Preparation of an anti-acid sphingomyelinase monoclonal antibody for the quantitative determination and polypeptide analysis of lysosomal sphingomyelinase in fibroblasts from normal and Niemann-Pick type A patients

Journal oflmmunological Methods, 160 (1993) 199-206

199

© 1993 Elsevier SciencePublishers B.V. All rights reserved 0022-1759/93/$06.00

JIM 06646

Preparation of an anti-acid sphingomyelinase monoclonal antibody for the quantitative determination and polypeptide analysis of lysosomal sphingomyelinase in fibroblasts from normal and Niemann-Pick type A patients Robert Rousson a, Parviz Parvaz b, Jean Bonnet a, Claire Rodriguez-Lafrasse a, Pierre Louisot a and Marie T. Vanier a a Ddpartement de Biochimie, Inserm U.189, Facultd de Mddecine Lyon-Sud, 69921 Oullins Cedex, France, and b Laboratoire de Biochimie, H~pital Ste Eug~nie, C.H. Lyon-Sud, 69310 Pierre Bdnite, France

(Received 5 May 1992,revisedreceived2 November1992,accepted 19 November1992) An anti-acid sphingomyelinase monoclonal antibody has been prepared using an in vitro booster technique. The antigen, acid sphingomyelinase, was purified from human placentas by sequential chromatographic steps in the presence of the non-ionic detergent Nonidet P40. This monoclonal antibody (MAB 236) precipitates specifically the enzyme activity by immunoadsorption techniques and presents the same specificity to normal and mutated sphingomyelinase in Niemann-Pick type A patients. MAB 236 is the first antibody able to precipitate the protein in the presence of detergent thereby permitting the quantitative determination of normal and mutated sphingomyelinase in tissue and cell extracts. Polypeptide analysis and quantitative determination experiments using this monoclonal antibody showed no difference between patients and normal controls. Key words." Sphingomyelinase;Monoclonalantibodypreparation; In vitro booster; Quantitative determination;Polypeptideanaly-

sis; Niemann-Pickdisease

Introduction

Acid sphingomyelinase (SPM, sphingomyelin phosphodiesterase, EC 3.1.4.12) is the lysosomal phosphodiesterase that hydrolyses sphingomyelin to ceramide and phosphorylcholine. The genetic deficiency, of this enzyme activity results in the neuronopathic (type A) or the visceral (type B) forms of Niemann-Pick disease (Spence and Callahan,1989) an autosomal recessive disorder.

Correspondence to: R. Rousson, D~partement de Biochimie, Inserm U.189, Facult~ de M~decine Lyon-Sud,69921 Oullins Cedex, France.

Although the SPM gene has recently been sequenced (Schuchman et al., 1992), the biochemical characterization of the SPM protein remains fragmentary due to the strong hydrophobic behaviour of the enzyme (Callahan et al., 1981) which leads to incompletely resolved methodological problems. For example the number and masses of the different active polypeptides still remain controversial. In addition no satisfactory antibody has previously been available for SPM. Neither the antiSPM monoclonal antibody briefly described by Freeman et al. (1983), nor the polyclonal antibody produced by Weitz et al. (1985), against a partially purified SPM from urine, has not been

200 adequately documented. Overall, these antibodies, including our former polyclonal rabbit antibody (Rousson et al., 1987), were not able to precipitate the enzyme solubilised and stabilized in detergent containing buffer. These properties were underlined by Driessen et al. (1985), who explained such results as probably due to nonionic detergent inhibition of the SPM immunoprecipitation reaction. In the present study we describe the properties of a novel monoclonal antibody prepared against normal placental SPM able to precipitate normal enzyme in the presence of Nonidet P 40 (NP40). Its reactivity against SPM extracted from deficient Niemann-Pick fibroblasts has also been studied. For the first time, quantitative estimations of normal and mutant protein levels were performed. Materials and methods

Preparation of biological material Sphingomyelinase purification.

Acid sphingomyelinase was purified from human placentas according to our previous protocol based on sequential chromatographic steps on concanavalin A-Sepharose (Pharmacia), butyl-agarose (Miles), octyl-agarose (Miles), matrex gel Red A agarose (Amicon), polybuffer exchanger (Pharmacia) in the presence of 0.1% (w/v) non-ionic detergent NP40 (Fluka) during all the purification steps (Rousson et al., 1987). Fibroblast extracts. Secondary skin fibroblast cell lines were prepared from several normal subjects and five unrelated patients with type A Niemann-Pick disease (NPA) diagnosed in our laboratory. Cultures were made as previously described (Vanier et al., 1985). After harvesting, 5 x 107 cells were suspended in 500/zl of Tris-HC1 10 mM pH 7.4 buffer containing 0.1% of NP40, sonicated 30 s three times and homogenized by six strokes in a Potter Elvhejem homogenizer. Supernatants collected after centrifugation (150,000 x g for 30 min) contained sphingomyelinase activity.

Immunological methods Enzyme-linked immunosorbent assay (ELISA). This was performed in microtitre plates (Nunc)

coated with 200 /zl of a solution of partially purified sphingomyelinase (concanavalin A eluate) diluted to 25/xg/ml in carbonate buffer pH 9.6. Anti-sphingomyelinase antibodies were incubated for 1 h at room temperature followed by 6 washes with Tris-HC1 10 mM pH 7.2, NaC1 150 mM containing 0.05% Tween 20 (Sigma). Peroxidase conjugated anti-mouse and anti-rabbit goat antisera (Bio-Rad) diluted 1/100, were incubated for 30 min at 37°C followed by six washes as described above. Peroxidase activity was measured at 596 nm using orthophenylene diamine (OPD) (Sigma) substrate in acetate buffer pH 5.0 after incubation for 15 min at 37°C.

Immunoadsorption of sphingomyelinase actiuity from fibroblast extracts. The Bio-Rad Affigel protein A map II kit was used for this purpose. 150 /~1 of fibroblast extracts in 0.1% NP40 were mixed with 50 tzl of monoclonal anti-sphingomyelinase antibody and 200/zl of the binding buffer included in the kit in a 1.5 ml Eppendorf microtube. Samples were shaken overnight at 4°C. Immune complexes were adsorbed by addition of 50 /xl of Affigel protein A agarose diluted 1/1 (v/v) in the binding buffer and 1 h incubation at 4°C with constant shaking. Agarose was spun down for 30 s at 12,000 x g in a Hettich microfuge. For enzyme activity determination tests 20 pA of supernatants were mixed with 10/~I of three times concentrated reaction buffer.

Polypeptide analysis after immunoadsorption of sphingomyelinase in the presence of NP 40. Fibroblast extracts were first incubated for 2 h at 4°C in PBS containing 0.5% (w/v) NP40 with 100 /xl of an irrelevant antibody (anti-hexosaminidase) covalently bound to activated-Sepharose 4 B (Pharmacia) according to Axen et al. (1967). After centrifugation, supernatants were then shaken overnight at 4°C with 50 /zl of a suspension of an anti-sphingomyelinase antibody bound to Sepharose 4B. After extensive washing with Tris-HCl 125 mM, EDTA 10 mM, NaC1 500 mM, NP40 0.5%, pH 8.2, and with PBS, antigenic material was eluted by adding 100/xl of 5% SDS, 5% 2-mercaptoethanol in PBS and heating for 5 min in a boiling water bath. The eluate was centrifuged and electrophoresis experiments were performed on the supernatants. Others procedures. The Ouchterlony double

201 diffusion technique was carried out in 0.8% agarose (Sigma) containing 0.1% NP40. For quantitative Mancini assays, ascites fluid containing monoclonal antibody was diluted to 20 /~g protein/ml in 0.8% agarose containing 0.1% NP40. Precipitating ring diameters were squared and data plotted versus protein concentrations. In immunoblotting experiments ascites fluid containing monoclonal antibody was used as the antibody source. Immunostaining of the nitrocellulose membranes (Bio-Rad) was performed following a peroxidase anti-peroxidase procedure (De Bias and Cherwinski, 1983), all buffers containing 0.05% Tween 20.

Preparation of the monoclonal anti-sphingomyelinase antibodies Monoclonal antibodies were prepared in B A L B / c mice (Iffa-Credo, L'Arbresle, France) using purified placental sphingomyelinase as antigen. Immunization. In vivo immunization: female 6-week-old B A L B / c mice were primed by an intraperitoneal (i.p.) injection with 100 /zg of antigen emulsified with an equal volume of complete Freund's adjuvant (Behring)(total volume: 80 /zl/mouse). Three i.p. booster injections of 100 /zg of antigen with 4 week intervals were performed in incomplete Freund's adjuvant (Behring). The mice received a similar booster injection 6 months later. In vitro booster: was performed according to a modified technique of in vitro immunization described previously (Parvaz et al., 1989a). Briefly, mouse spleen cells were taken 2 weeks after the last injection with purified sphingomyelinase. Thymocyte conditioned medium (TCM) was prepared by co-culturing 3 x 108 thymocytes of M R L / l p r / l p r mice with 1.5 X 108 thymocytes of C57B1/6 mice (Iffa-Credo) in in vitro immunisation (IVI) medium (100 ml of RPMI 1640 (Flow) complete medium containing 20 mM Hepes (Sigma), 1% MEM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Flow), 2 mM L-glutamine (BioM6rieux), 3 g/1 glucose, 100 I U / m l penicillin, 50 /xg/ml streptomycin (BioM6rieux), supplemented with 100 /zM hypoxanthine (Sigma), 16 /zM thymidine (Sigma), 2% serum substitute S F / X (Costar) and 50 /zM 2-

mercaptoethanol (Sigma)). The cell suspension was divided between 2 x 75 cm 2 tissue culture flasks (Falcon) (50 ml/flask) and cultured in humidified 37°C incubator in 5% CO2. After 48 h the cell suspension was centrifuged for 10 min at 2500 x g and the supernatant was sterilized through a filter unit (Falcon 7107) and stored at 80oc. Cell fusion. Preparation of myeloma cells: S p 2 / O myeloma cells were cultured in RPMI 1640 complete medium supplemented with 10% heat-inactivated foetal calf serum (FCS) (Flow) and 20/.~M 8-azaguanine (Sigma). Preparation of immunized spleen cells: spleen cells from a selected donor mouse were washed twice and suspended in freshly prepared IVI medium (65 ml) supplemented with TCM (35 ml) as described above. After addition of 10/zg antigen per ml, the spleen cell suspension (2 x 106/ml) was incubated as for the thymocytes until fusion 3 days later. Fusion was performed according to Lane (1985) by slow addition (45 s) of 1 ml of fusogen (1 g PEG 4000 (Merck), 0.1 ml DMSO (Merck) in 1 ml PBS pH 7.4 solution) as previously described (Parvaz et al., 1989b). The fused cell suspension was distributed (100 /zl/ well) in the 96 micro well plates (Nunc) previously seeded with feeder cells (105 spleen cells in 100 /xl of the above mentioned RPMI 1640 medium, without 8azaguanine but containing 50 /zM hypoxanthine, 10 /zM azaserine (Sigma) and 20% FCS). After 20% of confluence was reached 100/xl of non-diluted hybridoma supernatants were screened by ELISA. An absorbance greater than 0.350 was considered as positive. Cloning. Selected hybridoma were cloned by the limiting dilution method. Immunoglobulin classes. Isotypes were determined by Ouchterlony double diffusion method using an anti-mouse immunoglobulin screening kit (Serotec). Ascites fluids. These were obtained as described previously (Parvaz et al., 1989b). -

General methods Enzyme activities. These were measured as formerly described (Rousson et al., 1987). Briefly activity of sphingomyelinase was expressed as the

202 amount of nmoles of labelled phosphorylcholine produced per min and per mg protein using [Met14C] choline sphingomyelin - 500 k B q / n m o l (NEN) - as substrate. For other hydrolases (/3hexosaminidase, /3-galactosidase, acidic phosphatase) activities were tested using appropriate 4-methyl-umbelliferyl derivatives as substrates. Polyacrylamide gel electrophoresis. This was performed in 10% gel slabs (Interchim) under denaturing conditions according to the Laemmli procedure (1970), except that sodium dodecyl sulphate concentration was raised to 0.5% in the gels and buffers to prevent non-specific polypeptide adsorption onto the gels. All the samples were adjusted to give approximately 25 /xg protein content and were heated for 5 min at 95°C in the presence of 1% SDS and 5% 2-mercaptoethanol. Polypeptide bands were stained using a silver stain procedure (Merril et al., 1981). In some experiments, electrophoretically separated polypeptides were blotted onto nitrocellulose m e m b r a n e s by liquid electrotransfer for 22 h under 0.5 V / c m 2 according to Towbin et al. (1979). Protein concentrations. These were determined according to Bradford (1976) or Schaffner and Weissman (1973) for diluted samples, using bovine serum albumin as a reference. All chemicals were of the purest quality available.

Results

Characterization of the antigens H u m a n placental sphingomyelinase was highly purified through our former six-step method (Rousson et al., 1987) as shown in Table I. The final product was free of other lysosomal hydrolase activities and acidic sphingomyelinase was over 200,000 times purified with an overall yield of 8.8%. Pure enzyme preparations had specific activities ranging between 100 and 150 t z m o l / h per mg protein and gave two constant silverstained bands with 70 and 57 kDa masses on acrylamide after electrophoresis under denaturing conditions. In fibroblast extracts, sphingomyelinase activities ranged between 75 and 104 n m o l / h per mg

TABLE I PURIFICATION SCHEME OF ACID SPHINGOMYELINASE FROM NORMAL HUMAN PLACENTA Crude homogenate was from 250 g placental tissue Step

Crude homogenate Supernatant fluid ConA-Sepharose Butyl-agarose Octyl-agarose Matrex gel RedA Polybuffer exchanger

Volume Protein (ml) (rag)

Specific activity (nmol/h/ mg prot)

Yield (%)

1,000

32,350

5.3

100

760

17,025

8.2

81

400 350 60

335 41 6.80

336 2,347 9,335

66 57 37

25

0.82

46,300

22

7

0.15

102,000

8.8

protein for normal controls and were less than 0.1% of the normal rate for affected cell lines.

Preparation of the monoclonal antibodies Immunization of mice and characterization of serum antibodies. Anti-sphingomyelinase antibody titres were determined 10 days after each injection by ELISA. The mouse showing the highest antibody titer for sphingomyelinase ( O D = 0.3 for 1/4000 dilution) was selected for the fusion. Fusion. After the fusion the spleen cells were seeded in 576 wells. 4 days after fusion all the wells contained hybridomas but this n u m b e r decreased with time. Of 348 wells tested for antibody secretion (60.4%), 17 were identified as being positive by E L I S A for anti-sphingomyelinase antibody (4.8%). The four hybridomas showing the highest antibody titers were selected for further characterization.

Characterization of the monoclonal antibody Using the Ouchterlony double diffusion technique in the presence of 0.1% NP40, only one monoclonal antibody (MAB 236) reacted with native purified placental sphingomyelinase. Under similar conditions no precipitin line could be detected with the three other monoclonal anti-

203 o4

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2

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Protein concentration (mg/ml) Fig. 1. Sphingomyelinase amounts in normal and NiemannPick type A fibroblast extracts determined by the Mancini technique in the presence of Nonidet P 40.

bodies and our former rabbit polyclonal antibody (Rousson et a1.,1987). Using the double diffusion technique MAB 236 was shown to be of the IgG2a subclass. Using M A B 286 quantitative experiments with the Mancini technique in agarose containing 0.1% NP40 gave a linear response to sequential antigen dilutions in 10 mM Tris-HC1 containing 0.1% NP40 either with purified SPM or with fibroblast extracts as shown in Fig. 1 (full line). In contrast no precipitating rings were obtained with the polyclonal antibody under the described experimental conditions and consequently no reliable measurements could be done using MAB 236 in absence of the detergent. Two constant bands, with apparent masses of 70 and 57 kDa, were detectable with our monoclonal antibody on immunoblots performed either with placental SPM preparations at various stages of the purification or with fibroblast extracts. However the relative staining of the two bands was variable for a given step of the purification scheme and from one experiment to another. These results were in good agreement with the silver staining of the polypeptides obtained after SDS P A G E of the purest sphingomyelinase preparations and with our former results obtained with the polyclonal antibody (Fig. 2).

Imrnunoadsorption of sphingomyelinase activity by MAB 236 in the presence of NP40. With pure ascites fluids, 80% of sphingomyelinase activity

Fig. 2. Immunoblots of placental sphingomyelinase at two stages of purification: lanes 1 and 3 were loaded with material obtained after butyl agarose chromatography;lanes 2 and 4 represent the final preparation. MAB 236 was used as described in the materials and methods section for immunostaining of lanes 1 and 2 while lanes 3 and 4 are stained with our previously described polyclonalantibody.

either of fibroblast extracts or post-concanavalin A-Sepharose eluates were precipitated. In contrast, three other enzymatic activities, fl-hexosaminidase, /3-galactosidase and acidic phosphatase, were not altered by the monoclonal antibody.

Polypeptide analysis after immunoadsorption of sphingomyelinase in the presence of NP 40. Electrophoretic analysis of the material eluted from immobilised monoclonal antibody revealed (Fig. 3, lanes 1 and 2) two bands of 70 kDa and 57 kDa in the pure sphingomyelinase preparations.

Ref

1

2 3 4 5

Fig. 3. Electrophoretic analysis of polypeptides immunoadsorbed on immobilised MAB 236 after elution. Lanes 1 and 2 were loaded with two different normal fibroblast extracts while lanes 3 and 4 were loaded with two Niemann-Pick type A fibroblast extracts. Lane 5 was loaded with material after the procedure was performed with a normal fibroblast extract omitting the MAB 236 immobilisation.

204 MAB 236 as a tool to study sphingomyelinase in fibroblasts from NPA patients. In Ouchterlony experiments using MAB 236 the precipitation arcs showed a common identity between normal and pathological fibroblast extracts and in such tissues, Immunoblot tests with MAB 236 confirmed our previous results with polyclonal antibody, revealing the presence of the two sphingomyelinase polypeptides found in normal tissues. The capacity of the monoclonal antibody to precipitate mutant sphingomyelinase permitted quantitative determination of this enzyme by the Mancini technique. As shown in Fig. 1 there was no significant difference between normal and pathological fibroblast extracts (p < 0.01). Finally, as shown in Fig. 3, electrophoretic results of polypeptide analysis after elution of mutant sphingomyelinase adsorbed on immobilised MAB 236 were essentially similar to those obtained with the normal enzyme.

Discussion

The solubilization of acidic sphingomyelinase, a lysosomal membrane protein, requires the use of a non-ionic detergent. Indeed, in aqueous solutions the enzyme is denatured and form aggregates. These hydrophobic properties do not permit qualitative and quantitative immunological studies in classical aqueous buffers and thus make it imperative to use a detergent for such experiments. Moreover it has been reported for lysosomal sphingomyelinase that detergents inhibited the formation of low affinity antigen antibody complexes for polyclonal (Driessen et al., 1985) and monoclonal (AI et al., 1986) antibodies. In this paper we describe the first monoclonal antibody able to bind and precipitate sphingomyelinase in the presence of the non-ionic detergent NP40. The antigen purification from human placenta, based on the repeated use of hydrophobic chromatographies, takes advantage of the properties of the sphingomyelinase and gives in the final step a fully representative preparation of the native enzyme. Immunization of mice with high doses of antigen at 4-week intervals between booster injections led to antibody response after

four injections. However the additional 6 month delayed booster dramatically increased the antibody titer. This anusual immunization scheme took advantage of our former experience with monoclonal antibody production using long-term immunization to obtain high antibody titres for weakly immunogenic antigens (Parvaz et al., 1989b). In the present study the long interval between the third and the fourth booster injections helped to establish a strong immunological memory and a high antibody response was obtained after the last in vivo injection. For the last booster before cell fusion, we used an in vitro technique, which was found to be much more efficient than the in vivo technique (unpublished data). The selected monoclonal antibody 236 was the only antibody able to precipitate significant amounts of sphingomyelinase in the presence of NP40. This antibody enabled us to perform different tests for the characterization and quantitative determination of sphingomyelinase in various tissue extracts. MAB 236 recognised two bands of 70 and 57 kDa in immunoblot experiments on placental preparations and fibroblast extracts. These two bands corresponded to the two silverstained polypeptides previously described on acrylamide gels for purified preparations and were also recognized by the rabbit antiserum. These results highlighted the still unsolved problem of the number and the molecular masses of the sphingomyelinase polypeptides. Initial estimates of the enzyme molecular masses in the presence of detergent ranged from 180 to 300 kDa (Pentchev et al., 1977). In later reports the number of separated stained bands varied from one (Yamanaka and Suzuki, 1982) up to 30 (Jones et al., 1981) with molecular masses widely spread between 28 and 98 kDa. Four groups (Jones et al., 1981; Sakuragawa et al., 1982; Yamanaka and Suzuki, 1982; Quintern et al., 1987) concluded that a major sphingomyelinase component with a molecular weight ranging between 70 and 90 kDa existed in placenta, brain and urine. Using a polyclonal antiserum raised against purified placental sphingomyelinase and immunoblots, Callahan and Jobb (1988) detected a polypeptide of about 80 kDa in urine and brain, a larger one (108-120 kDa) in fibroblasts, spleen, and liver

205 and both forms in kidney. Freeman et al. (1983) also described a monoclonal antibody against placental sphingomyelinase that gave the same mass for the placental enzyme as the polyclonal serum but these authors did not described the precipitation of active solubilised sphingomyelinase. However, the recent cloning by Schuchman et al. (1992) of a c D N A coding for a 70 kDa enzymatically active polypeptide strongly supports the existence of at least a 70 kDa sphingomyelinase form found also with our monoclonal antibody. It does not exclude the presence of an additional 54 kDa form. Precipitation results (a decrease of sphingomyelinase activity and polypeptide analysis of the precipitated material) in tissue extracts with MAB 236 suggested that there was a correlation between the 70 a n d / o r 54 kDa polypeptides and sphingomyelinase activity. Assuming that a monoclonal antibody is specific for a single epitope, the fact that MAB 236 recognised the two co-purified bands supports the concept of a common epitope on the two polypeptides. Experiments are in progress to elucidate the significance of these two bands. The faster one might represent a partially degraded form or a different maturational stage of the 70 kDa polypeptide or an alternative splicing product of the sphingomyelinase gene according to the work of Quintern (1989) who first described the occurrence of alternatively processed transcripts for SPM gene. This specific monoclonal antibody with high binding capacity and the ability to precipitate sphingomyelinase activity, provided a tool for studying sphingomyelinase expression in Niemann-Pick type A patients. Using immunological tests - Ouchterlony, immunoblots - no qualitative differences have been found between the normal and the pathological enzyme, confirming the presence of cross reactive material in N P A fibroblasts, previously established with our former polyclonal antibody (Rousson et al., 1987). For the first time we have shown by the Mancini technique and polypeptide determination of immuno-precipitated material that sphingomyelinase polypeptide(s) are present at a normal level in pathological tissues. These results are in agreement with the data of Levran et al. (1991) and Ferlinz et al. (1991) who demonstrated the existence of several single base mutations in

the SPM gene, all in exon 6, resulting in the N P A phenotype but with no effect on R N A transcription. The ability to specifically precipitate the normal and the pathologic enzymes in the presence of detergent in tissue extracts should permit further experiments to elucidate how a single base mutation can generate a non-functional sphingomyelinase in normal amounts and with substrate affinity not affected (Rousson et al., 1987).

Acknowledgments This work was supported by funds from INSERM.

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