Journal of Immunological Methods, 151 (1992)165-175

165

© 1992ElsevierScience PublishersB.V. All rightsreserved01122-1759/92/$05.00

JIM O631O

Production and characterisation of monoclonal antibodies against native and disassembled human eatalase Erik A.C. Wiemer ~.t Rob Ofman b, Esther Middelkoop ~, Mark de B.'~er ~, Ronald J.A. Wanders b and Joseph M. Tager a E.C. Slatcr Institute for Biochemical Research, Universityof Amsterdam, Amsterdam, Netherlands, h Department of Pediatrics, University Hospital, Unicersityof Amsterdam, Amsterdam, Netherlands, and c N. V. lnnogenetics, B-9052 Zwijnaarde. Belgium

(Received 16 August1991,revisedreceived12 December t99t, accepted 3l January 1992)

Catalase isolated from human erythrocytes was used to immunise mice, in order to generate hybridomas producing specific monoclonal antibodies to the enzyme. Hybridomas secreting anti-(catalase) antibodies were identified by a modified enzyme-linked immunosorbent assay (ELISA) using either monomer/dimer catalase or native, tetramcric enzyme. Three stable hybridoma clones were selected and the characteristics of the antibodies produced were investigated by ELISA, immunofluorescence, immunoprecipitation and immunoblotting experiments. One monoelonal antibody (17E10) was shown to interact with both native tetramer catalase and - to a lesser extent - with monomer/dimer catalase. Two monoclonal antibodies (10B12H9, 13A10) were found to react only with completely denatured catalase or with monomer/dimer catalase but not with native catalase. Key words: Catalase;Monoelonalantibody;,Peroxisome;Peroxisomalbiogenesis

Introduction Catalase (hydrogen peroxide: hydrogen peroxide oxidoreductase, EC 1.11.1.6) is a haemoprorein that catalyses the conversion of H 2 0 2 into

Correspondence to: E. Middelkoop, E.C. Slater Institute for Biochemical Research, Universirj of Amsterdam, Meibergdreef 15, 1105AZ, Amsterdam, Netherlands. I Present address: International Institute of Cellular and Molecular Pathology, Research Unit for Tropical Diseases, AvenueHippocrate 74, 1200Brussels~Belgium. Abbreviations: CAPS, 3-[eyclohe~lamino]-I-propanesulphonicacid; DMEM,Dulbecco'smudifieaEagle'smedium; ELISA, enz,/me-linked immunosorhentassay; FITC, fluorescoin isothiocyanate;PAGE, polyacrylamidegel electrophoresis; PBS, phosphate-buffered saline; PMSF, phenyl methylsulphonylfluoride;SDS,sodiumdodecylsulphate.

H 2 0 and 0 2 (Deisseroth and Dounce, 1970; Chance et al., 1979). Under suitable conditions and in the presence of suitable hydrogen donors the enzyme acts as a peroxidase (Deisseroth and Dounce, 1970; Chance et al., 1979). Catalase has been isolated and characterised from a number of different species of plants, yeasts and mammals (for reviews see Deisseroth and Dounce, 1970 and Lazarow and Fujiki, 1985). The mammalian enzyme has a molecular weight of approximately 240,000 Da and is composed of four identical subunits, each containing a haem group (Deisseroth and Dounce, 1970; Lazarow and Fujiki,

1989. "n mammalian tissues catalase is predominanLJy located in peroxisomes, in which it is the most abundant enz'~ne (De Duve and Baudhuin, 1966), and is therefore used as a marker enzyme

166 for this organelle. There are reports in the literature indicating the existence of a substantial pool of cytoplasmic catalase (Fukami and Flatmark, 1986). Moreover, in some species the bulk of catalase has been reported to be extra-peroxisomal (Holmes and Masters, 1972; Roels, 1976; Roels etal., 1977; Yamamoto et al., 1988). The biosynthesis of mammalian eatalase has been most extensively studied in rat liver. Ratliver catalase is synthesised on free ribosomes, after which it is post.translationally incorporated into peroxisomes (Goldman and Blobel, 1978; Robbi and Lazarow, 1978; Raehubinski et al., 1984). No detectable modifications of the protein occur during this process (Robbi and Lazarow, 1982). Formation of an active enzyme requires binding of haem and tetramerisation. The results of early studies by Lazarow and De Duve (1973a,b) led these investigators to suggest that both processes, binding of haem and oligomerisation, take place in the peroxisomes. Recently a group of peroxisomal disorders has been recognised in which functional peroxisomes are deficient (for reviews see Wanders etal., 1988 and Lazarow and Moser, 1989). Catalase has been found to be located in an enzymically active state in the cytoplasm in patients with these disorders (Wanders et al., 1984; Lazarew et ak, 1985; Santos el al.. 1985), thus implying that eatalase assembly does not necessarily have to occur in the peroxisomal compartment. In order to investigate the biogenesis of human catalasc in detail, especially in relation to several peroxisomal disorders, monoclonal antibodies have been produced that are able to discriminate between the fully assembled tetramerie form of human catalase and the partially assembled monomeric or dimeric form of the protein.

Materials and methods

Materials Dulbecco's modified E a g l e ' s m e d i u m (DMEM), penicillin, streptomycin and fetal calf serum were obtained from Gibeo (Glasgow, UK); streptavidin labelled with fluoreseein isothiocyanate (FITC) and biotinylated goat anti-mouse Ig were purchased from Amersham International

(Amersham, UK). Class and subclass of the monoclonal antibodies were determined using the INNO-line immunoassay developed by Innogenetics (Antwerp, Be[glum). Bovine liver eatalase was obtained from Boehringer Mannheim (Mannbe[m, Germany); dioctyl sodium sulphosuccinate was from Janssen Chimica (Beerse, Belgium) and 4-chloro-l-naphthol, 3,3',5,5'-tetramethylbenzidine (TMB), prestained molecular weight markers (SDS 7B) and the bacterial protease inhibitors leupeptin, pepstatin, ehymostatin, antipain and phenylmethylsulphonyl fluoride (PMSF) were obtained from Sigma Chemical Co. (St. Louis, USA). All other reagents used were of the purest grade available. Cell lines and culture media The routine myeloma ceil line NS-1 was used as a fusion partner for obtaining hybridomas. The NS-1 cells and hybrid cells were cultured in DMEM supplemented with penicillin (200 U/ml), streptomycin (200 ,~g/ml) and 10% heat-inactivated fetal calf serum. Cultured human skin fibroblasts were obtained from a patient with the cerebro-hepato-renal (Zellweger) syndrome (cell line W78/515) (Heymans et al., 1984) or from a healthy adult (85AD5035F). The fibroblasts were cultured as described by Wiemer et al. (1989). Isolation of human catalase Packed erythroeytes, isolated from fresh blood samples with citrate-phosphate-dextrose (CPD) as anticoagulant, were obtained from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service. Haemolysates were prepared as follows. The packed red cells were washed three times with 0.9% (w/v) NaCI solution. The buffy coat was removed and the cells haemolysed by resuspending the erythrocyte pellet in four volumes of a solution (4oc) of 10 mM sodium phosphate buffer (pH 7.4) containing 5 mM dithiothreitol (DTT) and homogenising the suspension using an MSE sonieator (3 X 20 s at maximal output) or an Ultraturrax homogeniser for 60 s. Subsequently the cell stroma was removed from the haemolysate by centrifugation at 25,000 × g for 1 h at 4°C in a Sorvall SS34 rotor. Catalase was partially purified from the haemolysate using a ehromatofocusing column es-

167 sentially as described for the purification of catalase from rat liver (Mainferme and Wattiaux, 1982). Chromatofocusing, which separates proteins according to their isoeleetric points, with Polybuffer exchanger (PBE) 94 and Polybuffer 96 (Pharmaeia Fine Chemicals, Uppsala, Sweden) was performed according to the recommendations given by the manufacturer. In brief the column used (2.6 × 10 era) consisted of the Polybuffer exchanger (PBE) 94 and was equilibrated to pH 8.3 with 25 mM Tris-CH3COOH. The sample (equivalent to 2.5 g of protein) was applied to the column which was doted with Polybuffer 96, adjusted to pH 6 0 with acetic acid, at a flow rate of 0.5 ml/min thereby generating a linear pH gradient ranging from 8 to 6. Approximately 70 fractions of 10 ml were collected. Protein concentration and catalase enzymic activity in the fractions were determined. Peak fractions containing catalase activity were pooled and subsequently dialysed overnight at 10°C against 25 mM sodium phosphate buffer (pH 7.4) containing 5 mM DTT. A similar batch of partially purified catalase was used to identify and isolate hybridomas secreting anti(eatalase) antibodies in a sandwieh ELISA. In order to use it for immunisation, the catalase preparation was further purified by ion-exchange chromatography essentially as described by Aebi et al. (1974) with the following modifications. The chromatofocusing preparation was concentrated on an Amieon filter to about a tenth of the original volume and applied to a DEAE-Sepharose column (2.6 × 9 cm). The column was eluted, at a flow rate of 0.5 ml/min. with a linear gradient (525 ml) of potassium chloride extending to 400 mM in 5 mM sodium phosphate buffer (pH 7.4) containing 5 mM DTT. 70 fractions of 7.5 mt were collected; those fractions containing the bulk of the catalase activity were pooled, concentrated on an Amicon filter and dialysed against 25 mM sodium phosphate buffer (pH 7.4) containing 5 mM D'IT. This preparation was used for immunisation.

lmmunisation For primary immunisation B A L B / c mice were injected intraperitoneally with approximately 50 ~ g of the partially purified antigen in an emul-

sion consisting ef equal volumes of the antigen in phosphate-buffered saline (FBS) and Freund's complete adjuvant. Two booster immunisatioos were performed on day 7 and day 14 as above, except that Freund's incomplete adjuvant was used. On day 28 the mice were immunised either with 50 p.g native catalase (mouse A) or with 50 txg denatured enzyme (mouse B), in Freund's incomplete adjuvant. Catalase was denatured by heating the preparation for 2 rain at 100~C in the presence of 0.1% sodium dodecyl sulphate (SDS). On day 42 the mice were given an intraperitoneal booster injection with 50 9,g of native catalase (mouse A) or 50 btg of denatured enzyme (mouse B).

Cell fusion 3 days after the last booster injection, splcnocytes from mice were fused with NS-1 murine myeloma cells at a ratio of 5:1 using 38% polyethylene glycol (mol. wt. 1500) in DMEM. The fused cells were suspended in DMEM containing 10% heat-inactivated fetal calf serum, 0.1 mM hypoxanthine, 0.01 mM aminopterin and 0.016 mM thymidine. The fused cells were then distributed between the wells of 96-well tissue culture plates. After 10-14 days the supcr-~'~mts were screened for specific antibody produL~.ion (see below).

Preparation of human liuer extracts, electrophoresis. immunoblotting, immunoprecipitation and immunofluorescence microscopy Liver extracts were prepared from frozen autopsy material by homogenisation in 25 mM sodium phosphate buffer (pH 7.5) containing 0.5% sodium dodecyl sulphate (SDS) and the following protease inhibitors: 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 50 ~ g / m l leupeptin, 50 p.g/ml antipain, 25 /~g/ml chymostatin and 25 p.g/ml pepstatin. Subsequently the samples were sonicated (MSE sonieator, 5 x 15 s at maximal output) and centrifuged for I rain at 12,000Xg in order to remove membrane fragments. The supernatants were aliquotted and stored at - 20°C or - 70°C. Polyacrylamide gel electrophoresis (PAGE) in the presence of SDS was carried out essentially as described by Laemmli (1970). Western blotting

168 to nitrocellulose was carried out using the semidry blotting method according to Kyhse.Ander. sen (1984). Catalase was detected by incubation of the filters with culture medium of seleeted hybridomas or partially purified IgG preparations from hybridoma culture medium in phosphatebuffered saline (PBS) containing 0.05% (v/v) Tween 20 in the presence of 0.5% (w/v) bovine serum albumin (BSA) or 20% (v/v) skimmed milk, followed by an incubation with goat antimouse lg antibodies conjugated to horseradish peroxidase (Bio-Rad, Watford, UK). Immune complexes were visualised either by incubation in 50 ml PBS to which were added 10 ml of 4ehloro-l-naphthol (3 mg/ml) in methanol and 30 /zl 30% (w/v) H 2 0 2 or by incubation in 50 ml of a 50 mM sodium acetate buffer (pH 5) containing 12.5 ml of a 4 m g / m l dioctyl sodium sulphosuccihate solution in ethanol, 250 /.tl of a 50 m g / m l 3,3',5,5'-tetramethyl-benzidine solution in acetone and 25/~1 of a 30% (w/v) H202 solution. A partially purified IgG preparation was obtained from the culture medium of hybridomas by ammonium sulphate precipitation. The IgG fraction was coupled to activated CNBr-Sepharose beads (Pharmacia, Uppsala, Sweden) according to the directions given by the manufacturer. Immunoprecipitations from crude haemolysates were routinely carried out in PBS containing 0.1% (v/v) Triton X-100 and 1% (w/v) BSA for 2-4 h at 10°C. Thereupon the beads were washed extensively with PBS containing 0.1% (v/v) Triton X-100. Precipitated proteins were analysed either on SDS-PAGE and subsequent immunoblotting or, in the ease of tetramer catalase, by measuring the enzymic activity attached to the beads. lmmunofluoreseenee experiments were carried out essentially as described previously (Wiemer et al., 1989). Sandwich enzyme-linked immunosorbent assay (sandwich-ELlSA) lgG from a polyelonal rabbit antiserum directed againsl eatalase (described by Tager et al., 1986) was coated to the wells of polyvinyl microtitre plates (Falcon products, Becton Dickinson Labware, Oxnard, CA, USA) by adding 250 ng of lgG in 50 ~1 of phosphate-buffered saline (PBS) (pH 7.4) to each well and incubating the

plates overnight at 4°C. Unbound IgG was removed by washing six times with PBS-Tween (0.05% (v/v) Tween 20 in PBS). Subsequently 50 /zl of a partially purified human erythrocyte catalase preparation were added to each well and incubated for 60 mln at 37°C. The catalase preparation was either directly diluted in PBS-Tween containing 1% bovine serum albumin (BSA) or was first incubated for 90 rain at 25"C in the presence of 200 mM 3-[cyclohexylamino]-l-propanesulphonic acid (CAPS) (pH 11.2) in order to disassemble catalase. After six washes with PBSTween, 50 ,~1 of hybridoma supernatant were added to the wells which were then incubated for 60 rain at 37°C. Following six washes with PBSTween, bound murine antibodies were detected by incubation with 50 ,ttl of a 1/1000 dilution of rabbit anti-mouse Ig immunoglobulins conjugated to horseradish peroxidase (Dakopatts, Copenhagen, Denmark) in PBS-Tween containing 1% (w/v) BSA. After six washes with PBS-Tween, bound peroxidase was revealed by the addition of 100 ,~1 of an assay mixture prepared by diluting 100 tzl of a solution of 6 m g / m l 3,3',5,5'-tetramethylbenzidine (TMB) in dimethylsulphoxide and 15 t~l of a 30% (w/v) H 2 0 2 solution to 10 ml with 0.1 M sodium phosphate buffer (pH 6.0). The reaction was stopped after approximately 15 min of incubation in the dark by the addition of 50 jzl of 2 M H2SO 4. The extinction was determined spectrophotometrically at 450 nm with a Flow Titertek Multiscan (Flow Laboratories, Irvine, UK). Determination of protein concentration and enzymic measurements Protein was determined as described by Bradford (1976) or by the BCA method (Smith et al., 1985). Catalase enzymic activity was measured polarographically according to Wanders et al. (1984).

Results and discussion

Immunisation The eatalase preparation used for immunisation had a rather low specific activity of 76/.~mol O 2 / m i n / m g protein because of inactivation of

169