HYBRIDOMA Volume 11, Number 3, 1992 Mary Ann Liebert, Inc., Publishers

Production and Characterization of a Monoclonal Antibody to Human Saposin C JAROSLAV J.

STASTNY,1

YASUO KISHIMOTO,2 JOHN S. CRAIG W. BEATTIE1·3

O'BRIEN,2 and

'Cancer Center, University of Illinois at Chicago, Chicago, IL 60612 Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093 'Present address: USDA-ARS, Meat Animal Research Center, Clay Center, NE 68933

2Departmeni of Neurosciences,

ABSTRACT

IgGl monoclonal antibody, termed 68-12, was produced against Immunoprecipitation and binding analysis indicated saposin C. that the antibody reacted only with saposin C. Dot blotting and Western analysis demonstrated that antibody 68-12 also reacted with prosaposin and a higher Solid molecular weight protein(s) in murine spleen and cerebral grey matter. phase competitive radioimmunoassay against 125I labeled saposin C (0.25 /ig/ml) and D up to 15 /¿g/ml. At a showed no cross reactivity for saposin A, and D cross reacted 21, 1.5, and 49% concentration of 50 /ig/ml saposin A, respectively. Monoclonal antibody 68-12 appears to have potential utility in the purification, detection and quantitation of human saposin C and its precursor. A

purified

murine human

INTRODUCTION The saposin group of sphingolipid activator proteins are derived from a single lysosomal precursor, prosaposin [(524aa, 68-73 kilodaltons (kDa)] by Saposins A, B, C, D are small (-12 kDa) heat-stable proteolytic processing. proteins, localized in lysosomes, that appear necessary for the hydrolysis of Prosaposin, unlike sphingolipids by specific lysosomal hydrolases (1-6). saposin(s), apparently does not promote the hydrolysis of sphingolipids (1) Saposin C which activates the hydrolysis of glucocerebroside by glucosylceramide/3-glucosidase and galactocerebroside by galactocerebroside-/3-glucosidase is found along with prosaposin in testes, seminal plasma, brain, kidney, spleen, liver and .

human milk (1). The rat Sertoli cells

of prosaposin, SGP-1, a major secretory protein of is a postranslationally glycosylated 67 kDa precursor postranslationally modified to 70 kDa prior to secretion into the extracellular The native secreted form has been reported to occur as a noncovalent space (1). homodimer of 140 kDa (1). SGP-1 is widely distributed in rat tissues and exhibits a tissue dependent proteolytic processing (9). As the quantification of saposins currently requires a minimum of HPLC (10) and an activating assay (11) rapid, highly specific assays that incorporate monoclonal antibodies (MAbs) could prove highly useful in isolating and We report here the production of a murine MAb quantifying these proteins. reactive with human saposin C and prosaposin as judged by electrophoretic

equivalent (7,8),

techniques. 351

MATERIAL AND METHODS

Antigens Saposins A, C, and D used in this study were isolated and purified as Gaucher spleen was homogenized in water and the homogenate was heated After lyophilization of and centrifuged as described (10) but on a larger scale. the supernatant and redissolving the residue in a small volume of water, the extract was fractionated by C4-HPLC (10) using a preparative column (0.46 cm 25 The effluents under the clustered peaks of saposins A, C, and D were cm; Vydac). collected together and the material obtained after evaporation of solvent was further fractionated by DEAE (DE-52; Whatman) column chromatography as described (5,6). Fractions containing saposins A, C, and D were separately collected and lyophilized. Each of the residues was finally purified by rechromatography on follows.

salts and other contaminants. the liver of a fucosidosis patient by for the other saposins. Saposin emerged from the C4 column earlier than the clustered peaks of saposins A, C, and D. Details of the preparation will be published separately. Extracts of human and mouse tissues were prepared by finely mincing each tissue, followed by homogenization with a Polytron homogenizer in 10 mM phosphate, 350 mM NaCl, pH 7.4 (PBS) at 4 °C. The supernatant obtained after centrifugation at 12,000 g for 20 min at 4 °C was used in this study. C4-HPLC

as

described above to

remove

was Saposin prepared from procedures similar to those described

of

Conjugation

saposin

C with BSA

Saposin C was covalently coupled to bovine serum albumin (BSA) with ethyldimethylaminopropyl carbodiimide hydrochloride (EDC) using a conjugation kit supplied by Pierce Chemical Co. (Rockford, IL). Briefly, a mixture of 0.4 mg saposin C, 0.5 mg BSA and 2.5 mg EDC, disolved in conjugation buffer, was incubated 2 hr at room temperature. After Sephadex G-25 gel filtration saposin CBSA conjugate fractions were pooled and individual aliquots stored at -70 °C until

use.

Antibody production BALB/c mice received two i.p. inoculations 3 weeks apart of 20 /ig conjugate in a mixture with Freund's adjuvant. The first injection was in 200 µ complete with the second in 200 µ Freund's incomplete adjuvant. Three days after the third injection, BALB/c splenocytes were fused with nonsecreting myeloma X63-Ag8.653 cells (12) as previously described (13, 14). The hybridomas obtained were screened against saposin C and BSA by indirect solid phase radioimmunoassay (RIA) using i25I-labeled rabbit anti-mouse IgG (RAMIgG) (13). Selected hybridoma cell lines were subsequently expanded in standard medium and formally cloned by limiting dilution (13, 14). MAb 68-12 was tested and further characterized either as culture supernatant or purified, from ascitic fluid (13) by affinity chromatography on protein G-agarose (Genex Corp., Gaithersburg, MD) according to the manufacturer's instructions. The isotype of MAb 68-12 was determined using micro Ouchterlony diffusion plates (Miles Scientific, Naperville, IL) and confirmed using an isotype-subtype specific enzyme-linked immunoabsorbent assay (ELISA) (Bio-Rad Laboratories, Richmond, CA). Antibody affinity was determined by Scatchard analysis (15) as described by Frankel and Gerhard (16). Female

saposin

C-BSA

Binding analysis Solid-phase RIAs tested for binding to different antigens by indirect solid-phase wells of polyvinyl chloride microtiter plates were incubated with 0.5 pg of antigen in 20 µ of PBS overnight at 4 °C. After blocking with 10% newborn calf serum (NCS) in PBS, test MAb was added and incubated for 1 hr. MAbs

RIA

were

(17). Briefly,

352

washed with 0.1% NCS in PBS and incubated with izsi-labeled RAMIgG for 1 hr. The wells were washed, cut out and bound radioactivity counted. All assays were performed in duplicate. Control wells were incubated with 10% NCS in PBS without test MAb. The values obtained from control wells were subtracted from the experimental wells. A direct solid-phase RIA was used in competition studies. Briefly, 20 µ of purified MAb at 50 µg/ml was applied to wells, incubated, washed and blocked with 10% NCS in PBS as described above. Twenty µ of izsi-iabeled saposin C (18) at 0.25 µg/ml alone or with unlabeled competing antigen in a range from 0.1 µg to 12 µg/ml was then added. After incubation for 1 hr, the wells were washed, dried, cut out, and bound radioactivity counted as described above.

The wells

were

(8x10^ cpm/well)

Immunoprecipitation Immunoprecipitation of 125I-labeled saposin C and saposin C-BSA conjugate by purified MAb bound to agarose-immobilized protein G was performed as previously described (19). Immune complexes were eluted from agarose with sample buffer, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (20), and visualized autoradiographically. Dot blot and Western analysis

Samples diluted in PBS were applied to Hybond nylon membrane (Ammersham Corp., Arlington Heights, IL) using a microsample filtration manifold (Schleicher and Schuell, Ine., Keene, NH). The nylon membrane was blocked with 10% NCS in PBS

(overnight; 4 °C), then incubated titer previously determined by

a

labeled

RAMIgG (105 cpm/ml)

temperature. The membrane

was

at room temperature with MAb 68-12 at RIA to give maximal binding (1:2,000). i25ithen added and incubated for 2 hr at room was washed and antibody-antigen binding was visualized

for 2 hr

by autoradiography. The specificity of MAb 68-12 was determined by Western blot analysis according to the method of Towbin et al.(21). Separated proteins were transferred from 7%, 1.5mm thick polyacrylamide gels to nitrocellulose (Schleicher and Schuell, Inc., Keene, NH) at 1.3 A for 3 hr in 25 mM Tris, 192 mM glycin, 0.1% SDS, 20% (vol/vol) methanol, pH 8.3. The nitrocellulose membrane was blocked with 10% NCS in PBS, incubated with MAb followed by incubation with l25I-labeled RAMIgG and antigen-antibody binding again visualized autoradiographically. RESULTS

Generation of MAb 68-12

Following fusion, supernatants from 1,152 culture wells with growing hybridomas were screened for reactivity with BSA by solid-phase indirect RIA. The supernatants which showed no binding to BSA (1,002 culture wells) were screened for reactivity with saposin C. Twelve clones with binding to saposin C up to 600 cpm and one clone with binding to 10,500 cpm (MAb 68-12) were expanded in culture and later stored in liquid nitrogen. Murine MAb 68-12 was further analyzed and characterized. Immunodiffusion analysis of MAb 68-12, indicated an isotype of IgGi with a binding affinity for saposin C of 3.2xl09 M"1. Specificity

of MAb 68-12

The specificity of MAb 68-12 for saposin C was tested by indirect solidRIA. MAb 68-12 did not crossreact with PBS extracts of neural tissues (neurosarcoma, Schwanoma), normal human skin, muscle and casein and whey fractions from different species (sheep, goat, bovine, pig) at 50 µg/ml (1 µg/well). MAb 68-12 exhibited low positive binding to PBS extracts of human liver and spleen which was less than 5% of binding to saposin C. The binding of MAb 68-12 by indirect solid-phase RIA was saturable for saposin C at 50 µg/ml (1

phase

353

One µg samples of saposin C (A), FIGURE 1. Dot blot analysis of MAb 68-12. prosaposin (B) 0.84 µg saposin C-BSA conjugate C) 80 µg protein (12,000xg supernatant) human liver (D), spleen (E) and 5 µ (200 µg protein) milk (F) was loaded into the first well (column) and diluted in PBS over a range of 1/2 to 1/2048 in the remaining 11 wells. ,

,

,

µg/well), prosaposin 12.5 µg/ml (0.25 µg/well) and the saposin C-BSA conjugate 3.75 µg/ml (0.075 µg/well) with half maximal binding at 5 µg/ml (0.1 µg/well), 0.4 µg/ml (0.008 µg/well) and 0.05 µg/ml (0.001 µg/well) for saposin C, prosaposin and the conjugate respectively. Dot-blot analysis (Fig.l) of prosaposin and the saposin C-BSA conjugate were in agreement with those obtained by RIA. Both antigens were detectable by MAb 68-12 at concentrations as low as 0.05 µg/ml (0.001 µg/well). Although MAb 68-12 detection of saposin C was less sensitive by dot blot (0.25 µg/well) than by indirect solid-phase RIA (0.001 µg/well), binding was clearly detected to whole human milk (110 µg protein), liver (10 µg protein) and spleen (2.5 µg protein). Immunoprecipitation

and Western

blotting

Saposin C and BSA used for conjugation were iodinated and separated on 7% SDS-PAGE (Figure 2). While saposin C showed only one band on 15% (data not shown) and 7% acrylamide gels (lane 1), saposin C-BSA conjugate revealed a major band at 68 kDa (albumin), a less prominent band at about 75 kDa and traces of saposin C and IgG (lane 2) A similar pattern was observed in 7% gels with unlabeled saposin C and saposin C-BSA conjugate stained with Coomassie Blue (data not shown). After immunoprecipitation of 125I-labeled saposin C and the i25I-labeled saposin C-BSA conjugate by MAb 68-12 followed by SDS-PAGE (Figure 2), the same binding pattern was observed for 125I-saposin C (lane 1) and immunoprecipitated 1251-labeled saposin C (lane 4). However, differences between l25I -labeled saposin C-BSA conjugate (lane 2) and immunoprecipitated 125I-labeled saposin C-BSA conjugate (lane 5) were evident. The major band of 68 kDa (lane 2) was not immunoprecipitated by MAb 68-12. MAb 68-12 immunoprecipitated the conjugate as a diffuse band in a range of 115 to 170 kDa, in addition to saposin C (lower edge of the gel) and a higher molecular weight conjugate (top of the .

gel).

Western blot analysis (Figure 3) of MAb 68-12 binding to saposin C, revealed a binding pattern similar to that described by immunoprecipitation MAb 68-12 binding to the saposin C-BSA conjugate was slightly (Figure 2). Western blotting revealed that MAb 68-12 was bound to different, however. saposin C (bottom) and a series of saposin C conjugates with Mrs >68 kDa. In samples of human milk and spleen, a band at 94 kDa was also recognized by MAb 68-12. Under nonreducing conditions MAb 68-12 also bound to saposin C dimers or aggregates which ranged from 170 kDa to 250 kDa in PBS extracts of mouse spleen and cerebral grey matter. -

354

12

5

4

3

i

•

150-

•

68-

FIGURE 2. Autoradiography of immunoprecipitated and SDS-PAGE (7% gel; nonreducing conditions) separated saposin C-BSA conjugate, 125I-labeled saposin C (lane 1), i25I-labeled conjugate saposin C-BSA (lane 2), i25I-labeled RAMIgG (lane 3), immunoprecipitate 125I-labeled saposin C (lane 4), and immunoprecipitated 1251-labeled saposin C-BSA conjugate (lane 5) by MAb 68-12 bound to agaroseimmobilized protein G.

3

4

5

7

I

8

220

—

150

ffc

—

68

Figure 3. Western blot analysis of MAb 68-12 specificity. Samples of 0.2 µg saposin C (lane 1), 0.04 µg saposin C (lane 2), 0.4 µg conjugate of saposin C-BSA (lane 3), 20 µ of human milk (lane 4), 12,000 g supernatant of human spleen (lane 5), liver (lane 6) mouse spleen (lane 7) and cerebral grey matter (lane 8), SDS-PAGE (nonreducing), transferred to nitrocellulose were separated using 7% membrane, treated with MAb 68-12 followed by incubation with 125I-labeled RAMIgG and visualized autoradiographically.

355

Binding

studies

In preliminary studies, which optimized saposin C concentrations in a solid-phase indirect RIA prior to titration analysis, a saposin C concentration of 7.25 µg/ml (0.125 µg/well) was found to be subsaturating (Figure 4). MAb 68-12 binding plateaued until a concentration of 0.5 µg/ml was reached and then

declined.

0.005

pg/mt

Figure 4. Avidity of MAb 68-12. Binding was determined at a fixed concentration saposin C (7.25 µg/ml) with increasing concentrations of MAb 68-12 (0.0039 µg/ml to 1 µg/ml). Counts were corrected by subtracting cpm in the absence of MAb 68-12 from total cpm. Values represent the mean of duplicate samples. of

The cross reactivity of saposins A, B, D, prosaposin and the saposin C-BSA conjugate was determined directly using 125I-labeled saposin C in a competitive binding RIA (Figure 5). Saposin A, B, and D did not compete with 125I-saposin C in binding to MAb 68-12 over a concentration range of 0.05 µg/ml to 15 µg/ml. However, competition between i25I-saposin C and unlabeled saposin C as well as prosaposin was significant. The binding of i25l-saposin was inhibited 50% by unlabeled saposin C and prosaposin at 0.25 µg/ml and 0.7 µg/ml respectively. Significantly less inhibition (- 18 %) was observed for the saposin C-BSA conjugate at a conjugate to 125I-saposin C ratio of 12/0.25. Although negligible binding of saposins A, B, and D to MAb 68-12 was demonstrable at lower physiologic concentrations (