Intrinsic enzyme activity associated with tropoelastin

Biochimica et Biophysica Acta, 446 (1976) 245-254

© Elsevier/North-Holland Biomedical Press BBA 37442 INTRINSIC E N Z Y M E ACTIVITY ASSOCIATED WITH T R O P O E L A S T I N

ROBERT P. MECHAM*, JUDITH ANN FOSTER and CARL FRANZBLAU Department of Biochemistry, Boston University School of Medicine, Boston, Mass. 02118 (U.S.A.)

(Received April 6th, 1976)

SUMMARY The presence of an enzyme(s) associated with purified tropoelastin has been established. Results indicate that the enzyme(s) remains closely associated with the soluble elastin throughout the entire purification procedure suggesting that it is very tightly bound. Enzymatic activity is optimum through the pH range 7-9 (37 °C) and can be inhibited by disodium ethylenediaminetetraacetate, N-ethylmaleimide, sulfite, soybean trypsin inhibitor and human a-l-antitrypsin. The fragmentation pattern appears to be specific and reproducible.

INTRODUCTION Several laboratories have clearly demonstrated that proteolysis of tropoelastin is a major hindrance to the isolation of a pure, homogeneous, well-defined soluble form of elastin [1-6]. This problem presents itself not only when dealing with animal tissues directly but also with studies in both tissue and cell culture systems where tropoelastin identification is sought [7, 9]. Consequently, incorporation of various enzyme inhibitors into isolation buffers as well as the use of low pH have become routine procedures in the isolation of tropoelastin. As yet there are no reports on the nature or specificity of the enzyme(s) responsible for this degradation of tropoelastin. The necessity of the presence of enzyme inhibitors throughout the isolation of soluble elastin is apparent. Using no inhibitors, Sykes and Partridge [10] have reported the isolation of a tropoelastin from lathyritic chick aortae with a predominant molecular weight of 57 000. On the other hand, Foster et al. [8] described a 72 000 dalton species when N-ethylmaleimide (MalNEt) and disodium ethylendiaminetetraacetate (EDTA) were added to the isolation buffers. The methodologies used in both investigations were identical. It was shown that the 57 000 dalton component isolated by Sykes and Partridge was missing the N-terminal region of the parent 72 000 dalton molecule. Cleavage apparently occurs during the isolation procedure. Tropoelestin has been shown to be a good substrate for various proteolytic enzymes [13-15]. The present communication describes the unusual properties of an enzyme system or systems which is present in our purified tropoelastin preparations. Under Abbreviation: SDS, sodium dodecyl sulfate. * To whom all correspondence should be addressed.

246 a p p r o p r i a t e conditions this enzyme(s) can be activated to degrade t r o p o e l a s t i n in a specific manner, n o t only cleaving the N - t e r m i n a l f r a g m e n t but m a k i n g successive welldefined scissions o f the p a r e n t molecule with time. MATERIALS AND METHODS

Isolation of tropoelastin T r o p o e l a s t i n was purified f r o m the a o r t a e o f lathyritic chicks as previously described by o u r l a b o r a t o r y [8]. A m i n o acid analyses o f this t r o p o e l a s t i n a n d o f aortic chick insoluble elastin are given in Table I. TABLE I AMINO ACID COMPOSITIONS OF CHICK SOLUBLE AND INSOLUBLE ELASTIN Compositions are expressed as residues per 1000 amino acid residues. Amino acid Lysine Histidine Arginine Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine lsoleucine Leucine Tyrosine Phenylalanine Allysine* A.C.P. ** Lysinonorleucine Isodesmosine Desmosine Merodesmosine

Tropoelastin fraction P

Control insoluble elastin

38.5

3.6

8.0 10.4 3.9 10.7 6.8 13.5 124.3 337.6 178.4 174.9 17.4 54.1 12.3 19,5 2.4 2.8

4.5 22.0 1.9 3.1 5.1 12.0 128.0 352.1 176.2 175.0 18.8 47.4 12.0 23.5 6.0 12.0 1.1 2.8 3.2 1.8

Allysine, determined as e-hydroxynorleucine the reduced derivative. ** A.C.P. is the aldol condensation product of two allysine residues determined as the reduced derivative. Cross-links are expressed as lysine equivalents.

Sodium doclecyl sulfate polyacrylamide gel electrophoresis The m e t h o d o f W e b e r a n d O s b o r n [11 ] was followed with the exception t h a t the gel buffer c o n t a i n e d 0.5 M urea [12]. Gels were m a d e 1 0 ~ in a c r y l a m i d e with a ratio o f N , N - m e t h y l e n e b i s a c r y l a m i d e to a c r y l a m i d e o f 1:74. Electrophoresis was carried o u t at 2-3 m A / g e l overnight. R a t tail collagen, bovine serum a l b u m i n and pepsin or ribonuclease were run with each set o f gels as standards.

Reduction and alkylation of tropoelastin 5 m g o f purified t r o p o e l a s t i n were r e d u c e d in 0.5 ml o f 8 M urea containing

247 50 mM dithioerythritol/0.1 M Tris (pH 8.5) at room temperature under a nitrogen atmosphere. After 4 h, 4.9 mg of iodoacetamide were added and the solution stirred at room temperature for 30 min. The reaction was terminated by the addition of an excess of 2-mercaptoethanol. Removal of excess reagents was accomplished by passing the mixture over a Bio-Gel P-2 column (1.5 × 75 cm) and eluting with 0.1 N acetic acid (flow rate ---- 85 ml/h).

Incubation-buffer solutions Incubations were normally carried out in 0.05 M sodium phosphate buffer at pH 8.0. For the pH studies, 0.05 M sodium phosphate buffer at various pH's were prepared by mixing appropriate volumes of 0.05 M dibasic or tribasic sodium phosphate with 0.05 M monobasic sodium phosphate until the desired pH was obtained. A stock solution of purified tropoelastin was made fresh in deionized water (1 mg/0.1 ml) before each experiment. 10 #1 of this solution were then added to 100 #1 of sodium phosphate buffer at the appropriate pH. Incubations were performed at 4, 25, and 37 °C for 16 h unless otherwise indicated. At the end of this incubation period, the incubation mixtures were examined by polyacrylamide gel electrophoresis in the following manner. To each incubation mixture, 100 #1 of the gel electrophoresis sample mixing solution [11] were added and the tubes placed in a boiling water bath for 5 rain. This stopped further enzymatic degradation. 100/A of this solution were then loaded on the SDS-acrylamide gels.

Inhibitor experiments Inhibitor studies were routinely carried out in incubation buffer volumes of 100 pl containing the desired inhibitor. The tropoelastin was added in a volume of 10 /zl. Sodium sulfite, sodium sulfide, MalNEt and EDTA were employed so that their final concentrations were 0.01 M or 0.001 M. Studies using soybean trypsin inhibitor and human a-l-antitrypsin (Worthington) were carried out by adding 10/~1 of inhibitor (concentration 1 mg/ml) to the incubation buffer. RESULTS Fig. 1 shows sodium dodecyl sulfate acrylamide gel patterns of lathyritic chick tropoelastin before and after incubation. The tropoelastin before incubation demonstrated a predominate molecular weight of approximately 70-72 000. The appearance of minor bands in the purified tropoelastin used in these studies is a result of apparent degradation which took place prior to this study. The tropoelastin when first isolated was a homogeneous band on SDS-gels with an apparent molecular weight of 70-72 000 [8]. Fig. 2 shows the effect of pH on the incubation of tropoelastin. The apparent pH optimum for enzymatic degradation of the tropoelastin occurs between 7 and 9. No appreciable differences in degradation products were noted between pH 2 to 6. While definite evidence of cleavage is seen at these pH values, it is far less than that demonstrated at the pH optimum. Choosing pH 8 buffer as optimum, a time study was conducted at 37 °C. As seen in Fig. 3, after 1 h the tropoelastin had undergone little degradation. At 5 and

248

Fig. 1. Polyacrylamide gel electrophoretic pattern of the tropoelastin used in this study (Gel A) showing a single band with an approximate molec :ular w<:ight of 70-72 000. Gel D shows the same tropoelastin incubated at 37 “C for 16 h. Gels B ar Id C sheJW tropoelastin which had been boiled (B) and reduced and alkylated (C) and then incubate :d. The direction of electrophoretic migration is from top to bottom.

249

~PH ,41. 7

8 9 10 12 Std

Fig. 2. Effect of pH on tropoelastin incubated at 37 °C for 16 h. Gel standard contains rat tail collagen, bovine serum albumin, and pepsin. The direction of electrophoretic migration is from top to bottom. 7 h, d e g r a d a t i o n is o b s e r v a b l e a n d at 14 h there is very little o f the 72 000 species remaining. I n h i b i t i o n studies on the e n z y m a t i c cleavage at p H 8 were carried o u t as described in the M a t e r i a l s a n d M e t h o d s section. Fig. 4 indicates the results o f the various

250

hrs. O 1 5

7 ......

I0

Fig. 3. Electrophoretic patterns showing the degradation of tropoelastin with time. Incubation conditions are pH 8 (38 °C). The direction of electrophoretic migration is from top to bottom.

inhibitors incubated with tropoelastin at 37 °C for 16 h. Inhibition was observed for sodium sulfite, sodium sulfide, M a l N E t and E D T A at concentrations o f 0.01 M and 0.001 M. Tropoelastin which has been reduced and alkylated also shows no enzymatic activity upon incubation (gel lc).

251

5td A

B

C D

E F

G

H

Fig. 4. Effects of the following inhibitors on tropoelastin degradation: 0.001 M sodium sulfide (A), 0.001 M sodium sulfite (B), 0.001 M N-ethylmaleimide (C), soybean trypsin inhibitor (D), human a-l-antitrypsin (E), and EDTA (F). Gel G shows tropoelastin incubated at 4 °C and Gel H shows tropoelastin incubated at 37 °C with no inhibitor. Gel standard contains rat tail collagen, bovine serum albumin and ribonuclease. Gels A-B and C-H were run on different days. The top bands in all gels correspond to the 70-72 000 dalton component of tropoelastin. The direction of electrophoretic migration is from top to bottom. DISCUSSION T r o p o e l a s t i n has been isolated from various animals a n d tissues. Most preparations c o n t a i n primarily a 72 000 a n d 57 000 d a l t o n c o m p o n e n t plus lower molecular weight species indicating degradation of the tropoelastin. W h e n enzymatic

252 inhibitors or acid extraction is incorporated into the purification procedure the predominent tropoelastin species isolated has a molecular weight of 72 000 [7, 8]. This species, however, can still undergo degradation when incubated under appropriate conditions in the absence of inhibitors (Figs. 1 and 2), suggesting the presence of an enzyme associated with and having an apparent high affinity for the tropoelastin. The presence of enzymatic activity is not simply an artifact of the tropoelastin employed in this study, nor is it species or tissue dependent, since enzymatic degradation has been observed in tropoelastin isolated from different animals as well as different tissues. Our laboratory has observed such degradation in tropoelastin from lathyritic chick aortae [8], chick gizzard, and pig lung (unpublished results). Tropoelastin heterogeneity has also been reported from copper-deficient pig aortae [6], lathyritic chick aortae [10, 16], and aortae from embryonic chick [7]. Also of interest is the fact that the ability to degrade is present in tropoelastins isolated by such contrasting purification procedures as extraction with organic solvents [6, ! 7] and neutral salt buffers [8, 10]. The observed cleavage of tropoelastin into relatively high molecular weight fragments (but lower molecular weight than the parent molecule) suggests restricted specificities of the proteases(s) for the soluble elastin substrate. This restricted specificity suggests the presence of only a limited number of enzymes associated with the tropoelastin, as well as a specific number of peptide bonds susceptible to enzymatic cleavage. We have also established that these discrete bands remain remarkably unchanged until the late stages of incubation. In addition to the parent molecule, five discrete polypeptide bands are usually observed on SDS gels with molecular weights of approximately 57 600, 45 600, 36 000, 24 700 and 13 000-14 000. After the 30 h digest, all of the resultant peptides ran with the tracking dye and, hence, had very low molecular weights. Sandberg has observed similar banding in tropoelastin from copper-deficient pigs [6]. He has further shown that the composition of each fraction (designated A, B, C and D) appears identical in amino acid content to that of whole tropoelastin with the exception of fraction C which has an elevated alanine and lower glycine and valine content. The demonstrated pH optimum of between pH 7 and 9 (Fig. 2) suggest optimal enzymatic activity over the range of physiological pH. Narayanan and Page [7] were able to isolate tropoelastin with a molecular weight of 72 000 exhibiting little enzymatic degradation by extracting with 0.5 M acetic acid. This pH would, of course, be below the pH optimum described above. Inhibition of enzymatic activity with various protein inhibitors is indicated in Fig. 4. Other investigators have reported that EDTA and MalNEt are effective in purification of tropoelastin with a molecular weight of 72 000 [6]. These observations, however, were based on an evaluation of the final product of purification rather than direct observation of enzyme activity. Inhibition by EDTA suggests that the enzyme requires divalent cations. Sulfite inhibition, along with no observable activity after reduction and alkylation, suggests the involvement of cysteine or cystine residues. Since tropoelastin contains no cystine or cysteine, these amino acids might be identified with enzyme(s) associated with the tropoelastin. It should be pointed out here that the tropoelastin preparation used in this study (see Table I) was judged to be approximately 90-95 ~ homogeneous by N-

253 terminal analysis and automated sequential analysis [8]. This would imply that the enzyme present would maximally be a 5-10 ~ contaminant on a molar basis and consequently is not identified in the amino acid composition of the tropoelastin preparation nor in the gel electrophoretic patterns. The data clearly demonstrates a critical problem in preventing proteolysis of the tropoelastin molecule during its purification. It is generally accepted that the addition of enzyme inhibitors to the extracting buffers was sufficient to insure protection of the tropoelastin. Our results reveal that an enzyme(s) remains closely associated with the soluble elastin throughout the entire purification procedure suggesting that it is very tightly bound. As pointed out earlier, this occurs in either the neutral salt or organic extraction procedures used to isolate tropoelastin. Once the inhibitors are removed, usually by dialysis, proteolysis can occur when the preparation is reconstituted in solution at the appropriate pH. It is interesting to note the inhibition of enzymatic activity by soybean trypsin inhibitor and a-l-antitrypsin. Such inhibition by the serum inhibitor a-l-antitrypsin suggests a possible in vivo control mechanism of elastin degradation. Robert [18] has demonstrated circulating elastin antibodies in experimental animals, leading to the suggestion that tropoelastin is degraded and the resultant peptides released into the blood. Tropoelastin turnover may be much greater than that of mature elastin suggesting that some elastin disease states might result from proteolytic degradation of soluble elastin before it is allowed to form crosslinks. Although one need not depend on a specific "tropoelastase", the enzyme observed in this study may serve such a degradative function in vivo. An alternative role for such enzyme systems described herein might be analogous to the procollagen to collagen conversion wherein soluble collagen molecules (tropocollagen) are formed by cleavage of amino and carboxyl terminal extension peptides from a procollagen by the enzyme(s) procollagen peptidase [19, 20]. These data also suggest the possibility that the proteolytic enzymes observed in this study might be necessary in vivo components to the formation of elastic fibers. ACKNOWLEDGEMENTS The authors acknowledge financial support of National Institutes of Health Grants HL-15964 and HL-13262. The data presented are taken in part from the Ph. D. dissertation of R.P.M. (in preparation). R.P.M. is grateful for Training Grant HD-00207. J.A.F. is an Established Investigator of the American Heart Association. REFERENCES 1 Weissman, N., Shields, G. S. and Carnes, W. H. (1963) J. Biol. Chem. 238, 3115-3118 2 Smith, D. W., Weissman, N. and Cames, W. H. (1968) Biochem. Biophys. Res. Commun 31, 309-315 3 Sandberg, L. B., Weissman, N. and Smith, D. W. (1969) Biochemistry 8, 2940-2945 4 Sandberg, L. B., Weissman, N. and Gray, W. R. (1971) Biochemistry 10, 52-56 5 Rucker, R. B. and Goettlick-Riemann, W. (1972) J. Nutr. 102, 563-570 6 Sandberg, L. B., Bruenger, E. and Cleary, E. G. (1975) Anal. Biochem. 64, 249-354 7 Narayanan, A. S. and Page, R. C. (1974) FEBS Lett. 44, 59-62 8 Foster, J. A., Shapiro, R., Voynow, P., Crombie, G., Faris, B., and Franzblau, C. (1975) Biochemistry 14, 5343-5347

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