Monoclonal antibodies to rabbit liver cathepsin B - Bioscience Reports

Bioscience Reports, Vol. 6, No. 7, 1986

Monoclonal Antibodies to Rabbit Liver Cathepsin B R. John Wardale, Rose A. Maeiewicz and David J. Etherington* Received July 7, 1986

KEY WORDS: cathepsin B; monoclonal; antibody.

Abbreviations: ELISA, Enzyme-linked immunoadsorbent assay; EIP, Enzyme immunoprecipitation;

PAGE, polyacrylamidegel electrophoresis; Ep-475~L-trans-epoxysuccinyl-leucylamido(~-methyl)butane; Z, benzyloxycarbonyl; NMec, N-methylcoumarin; PEB, phosphate-EDTA-Brij 35; IAA, iodoacetic acid; PBS, phosphate-buffered saline; DMEM, Dulbecco's Minimal Essential Medium; FITC, fluorescein isothiocyanate.

Three stable hybridoma cell lines (AF8, BC11, CE2) have been produced that secrete antibodies specific for cathepsin B. These have been characterized by ELISA, SDSPAGE immunostaining, immunoprecipitation and immunofluorescent staining. CE2 immunoprecipitated native cathepsin B with retention of enzymic activity, but failed to cross-react with the alkali-denatured enzyme. BC11 bound only to the denatured form of cathepsin B and AF8 cross-reacted with both native and denatured cathepsin B. However, unlike CE2-immunoprecipitated enzyme, activity could be detected only after dissociation of the antigen-AF8 antibody complex. No cross reaction was found with any lysosomal protein includihg the cysteine proteinases, catbepsins H and L.

INTRODUCTION Cathepsin B (EC3.4.22.1) is a lysosomal cysteine proteinase that has been well characterized from a variety of mammalian species and tissues (1). The enzyme appears to have an important role in normal cell activities such as protein turnover and in vivo proteolytic processing, as well as contributing to tissue damage in various pathological conditions (2). AFRC Institute of Food Research, Bristol Laboratory, Langford, Brist01, BS18 7DY, UK. * To whom correspondence relating to these monoclonal antibodies should be addressed. 639 0144-8463/86/t1700-0639505.00/0 "~ 1986PlenumPublishingCorporation

640

Wardale, Maciewicz,and Etherington

Several laboratories have reported the preparation of polyclonal antibodies against cathepsin B but in general these were to the alkali-denatured form of the enzyme and only in a few instances was some cross-reaction with native cathepsin B observed (3-5). Preliminary reports have been published of monoclonal antibodies to cathepsin B but these were found to cross-react either with cathepsin M (6) or with chromosomal proteins (7). There was no indication in any of these reports of an antibody that could bind specifically to native and active cathepsin B. Such an antibody would be a useful probe for both in vivo and in vitro studies where one requires only native enzyme to be recognized. Consequently we have attempted to prepare monoclonal antibodies against the native enzyme by using native rabbit liver cathepsin B complexed to the synthetic cysteine proteinase inhibitor, Ep-475 (8), as the antigen. Use of this enzyme-inhibitor complex would minimize the removal of antigen by serum proteinase inhibitors such as e2-macroglobulin and also possibly prevent enzyme denaturation at physiological pH. We report here the production of three monoclonal antibodies, each recognizing a different antigenic determinant of cathepsin B including one specific for the native enzyme.

MATERIAL AND METHODS

Preparation of Cathepsins Rabbit liver cathepsins B and L were purified to the Mono S step as described previously for spleen (9). Cathepsin H was purified by a similar method except that the first chromatographic step was changed to DEAE-Sepharose and chromatography on phenyl Sepharose was used in place of organomercurial Sepharose. The lysosomal extract was prepared as described (10). Enzyme concentrations were determined by active site titration using Ep-475 (11). The Ep-475 inhibited cathepsin B antigen was prepared as follows: cathepsin B (1.73 #M) was activated at 37~ for 15 rain in 100 mM sodium phosphate, 1 mM Na2EDTA, 1% w/v Brij 35, pH 6.0 (PEB buffer), plus 10 mM cysteine. Ep-475 was added to a final concentration of 2.60 #M and the mixture incubated at 25~ for 60 min. Cathepsins were labelled with [2-14C]-IAA (Amersham International plc) as follows. Each enzyme was activated as above and IAA added to a final concentration of 0.2mM (10.6 #Ci/ml). The reactions were allowed to continue for 30min at 37~ Unincorporated IAA was removed from the enzyme sample by gel filtration using Sephadex G15 in PEB buffer. Alkali-denatured IAA-labelled cathepsin B was prepared by adding 1 M NaOH to a final pH of 11 and incubating at 37~ for 30 min.

Preparation of Hybridomas An emulsion containing equal volumes of 1.73 #M Ep-475--inhibited cathepsin B and Freund's Complete Adjuvant (DIFCO) was prepared, dispensed into lml syringes and frozen in liquid nitrogen until required. BALB/c mice were immunized

Liver Cathepsin B Monoclonal Antibodies

641

(200/~1, i.p.) at 14 day intervals and the mouse showing the strongest immune response after three injections was sacrificed and hybridoma cell lines established essentially as described (12).

ELISA ELISA (13) was carried out using PVC microtitre plates (Dynatech), coated with 100 #1 (30 ng) per well purified rabbit liver cathepsin B in PBS, pH 6.0, for screening assays or at raised pH values of 7-10 for characterization experiments.

Immunofluorescence A secondary fibroblast line derived from rabbit muscle was grown to confluence in DMEM containing 10 % (v/v) fetal calf serum on multitest slides (FLOW). Slides were fixed at 20~ using 1:1 acetone:methanol for 10 rain followed by 15 min in PBS-1% (v/v) Triton X-100 (Sigma). Hybridoma supernatants were applied undiluted and incubated at 37~ for 2 h, followed by a goat anti-mouse IgG FITC conjugate (1:30 dilution, Sigma) for 20 rain at 37~ A thorough wash with PBS was used between each stage.

Enzyme Immunoprecipitation(EIP) Goat anti-mouse IgG (4.67 rag, Sigma) was coupled to tresyl Sepharose (1 g dry weight, Pharmacia) in 0.1 M NaHCO3, 0.5 M NaCI, pH 7.5, for 4 h at 20~ as described by the manufacturers. Unbound immunoglobulin was washed out with the coupling buffer and residual active groups on the Sepharose were deactivated using 0ol M Tris-HCt, pH 8.0. The antibody-Sepharose was washed and stored in PEB buffer. Diluted antisera or hybridoma supernatants containing 0.1% (v/v) Tween 20 were mixed with the complex overnight at 37~ Unbound material was removed by washing in PBS Tween. 50 pmoles of native, unlabelled, or 25 pmoles of native or alkali-denatured [14C]-IAA labelled cathepsin B in 2.5 ml PEB buffer, were added to the antibody-Sepharose complex and agitated for 4 hours at 20~ The complex was then washed in PEB buffer and the amount of bound cathepsin B was determined either by fluorimetric assay or by radioactivity.

SDS-PAGE and Immunostaining Proteins were separated electrophoretically in 12.5 % SDS-polyacrylamide gels (14) and transferred to nitrocellulose paper (pore size 0.45 pro, Schleicher and Schuel) (15). The transfers were blocked with PBS Tween and then probed with hybridoma supernatants dilated in PBS Tween, for 3 h at 20~ After washing, bound monoclonal antibody was detected with goat anti-mouse IgG alkaline phosphatase conjugate (1:1000 dilution, Sigma) (16). When radio-labelled cathepsins were used, the transfers were autoradiographed using preflashed Kodak RP4 X-ray film.

642

Wardale, Maciewicz, and Etherington

RESULTS All mice immunized with Ep-475-inhibited cathepsin B were positive as determined by immunofluorescence and EIP. Screening of hybridoma cell lines by a combination of techniques (ELISA, immunofluorescence and EIP) indicated the secretion of antibodies against cathepsin B by three clones--AF8, BCll and CE2. All three hybridomas gave positive ELISA results when the antigen was coated at pH 6,0, although CE2 gave a notably weaker reaction. When the antigen was coated at pH 7.0 or higher (conditions which denature cathepsin B (1)), CE2 binding was no longer detected, whereas AF8 and BC11 gave an undiminished positive result (data not shown). These two hybridomas produced a characteristic lysosomal staining pattern by indirect immunofluorescence after fixation (Fig. 1). CE2 failed to show any reaction in this assay possibly due to the antigen having changed its conformation or becoming denatured during fixation. These results suggested to us that CE2 might be directed only toward the native enzyme. To test this hypothesis, the ability of antibodies from CE2, BC11 and AF8 to bind native and alkali denatured cathepsin was measured by EIP (Fig. 2). Bound, native unlabelled cathepsin B was detected by its enzymic activity (Fig. 2a) and bound native and alkali-denatured [14C]-IAA cathepsin B were determined from their radioactivity (Figs 2b and 2c). The data clearly indicate that CE2 recognized only native cathepsin B, and, furthermore, the complexed enzyme was still able to cleave the synthetic substrate. In contrast, native and denatured radiolabelled cathepsin B were clearly bound by AF8 in the EIP assay but, with unlabelled, native cathepsin B, no enzyme activity could be detected in the complex. It was apparent that the enzyme had lost its ability to cleave synthetic substrate upon antibody binding, since enzyme activity could be restored upon dissociation of this complex under non-denaturing conditions (data not shown). BC11 appeared to bind only alkali-denatured cathepsin B. SDS-PAGE immunotransfer (Fig. 3) indicated that AF8 and BCll detected a protein of Mr 31,000 which corresponded to the position of [14C~-IAA labelled

Fig. 1. Indirect immunofluorescent staining of rabbit fibroblasts with monoclonal supernatants BC11 (a) and CE2 (b) (Scale bar = 20/~m). Similar results to (a) and (b) were obtained using AF8 and control monoclonal supernatants respectively.


643

5 !

o K

O

ra

4 3

1

A

4

I O

"

3 2

A

A

4 C

3 9"

x

2

E 1 U 0

25 50 75 ug monoclonal antibody. antFmouse IgG. trasyl Sepharose

100

Fig. 2. Enzyme immunoprecipitation of (a) native, active cathepsin B, (b) native [14C]IAA cathepsin B, and (c) alkali-denatured [14C]-IAA cathepsin B by monoclonal supernatants. 50 pmoles of native, unlabetled, or 25 pmoles of native or alkali denatured [14C]-IAA cathepsin B, were mixed with increasing amounts of CE2 (O), AF8 (m), BC111 (A) or control (V) monocional antibody anti-mouse IgG tresyl-Sepharose complex and assayed for binding by enzyme activity (a) or radioactivity (b, c).

cathepsin B. AF8 and BCll did not cross react with cathepsins H or L or any other lysosomal proteins. Although two bands were observed in the lysosomal extract lane with both antibodies, the weaker band at Mr 25,000 appeared to correspond to the larger of the two subunits of cathepsin B. The proportion of this band relative to the Mr 31,000 band could be increased by t he addition of DTT which dissociates cathepsin B into its two subunits (data not shown). As expected CE2 did not bind to cathepsin B in this assay since the antigen had been denatured. Cross reactivity of CE2 with


644

Fig. 3. SDS-PAGE immunotransfers of cathepsin B and related lysosomal proteins. Cathepsin B (ll.5pmoles, lanes 1 and 5), L (22.2pmoles, lanes 2 and 6), H (22.2pmoles, lanes 3 and 7), and a lysosomal extract (100#g, lanes 4 and 8), were probed with monoclonal supernatants BC11 (a) and CE2 (b). The relative positions [14C]-IAAlabelled cathepsin B (22.2 pmoles, lane 9), L (22.2pmoles, lane 10), and H (84.4 pmoles, lane 11), are shown by autoradiography (c). Lane 12 is a lysosomal extract stained with coomassie blue. Positions of molecular weight standards phosphorylase b (92,500), bovine serum albumin (69,000), ovalbumin (46,000), carbonic anhydrase (30,000), and lysozyme(14,300), are as shown. Similar results to (a) and (b) were obtained using AF8 and a control monoclonal supernatant respectively.

cathepsins H and L were tested in the activity E I P assay and no binding was observed (data not shown).

DISCUSSION We report here, for the first time, the preparation of monoclonal antibodies specific for both native and denatured cathepsin B. The previous lack of success in raising monoclonal antibodies to native cathepsin B can be attributed both to problems in the initial immunization procedure and the subsequent immunoassays. Our approach to antigen preparation was to reduce the loss of native cathepsin B upon injection. Two problems needed to be considered; the instability of the enzyme at physiological pH; and the removal of active cathepsin by serum inhibitors. We therefore tested if the enzyme would retain its native epitopes by blocking the active site with the cysteine-specific inhibitor, Ep-475. F r o m our results it appears that Ep-475 inhibition of cathepsin B reduced binding by serum inhibitors and afforded the proteinase some protection against denaturation at physiological pH, perhaps by mimicking an enzyme-substrate complex.


645

The standard immunoassays (ELISA, immunofluorescence and those based on nitrocellullose paper) appeared to be unsuitable for the detection of antibodies specific for native cathepsin B. Previous work has shown that cathepsins lose most of their activity when applied to nitrocellulose paper or ELISA plates (unpublished observation). The weak response from CE2 on ELISA and the negative result on immunotransfer must be attributed to denaturation of cathepsin B during the assay. Results from this work also indicated that cathepsin B was denatured during fixation for immunofluorescence. By using rapid assays, i.e. ELISA and immunofluorescence, which are suitable for screening the large numbers of supernatants initially produced, positive samples by these criteria could then be applied to the more time-consuming and laborious EIP assays. It was apparent that immunoprecipitation of the enzyme was the only sure method of showing whether the antibody bound to the native enzyme. The three monoclonal antibodies reported here appear to be specific for cathepsin B and directed against different antigenic determinants on the enzyme. CE2 recognized an epitope present only in the native enzyme. This epitope was probably not located near the active site as the enzyme antibody complex still retained enzyme activity. AF8 recognized both the native and denatured forms of cathepsin B. Since the complex of native enzyme and antibody failed to hydrolyse the peptide substrate, the epitope was probably near to the active site or binding of this antibody caused a conformational change that rendered the enzyme inactive. The third monoclonal antibody, BCll, bound only to denatured cathepsin B suggesting that the epitope became exposed or was formed on denaturation. Evidence from immunotransfers showed that the epitopes recognized by AF8 and BC11 were located on the Mr 25,000 subunit of cathepsin B.

ACKNOWLEDGEMENT

This work was supported in part by a grant from the Arthritis and Rheumatism Council.

REFERENCES I. Barrett, A. J. (1977). In: Proreinases in Mammalian Cells and Tissues (A. J. Barrett, Ed.), North Holland, Amsterdam, pp. 181-208. 2. Poole, R. A., and Mort, J. S. (1981). J. Histochem. Cytochem. 29:494-500. 3. Pierart-Gallois, M., Trouet, A., Tulkens, P. (1977). Acta. biol. reed. 9erm. 36:1887-1891. 4. Graf, M., Baici, A., and Striiuli, P. (1981). Lab. lnv. 45:587-596. 5. Burnett, D., Crocker, J., and Stocktey, R. A. (1983). Am. Rev. Respir. Dis. 128:915-919. 6. Erickson-Viitanen, S., McDermott, M., Hempstead, J., Balestreri, E., Morgan, J., and Horecker, B. L. (1985). Fed. Proc. 44:877. 7. Seeler, B., Horton, M., Sieling, P., and Szego, C. (1985). Fed. Proc. 44:875. 8. Barrett, A. J. (1980). In: Proteinases and Their Inhibitors (Turk, V., and Vitale, L. J., Eds). Proceedings of the international symposium, Pormroz, Yugoslavia, Mladinska Knjiga, Ljubljana and Pergamon Press, Oxford, pp. 103-107. 9. Maciewicz, R. A., and Etherington, D. J. (1985). Bioehem. Soe. Trans. 13:1169-1170. 10. Harikumar, P., and Reeves, J. P. (1983). J. Biol. Chem. 258:10403-10410.

646


11. Barrett, A. J., and Kirschke, H. (1981). In: Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., Eds.), Academic Press, New York, Vol. 80, pp. 535-561. 12. Galfre, G., Howe, S., Milstein, C., Butcher, G., and Howard, J. (1977). Nature (Lond.) 266:550-552. 13. Voller, A., and BidweI1, D. E. (1975). Br. J. Exp. Pathol. 56"338-339. 14. Laemlli, U. K. (1970). Nature (Lond.) 227:680-685. 15. Towbin, H., Staehelin, T., and Gordon, J. (1979). Proe. Natl. Aead. Sei. USA 76:4350-4354. 16. Penny, I, F., Taylor, M. A. J., Harris, A. G., and Etherington, D. J. (1985). Bioehim. Biophys. Aeta 829:244--252.