microtiter Plates, No. l-220-29). After incubation for 24 h at 25°C. the wells were
PROCEDURES
Materials. Cathepsin M (2), the endogenous cytosolic cathepsin M inhibitor (5), and aldolase (6) were pu-
i To whom correspondence
’ Abbreviations used: PBS, 10 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl; SDS, sodium dodecyl sulfate; IgG, immunoglobulin G; Hepes, 4-(2hydroxyethyl)-l-piperazineethanesulfonic acid.
should be addressed. 267
0003-9861/84 $3.00 Copyright All rights
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
268
PONTREMOLI
FIG. 1. Specificity of monoclonal anti-cathepsin M antibody. A solution (1.0 ml) containing washed lysosomal membranes (2 mg protein) solubilized in 0.1 ml 1 M NaCl, 20 mM sodium phosphate buffer, pH 7.2, 0.15 M NaCl (PBS) was treated with 0.1 ml (settled gel volume) Sepharose-coupled nonspecific antibody (monoclonal antibody directed against glycoproteins IIb and IIIa of human platelets). The suspension was rotated end-over-end for 30 min at 5°C. The gel was then removed by centrifugation, and the supernatant solution was treated with 0.1 ml Sepharose-coupled DNAse to remove actin-like substances. After removing the gel by centrifugation, the supernatant solution was treated with 0.05 ml Sepharose-coupled monoclonal anti-cathepsin M antibody and suspended in 0.05 ml PBS. The suspension was gently stirred for 1 h at 5”C, and the gel was collected and washed three times with l-ml portions of 0.1 M Tris-HCl, pH 8.0, containing 0.5 M NaCl, followed by three washings with PBS. The washed gel was suspended in 100 ~1 10% SDS and heated for 1 min at 100°C. Undissolved material was removed by centrifugation, and 50 ~1 of the supernatant solution was used for radioiodination (see Materials and Methods). Solutions (50 ~1) containing 5 Fg purified cathepsin M or 50 pg total lysosomal membrane proteins after solubilization with 1 M NaCl were also labeled with ‘%I as described for the labeling of the antibodies. Aliquots of each sample containing 25,000 cpm were suspended in 0.1 M sodium phosphate, pH 7.0, containing 2% SDS and 2% mercaptoethanol, and analyzed on 10% polyacrylamide slab-gels containing 0.1% SDS. After electrophoresis for 4 h in 0.1 M sodium phosphate buffer,
ET AL. washed two times with PBS. Each well then received 200 ~1 of 5% bovine serum albumin dissolved in the washing buffer, and was incubated for 2 h at 25°C. The albumin solution was removed and the wells were washed extensively with PBS. Aliquots (70 ~1) of each culture supernatant were then added to the treated wells and left for 4 h at 25°C. After washing five times with PBS, 70 pl (0.48 pg, 12 X lo6 cpm/mg) of ‘%Ilabeled rabbit anti-mouse IgG antiserum was added to each well. After 2 h at 25°C the wells were washed five times and cut apart, and the radioactivity in each was determined. A culture medium was considered positive when the bound radioactivity exceeded by at least fourfold the quantity detected in a control containing medium from P3X63Ag8Ul cells. Four of the wells were found to be positive for anti-cathepsin activity. One (CM 7), showing the highest activity, was selected and repeatedly cloned in soft agar as described by Coffino et al. (11). Subclone CM 7 was injected intraperitoneally into BALB/c mice pretreated with Pristane (2,6,10,14-tetramethylpentadecane). The ascitic fluids (45 ml) were collected, and the proteins were precipitated with 80% saturated ammonium sulfate. The precipitates were suspended in PBS and chromatographed on an Ultrogel AcA 34 column (2.5 X 120 cm) previously equilibrated with the same buffer. The fractions containing anti-cathepsin M were concentrated to 4.0 ml by ultrafiltration on an Amicon UMlO membrane. The antibody was shown to be of the IgG class by its molecular weight (160,000) and by Ouchterlony analysis with anti-w and anti-X antisera. For the preparation of “Clabeled anti-cathepsin M, CM 7 cells were incubated in culture medium containing [i4C]leucine (350 Ci/ mol) [‘“C]valine (200 Ci/mol), and [Wlthreonine (250 Ci/mol) (12). After 6 h the culture medium was collected, and the labeled antibody was recovered as described above for the unlabeled anti-cathepsin M. Methods. The monoclonal antibodies (CM 7.2 and PBM 6.4) were coupled to BrCN-activated Sepharose 4B by the procedure described by Pontremoli et al. (4). Proteins were labeled by iodination using chloramine T according to Greenwood et al (13). Gel electrophoresis in SDS was carried out in 10% polyaerylamide gels (14). Assays for cathepsin M (2) and cathepsin C (15) were as described. The heavy particle fraction was prepared from rabbit liver, and the membrane fraction recovered after freezing and thawing of the heavy particle fraction was solubilized with 1 M NaCl as described (15). Treatment of the heavy particle fraction with trypsin
pH 7.0, the protein bands were revealed by autoradiography. Lane 1, ‘%I-labeled lysosomal membrane proteins; Lane 2, ‘?-labeled proteins recovered from anti-cathepsin M-Sepharose; lane 3, ‘%I-labeled cathepsin M.
CATHEPSIN
M MONOCLONAL
ANTIBODY
BINDING
269
was as described (4), except that the incubation mixtures contained 10 IIIM sodium borate, pH 7.4, instead of Hepes buffer, pH 7.2. RESULTS
Characterization of the anti-cathepsin M mono&ma1 antibody. Binding of cathepsin M was demonstrated with the Sepharosecoupled antibody. A suspension of matrixbound antibody containing 40 pug protein was capable of binding 50 units (10 pg) of purified soluble cathepsin M, or the same number of units of cathepsin M from a preparation of solubilized lysosomal membranes. Fructose-1,6-bisphosphatase-converting enzyme activity (15) in the same preparation was not removed by the immobilized antibody (data not shown). Specificity of binding was confirmed in a similar experiment in which the mixture of proteins eluted from the lysosomal membranes was labeled by iodination (Fig. 1). In addition to other proteins, this mixture contains cathepsin M (M, = 30,000) (2) and fructose-1,6-bisphosphatase converting-enzyme (M,. = 70,000) (15). When the adsorbed proteins were recovered from the gel and analyzed by SDS-polyacrylamide slab-gel electrophoresis, only a single band was detected (Fig. 1, lane 2), which migrated at the same position as purified iodinated cathepsin M (Fig. 1, lane 3). No bands were detected corresponding to the converting enzyme or to other proteins present in the membrane eluate (cf. Fig. 1, lane 1). Binding of the mono&ma1 antibody to intact lysosomes. This was demonstrated by incubating the 14C-labeled anti-cathepsin M with intact lysosomes in isotonic sucrose solution (Fig. 2). Binding was specific for the anti-cathepsin M antibody, and was not observed to a significant extent with the radioactive monoclonal antibody directed against membrane glycoproteins IIb and IIIa of human platelets. Binding of anti-cathepsin M was not affected by the presence of the nonspecific antibody. Trypsin-treated lysosomes showed little binding of anti-cathepsin M. We have previously reported (4) that membrane-bound cathepsin M was inactivated by this treatment.
Heavy
particle
fraction
(pl)
FIG. 2. Binding of %-labeled anti-cathepsin M to intact lysosomes. The indicated volumes of heavy particle suspension (1.98 mg protein/ml) were diluted with 1 ml 0.25 M sucrose containing 10 mM sodium acetate, pH 6.5, 1 mg/ml of bovine serum albumin, and 4500 cpm of %-labeled monoclonal anti-cathepsin M alone (filled circles), or together with 25 c(g unlabeled nonspecific monoclonal antibody (open circles). Other reaction mixtures contained trypsin-treated lysosomes and W-labeled anti-cathepsin M (filled triangles), or untreated lysosomes and 5000 cpm nonspecific antibody, labeled as described for the monoclonal anti-cathepsin M antibody (open triangles). The suspensions were rotated end-over-end for 30 min at 5°C and the lysosomes were recovered by centrifugation at 80009 for 10 min. The pellets were washed with 2 ml of isotonic sucrose containing 10 mM sodium acetate, pH 6.5, and 1 mg/ml of bovine serum albumin, and the washed pellets were suspended in 0.5 ml formic acid; 0.2-ml aliquots were added to 10 ml of Pica-Fluor 30 (Packard) for determination of radioactivity.
The binding of anti-cathepsin M to intact lysosomes was also measured with unlabeled antibody. The presence of the antibody on the surface of the lysosomes was detected with lz51-labeled anti-mouse IgG (Fig. 3). No binding of anti-mouse IgG was observed unless the lysosomes were first allowed to bind the specific anti-cathepsin M antibody. Binding of lysosomes to immobilized mono&ma1 anti-cathepsin M. A suspension of anti-cathepsin M coupled to Sepharose 4B showed significant binding of intact rabbit liver lysosomes (Table I). This binding was evaluated by measuring the cathepsin C activity associated with the antibody-coupled resin. No binding was observed to Sepharose 4B itself or to
PONTREMOLI
270
Heavy
particle
fraction
(~111
FIG. 3. Binding of anti-cathepsin M antibody to intact lysosomes determined with ‘?-labeled antimouse IgG. The reaction mixtures containing the indicated volumes of the heavy particle suspension and 25 pg unlabeled anti-cathepsin M antibody were incubated, and the lysosomes were recovered as described in Fig. 2. The washed pellets were suspended in 2 ml isotonic sucrose containing 10 mM sodium acetate, pH 6.5, and 0.1 ml of a solution containing 2 X 10’ cpm ‘?-labeled rabbit anti-mouse IgG was added. The suspensions were stirred at 5°C for 30 min, and the particles were recovered by centrifugation and counted (see Fig. 2, filled circles). Control experiments were carried out with no anti-cathepsin M (open circles) or with anti-cathepsin M replaced by the nonspecific monoclonal antibody (see Fig. 1, open triangles).
Sepharose 4B coupled to a nonspecific monoclonal antibody. Lysosomes were also specifically adsorbed to Falcon tissue-culture plates that had been coated with monoclonal anti-cathepsin M (Table II). Little or no binding was observed when the dishes were treated with PBS alone, or with PBS containing either nonspecific monoclonal antibody against glycoproteins IIb and IIIa of human platelets or Cohn Fraction II human IgG. DISCUSSION
The results reported here further support the surface location of lysosomal cathepsin M by demonstrating the binding of 14C-labeled monoclonal antibody to intact lysosomes. That this is binding, and not uptake and internalization of the antibody, is confirmed by the reciprocal binding of lysosomes to immobilized mono-
ET AL.
clonal antibody and of labeled anti-mouse IgG to lysosomes that had been allowed to adsorb unlabeled monoclonal anti-cathepsin M. Lysosomal proteinases have generally been considered to function in the degradation of proteins, particularly extracellular proteins carried into cells as pinocytic vesicles. Their role in the degradation of native cytosolic proteins is less well understood. The finding that some lysosomal enzymes are located on the outer lysosomal surface and in this form are active at neutral pH suggests that they may play a role in regulating the activities of certain cytosolic enzymes. The activity of fructosel,&bisphosphate aldolase is decreased in livers of fasted rabbits (16), apparently due to modification in vivo by limited proteolysis that is similar to that observed when liver aldolase is incubated with purified
TABLE BINDING
I
OF LYSOSOMES TO ANTI-CATHEPSIN M-SEPHAROSE~
Cathepsin
Resin Sepharose 4B Anti-cathepsin M coupled to Sepharose 4B Nonspecific antibody coupled to Sepharose 4B
Units 0
0.11
0.005
C activity Percentage of total 0
21
0.7
’ Aliquots of Sepharose 4B (0.5 ml settled volume), of Sepharose 4B coupled to anti-cathepsin M, or to nonspecific antibody, as indicated, were washed with 0.25 M sucrose containing 10 mM sodium borate, pH 7.4. The resins were then suspended in 2 ml of the same buffered sucrose solution containing 1 mg/ml bovine serum albumin and 100 ~1 of the heavy particle fraction (0.99 mg/ml of protein). The mixtures were rotated end-over-end for 30 min at 5°C and the resins were collected by free sedimentation and washed six times with the same buffered isotonic sucrose mixture. The washed gels were suspended in 0.5 ml 0.5% Triton X-100 and, after 10 min at room temperature, the supernatant solutions were assayed for cathepsin C activity.
CATHEPSIN TABLE
M MONOCLONAL
ANTIBODY
II
BINDING OF INTACT LYSOSOMESTO PLASTIC DISHES COATED WITH MONOCLONAL ANTI-CATHEPSIN M” Solution added to the plastic dishes
Cathepsin activity
2.
C 3.
PBS Anti-cathepsin M monoclonal antibody Nonspecific monoclonal antibodyb Human IgG’
0.0 0.060 0.001 0.001
4.
” Falcon 3046 6-well tissue culture plates were coated with l-ml aliquots of the following solutions: 20 mM PBS, anti-cathepsin M monoclonal antibody (1 mg/ ml in PBS), nonspecific monoclonal antibody directed against glycoproteins IIb and IIIa of human platelets (1 mg/ml in PBS), or human IgG (1 mg/ml in PBS). After 24 h at 25°C the wells were washed with PBS, and 0.1 ml of a suspension of heavy particles (0.99 mg/ml of protein) diluted to 1 ml in 0.25 M sucrose containing 10 mM sodium acetate, pH 6.5, and 1 mg/ ml bovine serum albumin was added to each well. The mixtures were incubated at 5°C for 30 min, with gentle stirring every 5 min to maintain the heavy particles in suspension. The particles were then removed, and the wells were washed five times with 2ml portions of 0.25 M sucrose containing 10 mM sodium acetate, pH 6.5, and 1 mg/ml bovine serum albumin. Two-tenths milliliter of 0.2% Triton X-100 was then added to each well. After incubation at 25°C for 5 min with gentle stirring, the solutions were collected, and aliquots (0.1 ml) were assayed for cathepsin C activity. b Nonspecific monoclonal antibody prepared against glycoproteins IIb and IIIa of human platelets. ‘Human IgG was the Cohn Fraction II human IgG from Sigma Chemical Company.
cathepsin M in vitro (17). It remains, however, to be determined whether this modification is indeed due to the action of the surface-oriented lysosomal proteinases.
6.
7.
8.
9.
10.
11.
12. 13. 14.
15.
ACKNOWLEDGMENT The Institute of Biological Chemistry of the versity of Genoa acknowledges support from the ian Consiglio Nazionale delle Ricerche Progetti alizzato Ingegneria Genetica e Basi Moleculari Malattie Ereditarie.
5.
UniItalFindelle
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16.
17.
BINDING
271
B. L. (1974) Arch. Biochem. Biophys. 164, 716721. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE, B., MICHETTI, M., AND HORECKER, B. L. (1982) Arch. Biochem. Biophys. 214,376385. PONTREMOLI, S., MELLONI, E., AND HORECKER, B. L. (1984) in Lysosomes in Biology and Pathology (Dingle, J. T., Dean, R. I., and Sly, W. A., eds.), Vol. 7, in press. PONTREMOLI, S., MELLONI, E., MICHETTI, M., SALAMINO, F., SPARATORE, B., AND HORECKER, B. L. (1982) B&hem. Biophys. Res. Commun. 106, 903-909. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE, B., MICHETTI, M., AND HORECKER, B. L. (1983) Proc. Nut1 Acad. Sci. USA 80,12611264. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE,B., MICHEITI, M., SINGH, V. N., AND HORECKER, B. L. (1979) Arch Biochem Bicrphys. 197, 356-363. DAMIANI, G., ZOCCHI, E., FABBI, M., BARGELLESI, A., AND PATRONE, F. (1983) Exp. Hematol. 11, 169-171. ZOLLINGER, W. D., DALRYMPLE, J. M., AND ARTENSTEIN, M. S. (1976) J ZmmunoL 117, 17881798. CORTE, G., MORETTA, L., DAMIANI, G., MINGARI, M. C., AND BARGELLESI, A. (1981) Eur. .I Zmmunol 11, 162-164. DAMIANI, G., FRASCIO, M., BENATTI, U., MORELLI, A., ZOCCHI, M. F., BARGELLESI, A., PONTREMOLI, S., AND DE FLORA, A. (1980) FEBS Z&t. 119, 169-173. COFFINO, P., BAUMAL, R., LASKOV, R., AND SCHARFF, M. D. (1979) J. Cell PhysioL 79,429440. DAMIANI, G., COSULICH, E., AND BARGELLESI, A. (1979) Exp. Cell Res. 118,295-303. GREENWOOD, F. C., HUNTER, W. M., AND GLOVER, J. S. (1963) Biochem. J. 89, 114-123. MAIZEL, J. V., JR. (1971) in Methods in Virology (Maramorosch, K., and Koprowski, H., eds.), Vol. 5, pp. 179-246, Academic Press, New York. MELLONI, E., PONTREMOLI, S., SALAMINO, F., SPARATORE, B., MICHETTI, M., AND HORECKER, B. L. (1981) Arch. Biochem. Biophys. 208, 175183. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE, B., MICHETTI, M., AND HORECKER, B. L. (1979) Proc. NatL Acad. Sci. USA 76,63236325. PONTREMOLI, S., MELLONI, E., MICHETTI, M., SALAMINO, F., SPARATORE, B., AND HORECKER, B. L. (1982) Proc. NatL Acd Sci. USA 79,51945196.