THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 14, Issue of May 15, pp. 10345-10350,1933 Printed in U. S. A.

Oxidation, Cross-linking, and Insolubilization of Recombinant Tropoelastin by Purified Lysyl Oxidase*

(Received for publication, July 27, 1992, and in revised form, January 4, 1993)

Debra Bedell-Hogan, Philip Trackman, William AbramsS, Joel RosenbloomS, and Herbert Kagang From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 021 18 and the *Department of Anatomy and Histology and Research Center in Oral Biology, School of Dental Medicine, University of Pennsyluania, Philadelphia, Pennsylvania 19104

The use of recombinant human tropoelastin (rTE) and selected variants thereof as substrates for the as- say of lysyl oxidase activity in vitro was explored. The possibility was also assessed that an insoluble elastin- like product could be generated from this elastin pre- cursor in the absence of other macromolecules found associated with elastin in vivo. rTE was more effi- ciently oxidized by lysyl oxidase than the insoluble chick aorta elastin substrate conventionally used. An- ionic amphiphilic elastin ligands strongly inhibited rTE oxidation consistent with the importance of elec- trostatic enzyme-substrate interactions previously noted with the insoluble elastin substrate. An rTE var- iant, rTEA26A, lacking the hydrophilic sequence coded by exon 26A, was a less effective substrate than rTE, largely due to an increase in K,, while the kinetic parameters for the oxidation of rTEA36, lacking the C-terminal polybasic sequence coded by exon 36, were quite similar to those for rTE. Incubation of rTEA26A with lysyl oxidase not only resulted in the generation of peptidyl a-aminoadipic-&semialdehyde and lysine- derived cross-linkages, but also yielded a product in- soluble in hot 0.1 N NaOH, consistent with the prop- erties of insoluble elastin. Thus, oxidation, cross-link- ing and insolubilization of elastin substrates by lysyl oxidase can occur in the absence of other macromole- cules implicated as being involved in this process in vivo, although such macromolecules may be essential to obtain the proper alignment between tropoelastin units for specifically placed cross-linkages and opti- mally functional elastic fibers.

The oxidative deamination of peptidyl lysine to peptidyl CY-

aminoadipic-hemialdehyde in tropoelastin is an essential posttranslational step in the synthesis of elastic fibers. Pep- tidy1 aldehydes generated in this manner are the precursors to the cross-linkages that covalently join individual tropoe- lastin molecules to each other, thus insolubilizing this protein. In addition to lysyl oxidase, other cellular and matrix com- ponents may also be involved in this process. For example, studies have pointed to the association of elastin with acidic glycoproteins of the microfibrillar network surrounding elas- tic fibers (l), with a cell surface elastin receptor (2), and with

Grants AR 18880, HL 46902, and HL 13262 (to H. M. K.) and AR * This research was supported by National Institutes of Health

20553 (to J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4064; Fax: 617-638-5339. S To whom correspondence should be addressed. Tel.: 617-638-

glycosaminoglycans (3). Moreover, the variation in the pri- mary structure of tropoelastin isoforms predicted by alterna- tive splicing of the primary transcript (4) could affect the properties of the resultant elastic fibers by changing the accessibility of the cross-link domains to oxidation by lysyl oxidase.

Prior studies have demonstrated that a purified preparation of tropoelastin can be cross-linked to a pre-existent insoluble matrix derived from chick embryo aortae (5). While this illustrates the potential for tropoelastin insolubilization in vitro, the complexity of the aortic preparation used in this instance did not permit the characterization of lysyl oxidase/ substrate interactions or the role of matrix components other than lysyl oxidase in the insolubilization of tropoelastin. In the present study, we have compared the ability of isoforms of recombinant tropoelastin to be oxidized to cross-linked products in an in vitro system in which the only macromolec- ular components are recombinant forms of tropoelastin and purified lysyl oxidase. The results reveal that insoluble, cross- linked products can be generated under these simple condi- tions and point to the use of recombinant tropoelastin as an efficient substrate form for the assay of this connective tissue enzyme.

MATERIALS AND METHODS

Preparation of Enzyme and Insoluble Chick Elastin Substrates- Lysyl oxidase was isolated from bovine aorta as a copurified mixture of the four ionic variants of this enzyme (6). The preparation exhib- ited a molecular weight of 32,000 when judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (7). Lysyl oxidase activity was also assayed from the conditioned media and the cell layers of neonatal rat aorta smooth muscle cell cultures prepared from aortic explants and cultured for 2 weeks in first passage, as described (8). The enzyme activity associated with the cell layer was extracted into 4 M urea, 16 mM potassium phosphate, pH 7.8, and then dialyzed against 16 mM potassium phosphate, pH 7.8, for subsequent assay. A tritiated, insoluble elastin substrate was prepared from aortae of 16- day-old chick embryos which had been pulsed in organ culture with [~-4,5-~H]lysine in the presence of BAPN,’ as previously described (9).

Preparation of Recombinant Tropoelastin-Plasmids expressing the complete human tropoelastin molecule (rTE), tropoelastin lacking the segment encoding exon 26A (rTEA26A) or tropoelastin lacking the segment encoding exon 36 (rTEA36) were constructed and trans- formed into the lysogenic host, Escherichia coli AR 120 as described (10). A 40-ml aliquot of an overnight culture of bacteria containing a specific expression vector was diluted 1:25 in into 1 liter of L broth containing 50 pg ml” ampicillin, and the freshly inoculated culture was incubated with shaking at 37 “C for 90 min. The cells were collected and then washed three times by centrifugation, the cell pellet suspended in lysine-free RPMI medium (Sigma) and incubated in this medium with shaking at 37 “C for 10 min. Protein expression

The abbreviations used are: BAPN, P-aminopropionitrile; rTE, recombinant tropoelastin.

10345

10346 Cross-linking of Recombinant Tropoelastin

was then induced by the addition of nalidixic acid to 60 pg ml-'. Following 2 h of further incubation at 37 "C, [~-4,5-~H]lysine (2 mCi/ liter; 88 Ci mmol") was added to the medium, and incubation was continued with shaking for 3 h or until the cells had undergone one population doubling. The labeled cells were isolated by centrifugation and washed twice with phosphate-buffered saline, and suspended in 50 mM Tris, 2 mM disodium ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 5% glycerol, pH 8.0, containing 0.1 mM phenylmeth- ylsulfonyl fluoride (buffer A). The cell suspension was incubated with lysozyme (200 pg m1-I) at 0 "C for 30 min, sedimented, and resus- pended in buffer A which was further supplemented with 0.05% deoxycholate. The suspension derived from 200 ml of bacterial culture was homogenized with a Dounce homogenizer and sedimented, and the pellet was suspended in a solution of 63 mg of CNBr in 4 ml of 70% formic acid and incubated with stirring overnight a t 25 "C. The solution was diluted with one-half volume of water and incubated uncovered on ice in the hood 4-5 h to allow excess HCN to escape. The pellet was removed by centrifugation, and the supernatant was exhaustively dialyzed against 0.1 M acetic acid and concentrated in vacuo. The concentrated recombinant protein product was further purified by reversed-phase chromatography on a Vydak CV, column (15 X 0.46 cm) eluting at 1 ml min" with a linear gradient between 0.1% trifluoroacetic acid, and 0.1% trifluoroacetic acid, 80% aceto- nitrile, as described (10). The peak of purified tropoelastin eluting at 48% acetonitrile was collected, freeze-dried and stored at -80 "C.

Assay of Lysine Oxidation-Rates of oxidation of peptidyl lysine were assessed using the tritiated, insoluble elastin or rTE as sub- strates. Unless otherwise noted, assay mixtures included 125,000 cpm of the elastin substrate suspended or dissolved in 0.1 M sodium borate, 0.15 M NaCl, pH 8.0, in a total volume of 0.75 ml, and were incubated for 2 h a t 37 "C. Tritiated water formed during the incubation was isolated by vacuum distillation and radioactivity in 0.5-ml aliquots of the distillates quantified by liquid scintillation spectrometry. All activities were corrected for enzyme-free controls and all enzyme activities reported are 2 90% inhibited by the inclusion of 50 FM p- aminopropionitrile as a specific inhibitor of lysyl oxidase (11) in the assay mixtures. Assay data obtained with the different isoforms of rTE were each corrected for the specific radioactivity of the various substrate preparations.

Cross-linkage Analyses-Aliquots (250,000 cpm) of the tritiated substrates were incubated with lysyl oxidase in 0.1 M sodium borate, 0.15 M NaC1, pH 8.0, for 24 h at 37 "C. The proteins were then reduced with 0.5 mg of sodium borohydride per 0.75-ml assay mixture for 30 min at room temperature, while maintaining the pH between 8 and 9. The reductions were terminated by titration to pH 2-3 with the addition of 50% acetic acid. The contents were dried under nitrogen and hydrolyzed at 110 "C for 18 h in 0.2 ml of 2 N NaOH in sealed alkali-resistant tubes or in 6 N HC1, as specified. The hydrol- ysates were analyzed with an amino acid analyzer equipped with a stream-splitting device to permit the monitoring of the effluent for radioactivity and ninhydrin reactivity. Positions of radioactive peaks were compared to those of authentic lysine-derived cross-linkages. Although oxidative deamination of peptidyl [~-4,5-~H]lysine releases a tritium ion from position 5 by keto-enol tautomerization of the aldehyde product, carbon 4 of the aldehyde retains its label, permit- ting the isotopic identification of lysine-derived products from the labeled tropoelastin in fractions eluting from the amino acid analyzer. Split-stream amino acid analyses for desmosine-type cross-linkages were separately performed on samples of recombinant tropoelastin which had been incubated with lysyl oxidase in the presence or absence of 50 p~ BAPN, reduced with NaBH4, and then hydrolyzed in 6 N HCl under nitrogen gas.

Assay for Znsolubilizatwn of rTE-Samples incubated with lysyl oxidase in the presence or absence of 50 ~ L M BAPN were sedimented by centrifugation at 10,000 X g, and the pellets were washed with 16 mM potassium phosphate, pH 7.8, and then with water. Pelleted material was then incubated in 0.1 N NaOH at 95 "C for 45 min (12), the remaining insoluble material was washed with water by centrif- ugation, and the pellets were then hydrolyzed in 6 N HC1 under nitrogen gas. The hydrolysates were dried under nitrogen and redis- solved, and the total moles of amino acid residues in the hydrolysate was assessed by automated amino acid analysis. Internal standards established that the efficiency of sample recovery by this method was z 95%.

RESULTS

Comparative Efficiencies of PHIElastin and rH]rTE Sub- strates-Equal aliquots of smooth muscle cell fractions or of purified bovine aorta lysyl oxidase were assayed for 2 h using 125,000 cpm of the [3H]elastin or the [3H]rTE substrates. As shown (Fig. l ) , considerably more tritium was released from the soluble rTE than the insoluble elastin substrate with the crude enzyme obtained from the culture media (5-7-fold) and cell layer (5-fold), as well as by the purified bovine aorta enzyme (2.8-fold). The greater sensitivity of the tropoelastin substrate should be particularly advantageous for assays of the relatively low levels of lysyl oxidase activity often available in crude extracts of cell culture or tissue fractions, while it may also be advantageously employed in kinetic studies of the pure enzyme with a natural substrate. Certainly, the solubility of the rTE substrate presents a significant advan- tage over the insolubility of the aortic substrate.

Kinetic Characterization of rTE Substrates-Three iso- forms of recombinant tropoelastin were kinetically compared as substrates for lysyl oxidase. These substrates included two recombinant forms of naturally occurring human tropoelas- tins, one of which (rTE; specific activity, 1.21 x lo4 dpm 3H/ pg) contained and the other of which (rTEA26A; specific activity, 1.4 x lo4 dpm 3H/pg) lacked the unusual hydrophilic sequence corresponding to exon 26A, i.e. GADEGVRRSLS- PELREGDPSSSQHLPSTPSSPR (4), while a third recom- binant form (rTEA36; specific activity, 9.77 X lo3 dpm 3H/ pg) lacked the C-terminal polybasic amino acid sequence encoded by exon 36 (4), i.e. GGACLGKACGRKRK, normally present in human tropoelastin (4). As shown (Fig. 2), the time dependency of tritium release from these substrates catalyzed by purified bovine lysyl oxidase is approximately linear for at least the first 60 min of assay at 37 "C, with the rTEA36 showing the greatest deviation from linearity with longer incubation.

When assay times were limited to 60 min to maintain linear rates, it was observed that each recombinant tropoelastin exhibited Michaelis-Menten, saturation kinetics, as shown in Lineweaver-Burk plots (Fig. 3). The K,, V,,,, and K m / V m e x

values for each are summarized in Table I. The VmaX values of the three isoforms are similar, whereas the K, values of rTE and rTEA36 are similar to each other but significantly smaller than that for rTEA26A. Thus, the catalytic efficiency of these isoforms as substrates for lysyl oxidase is greatest for rTE and rTEA36, as indicated by the V/K values (Table I). It is of interest that the sequences encoded by exons 26A and

3000 - Y c

I

Media Matrix Purlfled Lysyl

Rat Smooth Muscle Cells Oxidase

FIG. 1. Comparison of rTE and insoluble chick aortic elastin as substrates for rat aorta smooth muscle cell and purified bovine aorta lysyl oxidase activity. The height of the bars rep- resents the mean of quadruplicate assays, with the vertical lines representing mean f S.D.

Cross-linking of Recombinant Tropoelastin 10347

'*O 135 t

Minutes at 37°C FIG. 2. Time-dependent oxidation of rTE variants by bo-

vine aorta lysyl oxidase. 0, rTE; A, rTEA26A; 0, rTEA36. Assay points are the means of triplicate assays the standard deviation of which did not exceed k 5%. These patterns were reproducible in duplicate experiments.

36, although differing from each other, are each hydrophilic in character. Notably, deletion of exon 26A increases the K,,, while deletion of exon 36 causes little change in K,, relative to that of rTE which contains both sequences. Thus, the changes seen in the apparent affinity of these isoforms for lysyl oxidase cannot be simply interpreted in terms of net changes in hydrophilicity and must reflect additional factors such as the specific changes in secondary or higher ordered degrees of structure of the tropoelastin isoforms or their aggregates. Nevertheless, these data do indicate that various forms of tropoelastin resulting from translation of alterna- tively spliced transcripts can have different reactivities as substrates for lysyl oxidase. Further studies to be described here employed rTEA26A as substrate in view of the fact that this is the most frequently occurring isoform of human tro- poelastin (10).

Effect of Assay Components-It has previously been shown that the oxidation of the insoluble elastin substrate by purified lysyl oxidase is markedly stimulated by cationic detergents, including dodecyltrimethylammonium bromide, and mark- edly inhibited by anionic detergents, including SDS and so- dium linoleate, at concentrations of these agents which do not alter the intrinsic catalytic activity of lysyl oxidase toward simple alkylamines (13). Similarly, the activity toward simple alkylamines is stimulated at 1.2 M urea, an effect attributed to the prevention of the tendency of lysyl oxidase to polymer- ize to catalytically inefficient aggregates (14). However, as shown in Fig. 4, the oxidation of rTEA26A by purified lysyl oxidase is not markedly influenced by concentrations of urea in the assay up to 1.2 M, by NaCl concentrations up to 0.8 M, or by 0.8 mM dodecyltrimethylammonium bromide. The lack of effect of urea may be due to urea-resistant enzyme-tropoe- lastin interactions which prevent aggregation of the free ca- talyst. Notably, however, oxidation of rTEA26A oxidation is strongly to completely inhibited by 0.8 mM concentrations of SDS or sodium linoleate, concentrations of these amphiphiles a t which the intrinsic catalytic activity of lysyl oxidase is not altered (13).

Effect of Lysine Oxidation on Polymerization of rTEA26A- rTEA26A (125,000 cpm) was incubated with purified bovine lysyl oxidase and samples were removed a t specified times for analysis by SDS-polyacrylamide gel electrophoresis and au- toradiography. As shown (Fig. 5), at the outset of the incu- bation (lane 1 ), the rTEA26A product consists of a prominent band at 72 kDa. The lower molecular weight bands seen in

-0.03 0.00 0.03 0.06 0.09 0.12

-0.03 0.00 0.03 0.06 0.09 0.12

0.60

-0.03 0.00 0.03 0.06 0.09 0.12

FIG. 3. Lineweaver-Burk plots of the oxidation of variants of rTE by purified bovine aorta lysyl oxidase, Substrate con- centrations, [SI, are in units of cpm per assay. The kinetic constants derived from these data are corrected for the specific radioactivities of each rTE preparation in Table I.

TABLE I Kinetic constants of rTE substrates of lysyl oxidase

Calculation of kinetic constants accounted for the differing specific activities of each rTE preparation, as given in the text.

pM enzyme (X 10-4) V-/Km dpm/h/4 pg

rTEA26A 14.9 2.41 2.2 rTE 4.6 1.81 rTEA36

8.7 4.0 2.17 7.2

Cross-linking of Recombinant Tropoelastin

NaCl (M)

A

FIG. 4. Effect of salt ( A ) , urea ( B ) , and hydrophobic elastin ligands (C) on the oxidation of rTE by purified bovine aorta lysyl oxidase. Each assay contained 4 pg of lysyl oxidase. Data are corrected for enzyme-free controls. DTAB, dodecyltrimethylam- moinum bromide; L i d , sodium linoleate.

A

1 2 3 4 5

B

-72 kDa

1 2 3 4 5

FIG. 5. Autoradiogram of SDS-gel electrophoresis of rTE incubated with bovine aorta lysyl oxidase. Incubations of rTE (17 pg; 45,000 cpm) and purified lysyl oxidase (0.5 pg) a t 37 "C: lane 1, 0 time; lane 2, 3 h; lane 3, 5 h; lane 4 , 24 h; lane 5, 24 h in the presence of 0.1 mM BAPN. A , autoradiogram was exposed to film for 5 days; B, autoradiogram was exposed to film for 18 h.

TABLE I1 Insolubilization of recombinant tropoelastin by lysyl oxidase

Hot alkali- Percent hot

residues insoluble Incubated sample ::&? insoluble rTE alkali-

nmoles nmoles rTEA26A + enzyme 32 19.1 60 rTEA26A + enzyme + BAPN 32 1.2 3.8

the autoradiogram appear to be present only a t trace levels since Coomassie Blue staining of SDS-polyacrylamide gel electrophoretograms of this rTE preparation revealed only

the 72-kDa species.* With increasing time of incubation, the density a t the position of the rTEA26A monomer decreases markedly. An excluded, polymeric form of rTE which is too large to enter the stacking gel appears at the top of the gel between 3 and 24 h of incubation with enzyme (lunes 2-4). It is of interest that the amount of the excluded polymer does not appear to increase with time of incubation in the presence of lysyl oxidase. As described below, insoluble products also develop during this period of incubation. Thus, i t may be that the solubility of the excluded polymeric species is limiting such that a steady state level of this polymer exists between the monomeric precursor and the insoluble, more highly po- lymerized product. Analysis of the reaction mixture contain- ing BAPN, enzyme, and rTEA26A which had been incubated for 24 h (lune 5 ) indicates that most of the monomer band at 72 kDa remains apparently unmodified, consistent with the inhibition of lysyl oxidase by BAPN.

Visual inspection of the incubation mixtures after 3 and 4 h of incubation with enzyme in the absence of BAPN indi- cated the presence of particulate material which was absent in the incubation mixtures containing lysyl oxidase and BAPN. To assess the degree of insolubility of the precipitated product, each incubation was sedimented by centrifugation, and any pelleted material was washed by resuspension and centrifugation in potassium phosphate buffer and then water. The washed precipitates were incubated in 0.1 N NaOH at 95 "C for 45 min according to the hot-alkali Lansingprocedure (12) as an index of insoluble elastin formation. As seen (Table 11), 60% of the sample of the rTEA26A incubated with lysyl oxidase in the absence of the enzyme inhibitor resisted solu- bilization by hot alkali, compared to the negligible level of insoluble material in the sample incubated in the presence of BAPN. The amino acid compositions of 6 N HCl hydrolysates of the alkali-insoluble product and of the soluble rTEA26A are given in Table 111. As seen, the residue composition of the alkali-insoluble product is consistent with that of the rTEA26A precursor and is highly similar to that of a previ- ously described preparation of tropoelastin isolated from pig aorta, also shown in Table 111. Thus, the decrease in material entering the running gel with time and the appearance of material excluded from the gel as seen in Fig. 5 is consistent with the lysyl oxidase-dependent formation of high molecular weight polymers and, as the hot-alkali procedure reveals, of an insoluble elastin product.

Cross-link Analyses-Consistent with the development of insolubility, increased levels of the aldehyde product of lysine oxidation, the aldol condensation product, anhydrolysinon- orleucine, and desmosine cross-linkages are generated in rTEA26A incubated with lysyl oxidase for 24 h a t 37 "C (Table IV). Notably, low levels of a-aminoadipic-fi-semialdehyde, measured as c-hydroxynorleucine, the borohydride-reduced

* Western blotting (not shown) of the incubations described in Fig. 5 indicated that all of the isotopically labeled bands seen by autora- diography, including the excluded polymeric bands shown in Fig. 5A, were reactive with antibody specific for tropoelastin. The reactivity of the smaller bands with anti-elastin antibody has also been reported previously (10). The trace levels of smaller peptides likely represent breakdown products of rTE, either as a result of proteolytic action or as a result of the exposure to 70% formic acid during the purification of the rTE with CNBr, as described. Since these smaller tropoelastin- derived peptides decrease after 24 h in the presence of lysyl oxidase, it appears that these peptides are slowly incorporated into the high molecular weight aggregates. It also appears that the background level of cross-linking seen in the 24-h control (lane f i ) is insufficient to result in polymeric forms, suggesting that these cross-linkages are intramolecular. Indeed, intermolecular cross-linkage formation likely requires the oxidation by lysyl oxidase of specifically juxtaposed lysine residues.

Cross-linking of Recombinant Tropoelastin 10349

TABLE 111 Amino acid composition of tropoelastin preparations

Residues/1000 residues

Residue rTE

Alkali-

rTE tropoelastina Pig aorta

Aspartic Glutamic Serine Glycine Histidine Arginine Threonine Alanine Proline Tyrosine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine Lysine

9 26 15

285 1.9

12.7 15.4

184 135

117 18.6

1.2 1.4

23.9 75.6 24.4 56.2

12 32 23

291 0

13 12

203 138 18

107 0 0

22.5 69.3 24.6 24

10 18 15

320 0 7

16 230 114 16

122 0 0

15 49 29 38

From Ref. 18.

TABLE IV Cross-linkages i n recombinant tropoelastin

The abbreviations used are: ACP, aldol condensation product; AAS, lysinonorleucine; deHNL, dehydrolysinonorleucine; Des, des- mosine plus isodesmosine. Each oxidation and cross-linkage product were measured in rTE samples which had been reduced with NaBHl after incubation with lysyl oxidase. AAS and ACP were assessed from based hydrolized rTE, and deHNL and Des were determined in acid- hydrolized rTE samples.

Incubation Sample

Residues/1000 residues time, h at

37 "C ACP AAS deHNL Des Total rTE + LO 24 rTE 24

1.5 1.3 9.5 4.7 17.0

rTE 0.7 0.4 3.9 1.5 6.5

0 0.3 0.9 3.5 0.3 5.0 Insoluble ligament 12 6 1 8* 27

elastin" Data of Ref. 18.

* Sum of desmosine, isodesmosine, and merodesmosine crosslin- kages (18).

derivative of AAS, as well as lysine-derived cross-linkages exist in the rTEA26A prior to incubation with lysyl oxidase, while the quantity of the endogenous aldehyde decreases and thus appears to be converted to cross-linkages during incu- bation in the absence of enzyme. The origin of the endogenous aldehyde is not known. However, it is of interest to note earlier evidence that lysyl oxidase-like activity was noted in E. coli extracts (15, 16), raising the possibility that the recom- binant protein may have undergone a limited degree of enzy- matic oxidative deamination in the host cells in which it was expre~sed .~ Nevertheless, comparing the sums of all aldehyde

This possibility was assessed in the present study. For this pur- pose, E. coli transformed with the rTE plasmid as described was cultured overnight in 500 ml of L broth without isotope. The har- vested cells were washed and sequentially extracted at pH 7.8 with 6 ml each of 0.15 M NaC1, 16 mM potassium phosphate, with 16 mM potassium phosphate, and finally with 16 mM potassium phosphate, 4 M urea. Each extract was exhaustively dialyzed against 16 mM phosphate buffer. Aliquots (300 p1) of the dialyzed NaCl extracts and of the urea extracts were each assayed in quadruplicate with 125,000 cpm of the tritium-labeled rTEA26A substrate using the conditions described for lysyl oxidase assays. The salt extract catalyzed the BAPN-inhibitable release of 99 f 14 cpm (2200 f 311 cpm/mg of cell protein) of 3H20, while the urea extract released 695 f 13 cpm (3861 & 72 cpm/mg of cell protein) of 3Hz0. These results are consistent

and cross-linkage compounds identified in these analyses in the enzyme-supplemented, enzyme-free, and zero time, en- zyme-free incubations reveals the enzyme-dependent increase of nearly %fold in these cross-linkages and cross-linkage precursors. Thus, the insolubilization of the recombinant tropoelastin reflects the formation of cross-linkages newly generated in the incubation with lysyl oxidase. Moreover, as the amino acid compositions in Table 111 reveal, the alkali- insoluble product contains approximately 32 fewer lysines per 1000 residues than the rTEA26A precursor. This value ap- proximates the net decrease in lysine equivalents (27 residues per 1000 residues) accounted for by the increase of the alde- hyde and cross-linkage residues in the alkali-insoluble product in the 24-h incubation of rTEA26A with lysyl oxidase in comparison to the 24-h, enzyme-free incubation mixture. It is of interest that the enzyme-free control at 24 h contained a somewhat higher content of cross-linkages than the zero time control, consistent with the spontaneous condensation of the endogenous aldehyde functions in the isolated rTE. Never- theless, high molecular weight polymers (Fig. 5) do not de- velop in this 24-h sample under the incubation conditions used. It seems likely, therefore, that the significantly greater level of cross-linkages in the 24-h, enzyme-supplemented in- cubation is necessary to form these polymers. It is also pos- sible that the formation of intermolecular cross-linkages, re- quired for the formation of tropoelastin polymers, requires the presence and function of lysyl oxidase.

DISCUSSION

The present report demonstrates that purified tropoelastin generated by recombinant DNA technology is an efficient substrate for lysyl oxidase. Moreover, the formation of cross- linkages and the apparent insolubilization of the rTEA26A product indicates that lysine residues within tropoelastin are readily susceptible to oxidation by this catalyst and that orientations occur between oxidized lysines products and/or oxidized lysine and unmodified lysines which produce cross- linkages characteristic of insoluble elastin. This process can progress to yield high molecular weight, soluble polymeric forms of tropoelastin and, ultimately, insoluble, cross-linked forms of this molecule. While the fibrillar arrangement of the product has yet to be detailed and may differ from the inter- molecular organization of tropoelastin units within mature, elastic fibers formed i n uiuo, it is clear that oxidation, cross- linking, polymerization, and insolubilization of tropoelastin can occur in the absence of other macromolecules which may be involved in this process in uiuo. This in turn raises the possibility that improperly functional, although insoluble forms of elastin may accumulate in pathological conditions in which one or more of the macromolecules putatively involved in elastic fiber formation may be deficient.

The substrate properties of rTE clearly differ from those of the conventional elastin substrate. Importantly, a far greater percentage of peptidyl lysine is oxidized per unit time than in the insoluble substrate, as expected from the greater degree of accessibility and the greater number of unmodified lysine residues present in the soluble precursor compared to the highly cross-linked elastin product. Differences are also noted with respect to the effects of specific agents. For example, dodecyltrimethylammonium bromide strongly stimulates the oxidation of insoluble elastin but has little effect on the rTE substrate. This may well reflect the much higher degree of positive charge in the precursor molecule than in the insoluble

with the earlier suggestions that a lysyl oxidase-like activity exists in E. coli (16, 17). Further investigation of this enzyme activity is in progress.

10350 Cross-linking of Recombinant Tropoelastin

elastin which has already lost most of its available €-amino groups because of the action of lysyl oxidase in uiuo. Indeed, lysyl oxidase strongly prefers highly cationic substrates (17) and thus the cationic detergent binds to and enhances the cationic charge of the minimally charged insoluble elastin but may have little influence on tropoelastin which is intrinsically cationic. However, the neutralization of the charge comple- mentarity between the anionic enzyme and cationic sites in the substrate by the binding of negatively charged detergents to the substrate forms strongly inhibits the oxidation of both the soluble and insoluble forms. These results point to the possibility that naturally occurring anionic ligands such as free fatty acids and glycosaminoglycans previously implicated as ligands of insoluble elastin (13) and tropoelastin (3) may be able to regulate the oxidation of all forms of elastin in uiuo.

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