Immunological characterization of bovine lysyl oxidase

Comp. Biochem. Physiol. Vol. 81B, No. 4, pp. 845-849, 1985 Printed in Great Britain

0305-0491/85 $3.00+ 0.00 © 1985 Pergamon Press Ltd

IMMUNOLOGICAL CHARACTERIZATION OF BOVINE LYSYL OXIDASE P. D. BURBELO,* H. M. KAGANt a n d C. O. CHICHESTER* *Department of Pharmacology and Toxicology, University of Rhode Island, Kingston, RI, USA; and tDepartment of Biochemistry, Boston University School of Medicine, Boston, MA, USA (Tel: 401-792-2362) (Received 2 January 1985) Abstract--1. Antibodies to homogeneously purified bovine aortic lysyl oxidase were prepared in chickens. 2. The chicken anti-lysyl oxidase antiserum effectively inhibited bovine aortic lysyl oxidase activity. Non-immune antiserum from chickens, goats and humans was found to enhance bovine aortic lysyl oxidase activity, while non-immune rabbit serum inhibited enzyme activity. 3. A competitive ELISA was developed to monitor immunoreactive lysyl oxidase during purification. 4. Chromatography of bovine lysyl oxidase on Sephacryl S-200, the final step in purification, revealed two peaks of immunoreactive lysyl oxidase. The large molecular weight peak appears to contain inactive multimeric forms of the enzyme.

INTRODUCTION T h e tensile strength of connective tissue in organs such as the a o r t a largely reflect the presence o f intermolecular lysine-derived crosslinks in collagen a n d elastin. Lysyl oxidase catalyzes the f o r m a t i o n o f ~t-aminoadipic-f-semialdehyde, the reactive precursor to the crosslinkages, from specific lysyl a n d hydroxylysyl residues in collagen a n d m o s t o f the lysyl residues in elastin (Pinnell a n d Martin, 1968). Purification of the enzyme usually involves its solubilization by d e n a t u r i n g solvents such as urea, followed by a c o m b i n a t i o n o f collagen affinity, ionexchange a n d gel filtration in urea solutions (Siegel, 1979). The use o f urea m a y cause irreversible inactivation o f a certain p r o p o r t i o n o f enzyme molecules a n d removal o f urea m a y b e necessary for assay ( H a n a n d Tanzer, 1979). In this study we describe the use o f antibodies to bovine aortic lysyl oxidase in a n i m m u n o a s s a y to quantify lysyl oxidase. MATERIALS AND METHODS Assay and purification o f lysyl oxidase Lysyl oxidase activity was determined using the tritium release assay utilizing a chick aortic substrate (Pinnell and Martin, 1968). Assays included the aortic substrate suspension equivalent to 125,000 cpm (0.03-0.1 ml), enzyme (0.05 ml) and 0.1 M sodium borate, 0.15 M NaC1, pH 8.0. The final assay volume was 1.0ml and the assay was incubated with shaking at 37°C for 2 hr. The reaction was stopped by the addition of 0.1 ml of 50~ trichloroacetic acid and the tritiated water was collected by vacuum distillation. The distillate (0.8 ml) was added to 5 ml of Atomlight (New England Nuclear, Boston, MA, USA) and the radioactivity determined by liquid scintillation spectrometry at a counting efficiency of 28~. Protein was measured by the method of Lowry et al. (1951). Bovine lysyl oxidase was purified by a modification of the procedure of Kagan et al. (1979), which employed ~-elastin chromatography in place of DEAE cellulose chro*PBS-Tween 20:8.0 g of NaC1, 0.2 g of KH2PO4, 1.7 g of Na2HPO4, 0.2 g of 0.5 ml of Tween 20 per liter, pH 7.4. 845

matography. The ct-elastin affinity column was prepared by reacting one gram of ~-elastin (Keller and Mandl, 1971) to 1 1 of Sepharose CL-4B, which had been previously activated by treatment with cyanogen bromide (March et al., 1974). Following gelatin-Sepharose 4B chromatography, fractions containing lysyl oxidase activity were dialyzed in 0.016 M KPO4, pH 7.7, and then applied to the ct-elastin Sepharose 4B affinity column. The column was washed with 0.016M KPO4, pH 7.7, until the absorbance at 280nm dropped to zero, and lysyl oxidase was eluted with 6 M urea, 0.016M KPO4, pH 7.7. The fractions with lysyl oxidase activity were pooled, concentrated with a YM-10 ultrafiltration membrane (Amicon Corp., Boston, MA, USA) at 20 psi and applied to a Sephacryl S-200 column. Using this method, 0.49 mg of highly purified lysyl oxidase with a specific activity of 1.37 x 106 units/mg (an approximate 900-fold purification of crude urea extracts) was obtained, which corresponded to the small protein peak on the Sephacryl S-200 containing all the lysyl oxidase activity. The preparation was homogenous upon SDS-polyacrylamide gel electrophoresis with a molecular weight of approximately 32,000 daltons (Neville, 1971). Enzyme purified in this manner was used for all immunizations and as standard in the ELISA. Production o f anti-lysyl oxidase antibody Antibodies to bovine aortic lysyl oxidase were produced in a Rhode Island Red rooster. The chicken was injected in the thigh subcutaneously with 300/tg of purified enzyme in Freund's complete adjuvant. The chicken received booster injections at 2 week intervals of 500/zg of purified lysyl oxidase in incomplete adjuvant. Following the 8th injection, a 1:28,000 titer was obtained as determined by the ELISA method (Engvall and Perlmann, 1972). Analysis of the antisera by double immunodiffusion against crude and purified samples of lysyl oxidase demonstrated a single line of identity. Competitive E L I S A The competitive ELISA for lysyl oxidase is similar to the method used to quantitate collagen as described by Rennard et al. (1980). Lysyl oxidase was diluted from stock 6 M urea, 0.16 M KPO4 solutions into 20 mM carbonate buffer, pH 9.6 and allowed to absorb to polystyrene tubes at 4°C for at least 24 hr. All further dilutions and washes (three times) were made with PBS-Tween 20.* The lysyl oxidase-coated

P.D. BUgnELO et al.

846 lec

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ix

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4(

~0

Amount of Antiwra (ul)

Fig. 1. Inhibition of lysyl oxidase activity by chicken anti-lysyl oxidase antisera. Various amounts of non-immune serum (©) or anti-lysyl oxidase antisera (A) were incubated with purified lysyl oxidase for 30 min at 37°C and then the standard tritium release assay was conducted. tubes were washed before use. The sample containing enzyme to be titrated and antisera were mixed and incubated for 1 hr at 37°C prior to the transfer of 0.5 ml of the mixture to lysyl oxidase-coated tubes. This incubation was allowed to proceed for 30min and the mixtures were aspirated off and the tubes washed. A constant amount of peroxidase-linked rabbit anti-chicken conjugate (Avrameas and Ternynck, 1971) was added to each tube and the incubation was continued for 90 min at room temperature. The tubes were again washed and the bound peroxidaselinked rabbit anti-chicken conjugate was assayed using 5-aminosalicyclic acid and H202 as substrate (Mills et al., 1978). RESULTS

Effect of various sera on bovine aortic lysyl oxidase activity Repeated attempts to generate antibody to lysyl oxidase in the goat and in the rabbit proved negative. Even using milligram quantities of bovine aortic lysyl oxidase as antigen, no antibodies were detected using inhibition of lysyl oxidase activity or the E L I S A as the screen. However, antibodies were successfully produced in chickens. Antisera produced in a chicken was found to inhibit bovine lysyl oxidase activity by 50~o with 194 #1 of undiluted antisera in the tritium release assay as seen in Fig. 1. Interestingly, non-immune sera from several species were found to stimulate or inhibit lysyl oxidase activity. Figure 2 shows the effect of non-immune serum from rabbits, goats and humans. Non-immune chicken, goat and human serum enhanced bovine aortic lysyl oxidase activity, yet did not possess any lysyl oxidase activity of their own. The enhancing activity is non-dialyzable and is stable, retaining 80~o activity at 60°C for 20 min. Rabbit serum was found to be inhibitory at all concentrations tested. Competitive E L I S A for lysyl oxidase The competitive E L I S A utilizes lysyl oxidasecoated polystyrene tubes as the solid support. It is

initiated by preincubation of lysyl oxidase with antilysyl oxidase antibody which results in a decrease in activity of the antibody toward the antigen-coated tubes. On the basis of a series of experiments with various dilutions of coating lysyl oxidase, antisera dilutions, and incubation times, a competitive E L I S A for lysyl oxidase was developed under nonequilibrium conditions. It was determined that 10/~g of lysyl oxidase added to the polystyrene tubes and a 1:600 dilution of antisera produced reproducible and sensitive results. A preincubation time of 1 hr at 37°C was sufficient for anti-lysyl oxidase antibody to react with soluble lysyl oxidase. Incubation of antibody with the antigen-coated tubes for 30 min gave reproducible binding without allowing sufficient time for the insoluble lysyl oxidase to compete for antibody binding sites previously blocked by soluble lysyl

•,~

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I

I

I

Amount

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of Serum (ml)

Fig. 2. Effect of non-immune sera from goats ( O ), humans ( - - - - - ) and rabbits ( *--) on lysyl oxidase activity. Sera was incubated with purified lysyl oxidase for 30 min at 37°C and then the tritium release assay was conducted.

Bovine lysyl oxidase

847

Table 1. Purification of lysyl oxidase from bovine aorta* 10 6 x Total activity (units)t 4 M Urea extract Gelatin affinity chromatography ~-Elastin affinity chromatography YM-10 Ultrafiltration concentrate Sephacryl S-200 chromatography

4.53

Total protein (mg) 2867

10 5x Specific activity (units/mg)

Purification (fold)

Immunoreactive lysyl oxidase:~ (mg)

100

0.015

1

18.35

100

0.466

31.1

9.37

Yield (~)

4.52

97.01

2.78

10.51

61.4

2.640

176.0

5.21

0.98

7.76

21.6

1.262

84.1

4.46

0.67

0.49

14.7

13.673

911.5

0.49

*Purification of aortic lysyl oxidase began with 190 g of aorta. tOne unit of enzyme activity is defined as 1 cpm per 0.8 ml of assay reaction mixture released in 2 hr at 37°C with 125,000 cpm of substrate. :~Determined by the competitive ELISA.

oxidase. A typical standard curve for the competitive ELISA for lysyl oxidase is shown in Fig. 3, in which the results are expressed as percent of maximum binding. The competitive ELISA allowed detection of as little as 95 ng of immunoreactive lysyl oxidase.

Detection of immunoreactive lysyl oxidase during purification The competitive ELISA was used to follow immunoreactive lysyl oxidase during purification. A summary of the purification data is presented in Table 1. Gelatin-affinity chromatography bound only 51.5~ of the immunoreactive lysyl oxidase protein from the crude extract. Utilizing the ct-elastin affinity column a 5.7-fold purification was obtained over the gelatin affinity purified enzyme. The ct-elastin affinity column was effective in binding 61.5% of the lysyl oxidase activity applied to it, while binding 55.6~o of the immunoreactive lysyl oxidase. Ultrafiltration concentration of the ct-elastin column effluent caused a marked reduction in lysyl oxidase activity. This step resulted in the loss of 64.7~ of the lysyl oxidase activity with only a 14.4~ loss of immunoreactive lysyl oxidase. This step caused a marked reduction in specific activity. Figure 4 shows the resolution of the YM-10 concentrate on Sephacryl S-200 demonstrating two peaks of immunoreactive lysyl oxidase. The first peak contained the majority of the immunoreactive lysyl oxidase, yet was devoid of lysyl oxidase activity. The second smaller peak contained all the lysyl oxidase activity. Figure 5 shows SDS-polyacrylamide gel electrophoresis of the large inactive peak in the presence of mercaptoethanol revealing multiple molecular weight species with some protein corresponding to 32,000 daltons.

A less sensitive ELISA was developed based on the interaction of lysyl oxidase with its substrate elastin (data not shown). This ELISA is similar to the ELISA for fibronectin utilizing collagen as the immobilized support (Engvall and Ruoslahti, 1977). Lysyl oxidase is added to ~t-elastin immobilized to polystyrene, and the bound lysyl oxidase is then quantitated directly by the addition of anti-lysyl oxidase antibody. A PBS-BSAt solution was used to eliminate non-specific binding in this assay. Neutral detergent was not effective, since it prevented lysyl oxidase binding to the ct-elastin. This ELISA was found to be useful only with purified lysyl oxidase preparations due to the large amount of non-specific binding. Lysyl oxidase activity was stimulated by a nondialyzable, heat stable component(s) found in serum from goats, chickens and humans. Furthermore, the enhancing activity present in serum appears to possess no inherent lysyl oxidase activity and may act by changing the kinetic properties of lysyl oxidase. The enhancer did not appear to be pyridoxal phosphate, a cofactor of lysyl oxidase (Carrington et aL, 1984), since the addition of pyridoxal phosphate to bovine

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40

DISCUSSION

This report describes the production of antibodies against bovine aortic lysyl oxidase. A specific competitive ELISA for lysyl oxidase was developed which can be used to measure lysyl oxidase protein in crude or purified samples. The competitive ELISA is sensitive to a minimal level of 95 ng. t P B S - B S A : 8 . 0 g o f NaC1, 0 . 2 g o f KH2PO4, 1.7g o f N a 2 H P O 4, 0.2 o f KC1 and 1 g o f b o v i n e serum a l b u m i n per liter, p H 7.4.

Nonogroms of Lysyl Oxidase

Fig. 3. Standard curve for the competitive ELISA for lysyl oxidase. Increasing amounts of soluble lysyl oxidase antigen inhibits the amount of antibody bound to lysyl oxidasecoated polystyrene tubes. Ten #g of lysy! oxidase was adsorbed to the polystyrene tubes and a 1:600 dilution of antisera was added.

848

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400

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Fig. 4. Chromatography of the ultrafiltration concentrate of ~-elastin purified lysyl oxidase on Sephacryl S-200. (A) Lysyl oxidase protein was determined by the competitive ELISA (--). (B) Protein absorbance at 280 nm (--) and lysyl oxidase activity ( - - - ) .

400'

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4,

200,

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400

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DISTANCE MIGRATED (cm)

Fig. 5. SDSpolyacrylamide gel electrophoresis of the lysyl oxidase protein from Sephacryl S-200. Electrophoresis of highly purified lysyl oxidase is shown in (A), while the peak containing inactive lysyl oxidase protein is shown in (B).

Bovine lysyl oxidase aortic lysyl oxidase did not increase enzyme activity (data not shown). Rabbit serum did not enhance lysyl oxidase activity, which may be due to the presence of large amounts of inhibitors in the serum. Ultrafiltration inactivated a portion of the lysyl oxidase molecules, since the loss of enzymatic activity was not due to loss of lysyl oxidase protein. The inactivation of lysyl oxidase may have been caused by pressure denaturation of the enzyme. Catalytic oxidation of e-amino group of lysine in the enzyme may cause crosslinking of lysyl oxidase. SDSpolyacrylamide gel electrophoresis substantiates this data, since the larger molecular weight peak on Sephacryl S-200 upon reducing showed some protein corresponding to the molecular weight of 32,000 daltons. Since several other higher molecular weight forms were noted with electrophoresis, it appears possible that some of the lysyl oxidase may be unable to return to the monomeric active form, due to covalent crosslinking. Immunoblotting after SDS-polyacrylamide gel electrophoresis showed the presence of multiple reactive bands. Contamination of the antibody was ruled out, since immunoabsorption of antibody reactive only to the 32,000 molecular weight band upon reblotting still showed reactivity to the high molecular weight species (D. J. Reiss and H. M. Kagan, personal communication). The presence of inactive large molecular weight species of lysyl oxidase could be attributed to polymer formation, but may be due to the presence of large molecular weight precursors.

REFERENCES

Avrameas S. and Ternynck T. (1971) Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry 8, 1175-1179. Carrington M. J., Bird T. A. and Levene C. I. (1984) The

849

inhibition of lysyl oxidase in vivo by isoniazide and its reversal by pyridoxal. Biochem. J. 221, 837-843. Engvall E. and Perlmann P. (1972) Enzyme-linked immunosorbent assay, ELISA 3. Quantitation of specific antibodies by enzyme labeled anti-immunoglobulin in antigen-coated tubes. J. lmmunol. 109, 129-135. Engvall E. and Ruoslahti E. (1977) Binding of soluble form of fibroblast surface protein, fibronectin, by collagen. Int. J. Cancer 20, 1-5. Han S. and Tanzer M. L. (1979) Collagen crosslinking. J. biol. Chem. 254, 10438-10442. Kagan H. M., Sullivan K. A., Olsson T. A. and Cronland A. L. (1979) Purification and properties of four species of lysyl oxidase from bovine aorta. Biochem. J. 177, 202-214. Keller S. and Mandl J. (1971) Solubilized elastin as a substrate for elastase and elastase inhibitor determination. Biochem. Med. 5, 342-347. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. March S. C., Parikii I. and Cuatrecasas P. (1974) A simplified method for cyanogen bromide activation of agarose for affinitychromatography. Analyt. Biochem. 60, 149-152. Mills K. E., Gerlach H. E., Bell J. W., Farkas M. E. and Taylor R. J. (1978) Serotyping herpes simplex virus locates by enzyme-labelledimmunosorbent assays. J. din. Micro. 7, 73-76. Neville D. M. (1971) Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J. biol. Chem. 246, 6328-6334. Pinnell S. R. and Martin G. R. (1968) The crosslinking of collagen and elastin: enzymatic conversion of lysine in peptide linkage to ~-aminoadipic-3-semialdehyde (allysine)by an extract from bone. Proc. natn. Acad. Sci. U.S.A. 61, 708-716. Rennard S. J., Berg R., Martin G. R., Foidart J. M. and Robey G. P. (1980) Enzyme linked immunoassay (ELISA) for connective tissue components. Analyt. Biochem. 104, 204-215. Siegel R. C. (1979) Lysyl oxidase. Int. Rev. Conn. Tiss. Res. 8, 73-118.