Monoclonal Antibodies to Human Lysyl Oxidase

Collagen Res. ReI. Vol. 6/1986, pp. 153-162

Monoclonal Antibodies to Human Lysyl Oxidase PETER D. BURBELO, ANDREA MONCKEBERG and CLINTON O. CHICHESTER Department of Pharmacology and Toxicology, University of Rhode Island, Kingston, RI 02881, USA.

Abstract Hybridoma antibodies against human Iysyl oxidase were produced by fusing Sp. 2.O-Ag 14 myeloma cells with spleen cells from mice hyperimmunized with lysyl oxidase isolated from umbilical cords. Hybridomas positive by enzyme-linked immunosorbent assay (ELISA) for human Iysyl oxidase were cloned by the dilution method. Eight hybridomas producing antibodies were isolated, three of which also recognized purified bovine aortic lysyl oxidase in an ELISA. One of these antibodies, monoclonal antibody I, was purified and attached to Sepharose CLAB. The immobilized antibody was effective in binding an enzymatically active 30,000 dalton species. Immunoblot analysis of four of these antibodies showed reactivity against the 30,000 dalton catalytically active enzyme and the 24,000 dalton fragment of lys)' 1 oxidase. These monoclonal antibodies should be useful tools for studying the localization and biosynthesis of lysyl oxidase. Key words: immunoaffinity chromatography, lysyl oxidase, monoclonal antibodies. Introduction Lysyl oxidase (protein lysine oxidase, E.e. 1.4 3.13) catalyzes the oxidative deamination of specific-f-amino groups of Iysyl and hydroxylysyl residues in collagen and lysyl residues in elastin. The resulting aldehydes undergo aldol condensation or Schiffbase reactions to yield the covalent crosslinks of these connective tissue proteins. Purification of lysyl oxidase in 4-6 M urea from bovine aorta (Kagan et aI., 1979), chicken cartilage (Stassen, 1976) and human placenta (Kuivaniemi et aI., 1984) has shown enzyme activity to be associated with a molecular weight species of approximately 30,000 daltons. The use of urea, a strong denaturant, in die purification of the enzyme may disrupt subunit-subunit interactions of the enzyme and may irreversibly inactivate a certain portion of lysyl oxidase molecules. Removal of urea produces large aggregates of enzyme ranging in molecular weight from 60,000 to 1,000,000 daltons (Jordan et ai., 1977). . Polyclonalantibbdies raised against human lysyl oxidase recognize several forms of the enzyme, including the 30,000 dalton active form, a 60,000 dalton dimer and a 22,000 dalton fragment (Kuivaniemi et aI., 1984). Our work with polyclonal anti-

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P. D. Burbelo, A. Monckeberg and C. O. Chichester

bodies against bovine aortic Iysyl oxidase showed these antibodies reacted against at least two different molecular weight forms of the enzyme (Burbelo et aI., 1985 ). These antibodies showed reactivity against the catalytically active 30,000 dalton enzyme, as well as against catalytically inactive, large molecular weight protein(s) . Since polyclonal antibodies recognize multiple antigenic determinants, the exact nature of the reactivity was not resolved, but may have been due to inactive aggregates of enzyme or inactive precursors. In the present study we describe the preparation of monoclonal antibodies to human Iysyl oxidase. Since monoclonal antibodies recognize a single epitope, these antibodies have the potential to characterize distinct regions of the Iysyl oxidase molecule.

Methods Purification of Lysyl Oxidase from Umbilical Cords

Lysyl oxidase was purified from umbilical cords generously provided by Dr. D. Singer from Women and Infants Hospital, Providence, RI. A combination of Cibacron Blue-Sepharose 4B, DEAE-cellulose, and Sephacryl S-200 chromatography was used to purify the enzyme. The tritium release assay, utilizing a chick aortic substrate, was used to follow lysyl oxidase activity during the purification (Burbelo et aI., 1985). Protein was measured by the method of Lowry et al. (1951). The purification procedure was performed at 4°C and 1 mM phenylmethylsulfonyl fluoride was added to all buffers. Umbilical cords were minced and twice homogenized in a blender with 2.5 volumes (wi v) of 16mM potassium phosphate, 0.15 M NaC!, pH 7.7. The homogenate was centrifugated at 11,000 x g for 20 min and the supernatant discarded. The pellet was then extracted in 16 mM potassium phosphate and recentrifuged. The pellet was resuspended in 1 M urea. 16 mM potassium phosphate and allowed to stir for 30 min before further centrifugation. These extracts contained negligible Iysyl oxidase activity. The pellet was then extracted four times with 4M urea, 16mM phosphate, pH 7.7. The 4 M urea extracts containing Iysyl oxidase activity were combined, diluted to 1 M urea and applied to the Cibacron Blue-Sepharose column equilibrated in 1 M urea, 16 mM phosphate, pH 7.7. The column was washed with 16 mM phosphate, 150 mM NaC! and eluted with 6 M urea, 16 mM phosphate. The Cibacron Blue-purified enzyme was applied to a DEAE-cellulose column in 6 M urea, 16 mM phosphate and subsequently washed with the same buffer. The column was eluted with 6 M urea, 16 mM phosphate, 0.5 M NaC!. The DEAE-cellulose enzyme was concentrated to 6 ml by ultrafiltration on a YM-10 membrane (Amicon Corp.) at 20 psi and applied to a Sephacryl S200 column. Lysyl oxidase activity was shown to elute at a volume corresponding to 30,000 daltons on Sephacryl S-200 (Figure 1). This procedure resulted in a final 230~ fold purification over the initial urea extract. Purity of the enzyme preparation was checked by SDS-polyacrylamide gel electrophoresis (Laemmli et aI., 1970). Enzyme purified in this manner was used for all immunizations and as standard in the ELISA. To test whether the Sephacryl-purified enzyme preparation contained differing ionic variants (Williams and Kagan, 1985), anion-exchange, high performance liquid chromatography was performed. Lysyl oxidase (5 mg) was applied to Mono Q 5/5 column (Pharmacia) in 6M urea, 16mM KH 2 P0 4 and washed with the same buffer. Enzyme activity was eluted with a salt gradient from 0.0 to 1.0 M NaC!.

Monoclonal Antibodies to Lysyl Oxidase

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Preparation of Mouse Hybridoma Cells Balb/C mice were initially immunized intraperitoneally with 50 Ilg of Sephacryl S200 purified lysyl oxidase in Freund's adjuvant and subsequently injected at three week intervals with enzyme in incomplete adjuvant. One month following the fifth injection the mice were boosted with 50 Ilg of Sephacryl S-200 purified lysyl oxidase. On the fourth day following the last injection, the spleens were removed and the lymphocytes were isolated for fusion . SP2.0-Ag14 myeloma cells (American Type Culture Collection) were expanded and maintained in logarithmic growth prior to fusion. The myeloma cells were collected, washed in serum free media and mixed in a 1 : 10 ratio with spleen cells. The cells were fused for 2 min in 1.0 ml of 40% polyethylene glycol 1000, pH 7.4 in Hank's calcium free buffer. The cell suspension was diluted over an additional five min with 50 ml of serum free media. The cells were spun down and resuspended in medium containing hypoxanthine 0.1 mM, aminopterin 0.4 11M and thymidine 16 mM with 20% fetal calf serum and plated at a density of 2 x 105 cells/well in 96-well plates. After four days, half the medium was removed and replaced with media containing hypoxanthine, thymidine and 15% fetal calf serum, Ten days later the wells were observed for hybridoma growth. ELISA Screen and Cloning ELISA was performed as described by Engvall and Perlmann (1972). The wells of microtiter plates were coated with 100 III of purified lysyl oxidase (100 Ilg/ml) in buffer containing 20 mM carbonate, pH 9.6 and were allowed to stand overnight at 4 °C. Excess protein binding sites were blocked with the addition of PBS-3 % BSA for 2 h at room temperature. The plates were than washed 3 times with PBS-Tween. The supernatants from hybridoma media (100 Ill) were then added for 2 h at room temperature. Following incubation the supernatants were removed and the plates washed with PBSTween. After extensive washing, affinity-purified anti-mouse peroxidase (Hyclone Lab) diluted 1: 6000 in PBS-Tween was added for 90 min. The plates were again washed and the bound peroxidase-linked anti-mouse conjugate was assayed using 5aminosalicylic acid and HzO z as a substrate (Mills et al., 1978) .. Positive hybrids were cloned by limiting dilution in 96-well plates and rescreened by ELISA. After the first cloning the cells were weaned off media containing hypoxanthine and thymidine. Hybrids were recloned until stable antibody-producing lines were obtained. Isotyping and Isolation of Monoclonal Antibodies Isotyping of the monoclonal antibodies produced by the hybrid lines was determine~ using an ELISA kit (Hyclone Lab) specific for each of the murine immunoglobulin classes: IgG1, IgG2a, IgG2b, IgG3 , IgM and IgA. The source of the monoclonal antibodies for isotyping and purification were the supernatants of hybridoma cultures and ascites fluid of athymic mice (nu/nu) injected intraperitoneally with 5-10 x 10 6 hybridoma cells. Antibody preparations were purified using 50% ammonium sulfate fractionation, chromatography on Cibacron Blue-Sepharose 4B to remove albumin (Travis et al., 1976) and a final separation on DEAE-cellulose using a 0.0 to 0.4 M NaCl gradient. Alternatively, antibodies were purified on Protein A-agarose using the MAPS procedure (BioRad).

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Immunoblotting Western blots were performed as described by Towbin et al. (1979) . Samples were electrophoresed in 8 or 12 % SDS-polyacrylamide slab gels according to the method of Laemmli (1970) and the separated proteins were then electrophoretically transferred to a 0.45 11m nitrocellulose sheet (BioRad). The blot was incubated with PBS-3% BSA for 6 h and then washed with PBS-Tween. The blots were then incubated with ascites fluid diluted 1: 50 in PBS-Tween for 12 h 4 °C. Following washing with PBS-Tween, the blots were incubated with an anti-mouse-avidin and biotin-peroxidase kit (Bethesda Research Lab.). Color detection was performed using 4-chloronapthol as substrate.

Immobilization of Antibody for Immunoabsorption Purified monoclonal antibody I from tissue culture hybridoma supernatant was dialyzed into 50 mM borate, 0.5 M NaCl, pH. 9.0. The immunoglobulin was reacted with cyanogen bromide-activated Sepharose-CL 4B (Sigma) at a ratio of 6.4 mg of immunoglobulin/ml of gel and gently agitated at room temperature for 2 h. The unreacted sites were blocked with 1 M ethanolamine in the same buffer. Ninety-four percent of the total immunoglobulin was bound.

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Fig. 1. Sephacryl S-200 Chromatography of DEAE-Cellulose-Purified Lysyl Oxidase. Chromatography of the ultrafiltrate concentrate of DEAE-cellulose partially purified Iysyl oxidase on Sephacryl S-200. Protein absorbance at 280 nm (- 0 - 0 - ) and Iysyl oxidase activity (-e-e-).

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Results and Discussion Umbilical cords were found to be an excellent source of human lysyl oxidase. Previous workers have isolated human lysyl oxidase from placenta (Kuivaniemi et ai., 1984). Umbilical cords were chosen over placenta as a source of the enzyme because the crude urea extracts of umbilical cords were found to have a specific activity 9 times greater than that reported for placenta and had a specific acitivity 28% that of bovine aorta when expressed per unit of tissue wet weight. Purification of human lysyl oxidase employing chromatography on Cibacron Blue-Sepharose, DEAE-cellulose and Sephacryl S-200 resulted in a 230-fold purification over the initial urea extract. The final specific activity of the human lysyl oxidase preparation was 0.7 x 105 units/mg compared to 13 x 105 units/mg obtained for purified bovine aortic lysyl oxidase (Burbelo et ai., 1985). Mono Q high performance anion-exchange chromatography of the purified human lysyl oxidase showed the perparation to contain four peaks of lysyl oxidase activity when eluted with a salt gradient from 0 to 1.0 M NaCi, similar to what has been reported for human placentallysyl oxidase on DEAE-cellulose (Kuivaniemi et ai., 1984). Eight monoclonal hybridomas were generated from a total of four fusions. A typical fusion from a single spleen resulted in a total of 480 microtiter wells plated. Hybrid growth usually was observed in 90% of the wells and initial ELISA screen indicated that approximately 20% of the wells contained antibody against the immunizing antigen. Reassay by ELISA resulted in few clones remaining positive. Eight monoclonal hybridomas were isolated, all derived from a different primary clone following at least three subclones. These clones have remained stable producers of antibody for the last 6 months. Typical titers from ascites fluid have ranged from 1: 1,000 to 1 :20,000. Isotyping analysis indicated that all of the clones were IgG, except B7 and D7 which were IgM (Table I). Three of the eight clones were also capable of reacting against purified bovine aortic lysyl oxidase in an ELISA. The nature of the antigen recognized by monoclonal antibodies was further investigated by immunoblotting. Four of the eight antibodies did not react with lysyl oxidase Table 1. Immunoglobulin Type and Reactivity Demonstrated by Monoclonal Antibodies to Human Lysyl Oxidase. Monoclonal Antibody

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P. D. Burbelo, A. Monckeberg and C. o. Chichester

in immunoblotting when SDS-polyacrylamide gel electrophoresis was used to separate the antigen. Negative results in immunoblot suggests that the epitopes for these antibodies may be conformationally dependent (Williams and Woollett, 1985). The four immunoblot positive monoclonal antibodies (I, C3, F6 and G7) recognized both the 30,000 dalton active enzyme and a fragment smaller than the enzyme of approximately 24,000 daltons (Fig. 2). The recognition of this fragment has also been reported by conventional antisera (Kuivaniemi et aI., 1984). The 24,000 dalton component appears to be the major proteolytic fragment of the enzyme, which increases with storage of the enzyme (Sullivan and Kagan, 1982). The antigenic determinants for these four antibodies are located on the 24,000 dalton portion of the enzyme, since the antibodies reacted with both the 30,000 and 24,000 dalton component. Previous work with polyclonal antibodies against bovine aortic lysyl oxidase showed enzymatically inactive, immunoreactive protein eluting from the Sephacryl S-200 at molecular weights ranging from 200,000 daltons to 60,000 daltons (Burbelo et aI., 1985). Similar results were found in this study when a competitive ELISA using monoclonal antibodies was used to screen DEAE-cellulose-purified enzyme chromatographed on Sephacryl S-200. To further investigate this reactivity, immunoblotting with monoclonal antibody I was performed on the enzymatically inactive large protein

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Fig. 2. Immunoblotting of Human Lysyl Oxidase. Lysyl oxidase was electrophoresed on a reducing 12 % SDS-polyacrylamide slab gel, blotted onto nitrocellulose and stained with amido black (Lane A) or immunostained with monoclonal antibodies F6 (B), G7 (C), I (D) and C3 (E). Molecular weight standards on an adjacent strip were visualized by staining with amido black.

Monoclonal Antibodies to Lysyl Oxidase

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peak of the Sephacryl S-200. Weak reactivity was found with a 48,000 dalton component, while strong reactivity was found with a 66,000 dalton component (Fig. 3). These two components may represent precursor forms or covalently linked derivatives of the enzyme. Sullivan and Kagan (1982) have identified a catalytically inactive 60,000 dalton protein from bovine aorta, which had peptide profiles similar to lysyl oxidase. It is not yet clear whether this protein has biological relevance to lysyl oxidase or whether it is an artifact generated during enzyme purification. Monoclonal antibody I immobilized to Sepharose CL-4B was found to be an effective method of purifying lysyl oxidase. Immunoaffinity chromatography was performed by applying 13 mg of DEAE-cellulose partially purified lysyl oxidase in 16 mM potassium phosphate, 0.25 M NaCI to a column containing 5 ml of gel matrix. The column was washed with the same buffer and eluted with 6 M urea, 16 mM potassium phosphate, pH 7.7. The protein eluted was found to co~tain lysyl oxidase activity

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Fig. 3. Immunoblot Analysis of Enzymatically Inactive Protein from the Sephacryl S-200. DEAE·cellulose purified protein corresponding to a molecular weight of 150,000 daltons on Sephacryl S-200 was electrophoresed on a 8% SDS-polyacrylamide slab gel and transferred to nitrocellulose. Lane A, amido black stain of protein corresponding to 150,000 daItons on Sephacryl S-200. Lane B, immunostain of electrophoresed proteins with monoclonal antibody l. Molecular weight standards on an adjacent strip were visualized by staining with amido black.

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Fig. 4. Affinity Chromatography of Human Lysyl Oxidase Using Monoclonal Antibody 1Sepharose. DEAE-ceIIulose-purified lysyl oxidase was applied to the column in 16 mM KH zP0 4 , 0.25 M NaC!. After 200 ml of the extract had been applied, the column was washed with 80 ml of 16 mM KHzP0 4 , 0.25 M NaC!. The column was then eluted with 6 M urea, 16 mM KH zP0 4 • Protein was monitored at 280 nm (-0-0-) and lysyl oxidase activity determined by the tritium release assay (-e-e-).

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Fig. 5. SDS-Polyacrylamide Gel Electrophoresis of Immunoaffinity Purified Protein. Proteins were electrophoresed on 10% SDS-polyacrylamide gel and silver-stained. Lane A, protein applied to the column. Lane B, C, and D, protein purified on successive runs on monoclonal I-Sepharose affinity column. Lane E, affinity-purified protein concentrated by ultrafiltration.

Monoclonal Antibodies to Lysyl Oxidase

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(Fig. 4). This step resulted in a specific activity of 2.1 x 10) units/mg of protein which represented a 117-fold purification over the DEAE-purified protein. SDS-polyacrylamide gel electrophoresis of the affinity-purified protein showed a major 30,000 dalton band (Fig.5). Affinity-purified enzyme was shown to develop an additional 66,000 dalton component upon concentration by ultrafiltration (Fig. 5, Lane E). Immunoaffinity chromatography of the enzyme from tissue culture will be useful in characterizing both the initial translated from of the enzyme and that which is secreted from cells. The high specificity of these monoclonal antibodies will allow the development of a immunoassay for lysyl oxidase. Levels of lysyl oxidase will be examined in serum and tissue from normal and fibrotic individuals, in order to further understand the role of this enzyme in the fibrotic process. Finally, the monoclonal antibodies should be useful in the localization of the enzyme in cultured cells and tissues.

Acknowledgements This study was supported by the University of Rhode Island Foundation and the Rhode Island Foundation.

References Burbelo, P. D., Kagan, H. M. and Chichester, C. 0.: Immunological characterization of bovine Iysyl oxidase. Camp. Biochem. Physiol. 81B: 845-849, 1985. Engvall, E. and Perlmann, P. : Enzyme-linked immunosorbent assay, ELISA, 3. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen coated tubes. J. Irnrnunol. 109: 129-135, 1972. Jordan, R. E., Milbury, P., Sullivan, K. A., Trackman, P. C. and Kagan, H. M.: Studies on Iysyl oxidase of bovine ligamentum nuchae and bovine aorta. Adv. Exp. BioI. Med. 79: 531-542, 1977. Kagan, H. M., Sullivan, K. A., Olsson, T. A. and Cronland, A. L.: Purification and properties of four species of lysyl oxidase from bovine aorta. Biochern. J. 177: 202-214, 1979. Kuivaniemi, H., Savolainen, E. and Kivirikko, K. I.: Human placentallysyl oxidase. J. BioI. Chern. 259: 6996-7002, 1984. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-684, 1970. Lowry, O. H., Rosenbrough, N. J., Farr, A. L. and Randall, R. J.: Protein measurement with the folin phenol reagent. J. BioI. Chern. 193: 265-275, 1951. Mills, K. E., Gerlach, H. E., Bell, J. W., Faukas, M. E. and Taylor, R. J.: Serotyping herpes simplex virus locates by enzyme-labelled immunosorbent assays. J. Clin Micro. 7: 73-76, 1978. Stassen, F. L. H.: Properties of highly purfied lysyl oxidase from embryonic chick cartilage. Biochern. Biophys. Acta 438: 49-60, 1976. Sullivan, K. A. and Kagan, H. M .: Evidence for structural similarities in the multiple forms of aortic and cartilage lysyl oxidase and a catalytically quiescent aortic protein. J. BioI. Chern. 257: 13520-13526, 1982. Towbin, H., Staehelin, T. and Godron, J.: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. . Sci. USA 76: 4350-4354, 1979.

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Travis, J., Bowen, J., Tewksbury, D., Johnson, D. and Pannell, R.: Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma. Biochem. J. 157: 301-306, 1976. Williams, A. F. and Woollett, G. R.: What monoclonal antibodies see at cell surfaces. Biochem Soc. Trans. 13: 1-3, 1985. Williams, M. A. and Kagan, H. M.: Assessment of lysyl oxidase variants by urea gel electrophoresis: Evidence against disulfide isomers as bases of enzyme heterogeneity. Anal. Biochem. 149: 430-437, 1985. Dr. Clinton O. Chichester, Department of Pharmacology and Toxicology, University of Rhode Island, Kingston, RI 02881, USA.