©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of an Elastin Cross-linking Domain That Joins Three Peptide Chains
POSSIBLE ROLE IN NUCLEATED ASSEMBLY (*)

(Received for publication, February 27, 1995; and in revised form, May 8, 1995)

Patricia Brown-Augsburger (1) Clarina Tisdale (1) Thomas Broekelmann Carolyn Sloan (1) Robert P. Mecham (1) (2)(§)

From the  (1)Department of Cell Biology and Physiology, and (2)Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The alignment of elastin molecules in the mature elastic fiber was investigated by purifying and sequencing cross-link-containing peptides generated by proteolytic digestion of incompletely cross-linked insoluble elastin. Peptides of interest were purified by reverse phase and size exclusion high performance liquid chromatography and characterized by amino acid analysis and protein sequencing. One peptide, consisting of the cross-linking domain encoded by exon 10, contained a modified lysine residue that had not condensed to form a polyfunctional cross-link. Although this domain contains the characteristic paired lysine residues found in other cross-linking domains of elastin, protein sequence analysis indicated that the first but not the second lysine had been oxidized by lysyl oxidase. This finding suggests that lysine residues in an individual cross-linking domain may not have equal susceptibility to oxidation by lysyl oxidase. In a second peptide, we found that a major cross-linking site in elastin is formed through the association of sequences encoded by exons 10, 19, and 25 and that the three chains are joined together by one desmosine and two lysinonorleucine cross-links. Past structural studies and computer modeling predict that domains 19 and 25 are linked by a desmosine cross-link, while domain 10 bridges domains 19 and 25 through lysinonorleucine cross-links. These findings, together with the high degree of sequence conservation for these three domains, suggest an important function for these regions of the molecule, possibly nucleating the aggregation and polymerization of tropoelastin monomers in the developing elastic fiber.


INTRODUCTION

An important property of collagen molecules is their ability to self-assemble into fibrils under physiological conditions. Inherent in the primary structure of collagen is the information that directs alignment of collagen monomers in the appropriate register for fiber formation. Like collagen, elastin maturation in the extracellular matrix involves the assembly of a soluble precursor molecule (tropoelastin) into a highly cross-linked polymer. This assembly process, however, is more complex than for collagen because the ability to self-assemble does not appear to be an intrinsic property of tropoelastin. Instead, elastin assembly requires helper proteins to align the multiple cross-linking sites on elastin monomers in preparation for cross-linking. This most likely occurs through interactions between the carboxyl terminus of tropoelastin and protein components of microfibrils(1) .

Our understanding of collagen fiber assembly was greatly aided by the ability to extract soluble collagen monomers that would spontaneously reassemble into fibrils under physiological conditions. Unfortunately, elastin contains many more cross-links than collagen, which makes the mature protein impossible to dissociate into intact monomeric units that retain the ability to assemble in vitro. Other than understanding the chemical mechanism of cross-link formation(2) , nothing is known about how tropoelastin monomers interact one with another to form the functional polymer.

The first step in cross-linking of tropoelastin is the formation of the -aldehyde, allysine, through oxidation of lysyl -amino groups by the enzyme lysyl oxidase(3) . Once formed, the allysine side chain is thought to condense spontaneously with another allysine molecule via an aldol condensation reaction forming the bifunctional cross-link allysine aldol, or with an -amino group of an unoxidized lysine residue via a Schiff's base reaction forming dehydrolysinonorleucine. In some instances, specific aldol and dehydrolysinonorleucine cross-links further condense to form the tetrafunctional pyridinium cross-links desmosine and isodesmosine(4) .

Structural studies during the mid-1970s found that lysine residues, which eventually form the cross-links in elastin, are localized in specific domains that alternate with sequences rich in hydrophobic amino acids(5, 6) . This unit motif of cross-linking plus hydrophobic domain repeats 16 times in a single elastin molecule. The significance of this repeating structure is that for proper assembly to occur, elastin monomers must be correctly aligned so that all of the cross-linking domains are in proper register. Studies to determine how this alignment occurs, however, have not been productive. This is because mature elastin, which is highly insoluble, can only be solubilized by techniques that randomly cleave peptide bonds (such as limited acid or base hydrolysis) or through the use of enzymes like thermolysin or elastase that are relatively nonspecific in their cleavage patterns. The peptides that are generated by these procedures are generally small, in most cases containing the cross-link and 3-15 other residues(7) .

A second problem has been that, until recently, the complete amino acid sequence of elastin was not known, making it impossible to assign a particular peptide sequence to a precise location in the molecule. In the past few years, the primary sequence of tropoelastin has been determined for several animal species(8, 9) . These results have given us a domain map that now permits the unambiguous assignment of peptide sequences to specific regions of the protein.

In this study, we examined the question of domain alignment in elastin by isolating and sequencing cross-link-containing peptides from insoluble elastin obtained from copper-deficient pig aorta. Under conditions of copper deficiency, the action of lysyl oxidase on lysine residues is partially inhibited, resulting in elastin that contains more than twice the usual number of unmodified lysine residues and half of the usual number of cross-links(10) . As a result, copper-deficient elastin is more susceptible to proteolysis than fully cross-linked elastin. This has allowed us to use trypsin and chymotrypsin to generate cross-link-containing peptides that are large enough to identify individual domains that participate in the formation of a cross-link. Our results show that a major cross-linking site in elastin is formed through the interaction of domains()10, 19, and 25 with the potential of linking three tropoelastin molecules through this one site. The prevalence of this cross-linking motif in copper-deficient elastin suggests an important role in elastic fiber assembly with the possible function of nucleating alignment of other cross-linking domains.


MATERIALS AND METHODS

NaBH (specific activity 500 mCi/mmol) was obtained from Amersham Corp. Aortic elastin from copper-deficient pigs was a gift of Dr. Lawrence Sandberg (Loma Linda, CA). Bioprene and other sequencing reagents were purchased from Applied Biosystems. o-Phthaldialdehyde and all other reagents were from Sigma.

Enzymatic Purification and Reduction of Cross-linking Amino Acids

Elastin was purified from copper-deficient pig aorta by autoclaving(11) . Cross-links were radiolabeled by the addition of 16 mCi of NaBH to a suspension of 500 mg of purified protein in 50 ml of 1 mM EDTA, pH 9.0. The reaction was allowed to proceed for 20 min with the pH maintained at 9.0 by the addition of 0.01 M NaOH. Reduction was completed by the addition of 36 mg of cold sodium borohydride, after which stirring was continued for an additional 2 h. The reaction was terminated by the addition of glacial acetic acid until the pH reached 4.0. Excess NaBH was removed by sequential washes of the reduced protein with 0.01 M HCl, ethanol, and diethyl ether(10) .

Elastin peptides were generated by successive incubations of the insoluble protein with trypsin and chymotrypsin as described by Mecham and Foster(10) . Reduced elastin (100 mg) was suspended in 3 ml of 50 mM Tris, pH 7.5 containing 0.012 M CaCl. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (2 mg) was added, and the suspension was mixed for 24 h at 4 °C. The supernatant was removed by centrifugation, and digestion of the remaining pellet was repeated at room temperature. Following centrifugation, the insoluble residue was digested for 24 h at room temperature with 2 mg of chymotrypsin. An aliquot from each of the digestion reaction supernatants was counted for radioactivity in a Beckman LS8000 scintillation counter. Radioactivity in residual, undigested elastin was determined in a similar way by transferring the entire washed pellet to a scintillation vial.

Peptide Purification

Initial HPLC() purification of elastin peptides was performed on a 0.41 15-cm Hamilton PRP-3 reverse phase column at 0.5 ml/min flow rate. The column was developed using a linear 75-min gradient from 100% water, 0.05% trifluoroacetic acid to 50% acetonitrile, 0.05% trifluoroacetic acid followed by a 7-min gradient to 80% acetonitrile, 0.05% trifluoroacetic acid. Column fractions were collected at 1-min intervals and counted for radioactivity in a Beckman LS8000 scintillation counter to detect radiolabeled peptides. Peptide fractions containing radioactivity were dried by rotary evaporation, resuspended in water, and chromatographed on a 0.75 30-cm Altex Spherogel TSK size exclusion column developed with a 45-min isocratic gradient in 50% acetonitrile, 0.05% trifluoroacetic acid. Radioactive peaks were pooled, and the fractions were dried by rotary evaporation. Final purification was achieved by chromatography over a 0.46 22-cm Applied Biosystems Aquapore OD 300 (7-µm bead) column developed with the same gradient as the PRP-3 column. An alternate purification scheme used for some peptides consisted of two successive runs on the PRP-3 column as described above.

Amino Acid Analysis

The amino acid composition of each peptide fraction was determined by analysis on a Beckman model 6300 analyzer with a modified gradient for resolving cross-linking amino acids. Samples were prepared by hydrolysis in 6 N HCl in sealed tubes at 110 °C for 24 h. The program for resolving lysine-derived cross-links consisted of 0.2 M sodium citrate, pH 3.28 (Beckman buffer Na-E) for 14 min, 0.2 M sodium citrate, pH 4.25 (Beckman buffer Na-F) for 11 min, Beckman microcolumn citrate buffer (pH 5.26) for 10 min, and 1.0 M sodium citrate, pH 6.40 (Beckman buffer Na-D) for 22 min. Column temperature was maintained at 49 °C for the first 11 min and then elevated to 75 °C for the remainder of the run. Column flow rate was 20 ml/h. Peptide composition was normalized so that desmosine = 1.

Sequencing

Pulsed liquid sequencing was performed on an Applied Biosystems model 473A sequencer. For cross-linked peptides, o-phthalaldehyde (OPA) was used to achieve sequencing from a single amino terminus(12) . Peptides were spotted onto Bioprene Plus-treated glass fiber filter paper and initially sequenced to determine the location of proline residues. A second aliquot of the peptide was then sequenced with an OPA blocking cycle inserted when the desired proline residue was at the amino terminus. During the OPA cycle the filter was treated briefly with 12.5% trimethylamine, removed from the sequencer, and spotted with a solution containing 0.1 M OPA and 0.1 M 2-mercaptoethanol in acetonitrile. The filter was returned to the sequencer and after 10 min was washed with 12.5% trimethylamine for 5 min, acidified with trifluoroacetic acid, and rinsed with ethyl acetate. The filter was finally dried and sequencing continued.

Computer Modeling

Computer modeling was performed on a Evans and Sutherland ESV workstation using Sybyl version 5.5 (Tripos Associates Inc., St. Louis, MO). The cross-linking sequences in domains 19 and 25 were modeled as -helices as predicted by the primary structure. The Lys-Pro-Lys sequence found in both human and bovine elastin was used to model exon 10. Because of the restrained 60° turn induced by proline, this was thought to be the most restrictive case. Helical wheel representations were generated using the Protean module of the LASERGENE software package produced by DNASTAR, Inc., Madison, WI.


RESULTS

Purification and Characterization of Cross-link-containing Peptides

Our previous studies of cross-link-containing peptides established that elastin from copper-deficient animals could be used to identify cross-linking patterns in the insoluble molecule(10) . Copper deficiency partially inhibits the activity of lysyl oxidase so that less than half the number of cross-links form in elastin in these animals. Because of the higher number of unmodified lysine residues, copper-deficient elastin can be cleaved with trypsin and chymotrypsin, thereby generating larger peptides than those obtained with elastase or thermolysin cleavage (trypsin and chymotrypsin have little activity toward normal elastin). Using this approach, insoluble elastin purified from copper-deficient pigs was treated with sodium borotritide to radiolabel the reducible cross-links and was then exposed to successive treatments with trypsin and chymotrypsin.

Table 1shows the radioactivity released by each successive digest of [H]lathyritic elastin and the percent of the total material solubilized by each treatment. The majority of radioactivity (42%) was solubilized in the chymotrypsin digest. Lesser amounts (13% and 12%) were found in the first (T1) and second (T2) trypsin fractions. The remaining 32% of labeled cross-links were not solubilized by any treatment and remained associated with the undigested pellet. Amino acid analysis of the enzyme-solubilized fractions revealed both the presence of cross-linking amino acids and unmodified lysine residues. Desmosine (Des) to lysinonorleucine ratios were comparable to those found by Mecham and Foster(10) . Des and isodesmosine coelute from the amino acid analyzer following borotritide reduction and, thus, are collectively referred to as Des.



HPLC fractionation of the peptides solubilized by chymotrypsin digestion using a reverse phase PRP3 column yielded the chromatogram shown in Fig. 1. Cross-links were detected by scintillation counting, and those fractions containing the highest levels of radioactivity were selected for additional purification. Chromatograms of peptides()purified using two sequential PRP-3 column runs (peptides Chy46, Chy58, Chy60, and Chy62) or using the PRP-3 column followed by size exclusion and C18 chromatography (Chy61SZC18 and Chy36SZC18) are shown in Fig. 2. In all cases, radioactivity eluted with the main protein peak. Amino acid analysis of the isolated peptides was used to verify the presence of cross-linking amino acids and to determine the type of cross-link present. The results of these analyses are shown in Table 2.


Figure 1: HPLC separation of chymotrypsin digest of copper-deficient elastin. Peptides solubilized by chymotrypsin digestion were fractionated on a Hamilton PRP-3 reverse phase HPLC column using a linear gradient (dottedline) of water/0.05% trifluoroacetic acid and acetonitrile/0.05% trifluoroacetic acid. The flow rate was 0.5 ml/min. Shown is absorbance at 215 nm.




Figure 2: Purified cross-linked peptides. Cross-link-containing fractions from the first cycle of HPLC separation were rechromatographed as described under ``Materials and Methods.'' Columns were developed using an acetonitrile gradient as described in Fig. 1. Chromatograms of the final purified peptides are shown above. Absorbance is at 215 nm.





Sequencing Cross-linked Peptides

A major difficulty in sequencing cross-linked peptides is assigning the multiple amino acids obtained at each sequencing cycle to the two or more peptide chains that are being sequenced. To overcome this problem, we employed OPA to selectively block end-groups of cross-linked peptides so that only one peptide chain would be sequenced at a time(12, 13) . OPA reacts with primary amines, effectively preventing Edman degradation from the modified amino terminus. However, OPA does not react with secondary amines, permitting sequencing of chains with NH-terminal proline. Our strategy was to first sequence cross-link-containing peptides without OPA to determine the position of proline residues. A second aliquot of the peptide was then sequenced until a proline residue was exposed at the amino terminus of one of the chains. The peptide was then treated with OPA, washed, and sequencing resumed. Only one peptide arm of the cross-linked peptide was then accessible to sequential degradation (the one with proline at the amino terminus). The sequence of the second chain became immediately evident by subtracting the OPA determined sequence from the sequence of both chains obtained without blocking. If the cross-linked peptide contained more than two chains, multiple rounds of OPA blockage were necessary to establish an unambiguous sequence. Because porcine tropoelastin has not been completely characterized, the sequence of bovine tropoelastin was used for comparison in areas where the porcine sequence was unknown (Fig. 3).


Figure 3: Alignment of porcine tropoelastin fragments with sequence from bovine tropoelastin. Bovine sequence (toprow) is from cDNA and genomic clones(8) . Porcine sequences are from tryptic peptides(21) . Doubleunderlines indicate sequences obtained for peptide Chy62.



Sequence results for the peptides purified in this study are summarized in Table 3. In all cases, the sequence of each peptide begins in the hydrophobic domain that precedes the cross-linking domain. Consistent with chymotrypsin's preference for aromatic side chains, sequences in domains 18 and 24 result from cleavage of the Phe-Gly peptide bond at the beginning of these domains. Generation of the domain 10 sequence appears to have resulted from trypsin cleavage of the Arg-Phe bond at the beginning of domain 10. Although our sequencing results did not always continue through the cross-linking amino acid, it was clear from the compositional data that each peptide contained the hydrophobic domain followed by only one cross-linking domain.



Modification of Lysine in Domain 10

Peptide Chy36SZC18 differed from the other peptides in this study in that it contained radioactivity indicative of a reducible cross-link, but amino acid analysis revealed no desmosine or lysinonorleucine. The most likely explanation for this result is that the peptide contains a modified lysine residue that has not condensed to form a polyfunctional cross-link. The presence of a single modified lysine as opposed to a bifunctional cross-link was confirmed by protein sequencing, which demonstrated the presence of a single peptide chain with a modified lysine residue at position 5. A comparison of the derived sequence with the full-length sequence of bovine tropoelastin indicated that this peptide contained cross-linking domain 10 and that the first but not the second lysine had been oxidized by lysyl oxidase.

Sequence of Cross-linked Peptide

Domain 10 was also present in the other four cross-link-containing fractions we characterized in this study. In addition, these fractions contained cross-linking domains 19 and 25 (Table 3). The tight codistribution of the three sequences after several different chromatography steps suggests that the component peptides must be covalently cross-linked. Evidence that these chains were not separate peptides that copurify was best indicated by an identical repetitive yield for all chains during the sequencing reaction. A major difficulty in sequencing hydrophobic peptides like those found in elastin is that peptide washout is usually extremely high because of the organic reagents used in the sequencing reaction. Furthermore, the rate of washout, and hence the repetitive yield (i.e. the percent recovery at each sequencing step) is usually different for each peptide. If a sequencing run contains two separate peptides, the repetitive yield will most likely be different for peptide A than peptide B. If the two peptides are cross-linked, however, washout is greatly reduced and the repetitive yield will be identical for each chain. Table 4shows the repetitive yield for each of the peptides characterized in this study.



A key to understanding how peptides containing domains 10, 19, and 25 might be organized was the sequence and cross-link composition of fraction Chy62. This peptide contained sequences from all three domains with a cross-linking profile consisting of one desmosine and two lysinonorleucine residues. Several studies have shown that Des cross-links are only formed in alanine-rich regions of elastin from two lysines on each of two peptide chains(14, 15) . Because domain 10 does not contain the appropriate polyalanine sequence, the Des cross-link in Chy62 must form between domains 19 and 25 (a Des cross-link between these two domains has been documented by Gerber and Anwar; (15) ). Interestingly, domains 19 and 25 are the only two cross-linking domains in elastin that contain three lysine residues instead of two. Thus, we propose that the remaining lysine residue in each domain forms a bivalent lysinonorleucine cross-link with lysine residues in domain 10, as shown in Fig. 4.


Figure 4: Schematic model of cross-linked peptide formed by domains 10, 19, and 25. Domains 19 and 25, running antiparallel, are joined by a desmosine cross-link involving the last (from the amino terminus) two lysines in domain 19 and the first two lysines in domain 25. The third lysine in each domain joins with a lysine in domain 10 to form lysinonorleucine cross-links.



To determine whether domains 10, 19, and 25 can assume the correct spatial orientation to form the complex aggregate proposed in Fig. 4, we used a computer modeling program to emulate the proposed interactions. Assigning a helical structure to the polyalanine sequences of domains 19 and 25 (5) resulted in all three lysine side chains in each domain protruding on one face of the helix (Fig. 5). When the two helices are brought together, two of the lysines on each chain are perfectly positioned to condense to form a Des cross-link. The spatial positioning of the remaining lysines, one on each chain, precludes their direct interaction, but these residues are perfectly situated to interact with lysine side chains in domain 10.


Figure 5: Positioning of lysine side chain in domains 19 and 25. Helical wheel representations of domains 19 and 25 showing lysine and arginine side chains positioned on the same side of the helix. Diagrams were generated using the Protean module of the LASERGENE program from DNASTAR.




DISCUSSION

The conversion of tropoelastin molecules into a cross-linked polymer is a critical process in the assembly of a functional elastic fiber. The amino acid sequence of tropoelastin predicts multiple cross-linking sites that must be aligned during fiber assembly. Furthermore, each monomer must be cross-linked with more than one molecule to produce a three-dimensional network. With these constraints in mind, it is reasonable to assume that alignment of molecular domains between two or more tropoelastin molecules is not random, but requires the juxtaposition of specific cross-linking sequences.

The tertiary structure of tropoelastin undoubtedly imposes conformational constraints on how specific domains are positioned for interaction between and within molecules. Earlier studies have shown that the tetrafunctional cross-links desmosine and isodesmosine occur within alanine-rich sequences (KA domains)(6, 16) , where they link two peptide chains. Since desmosine is derived from four lysine side chains, the cross-link must be formed from two lysine residues on each of the two chains(14) . Sequence analysis confirmed that the lysines in these alanine-rich domains occur in pairs, separated by two or three alanine residues. Molecular modeling and structural analysis show these KA domains to be in an helical structure, which has the effect of positioning both lysine side-chains on the same side of the helix(5, 16, 17) . This conformation has been suggested to be critical to the formation of desmosine, whose biosynthesis is thought to involve the condensation of an allysine aldol formed between two lysine residues on one chain with an intramolecular dehydrolysinonorleucine on a second chain(18) . Modeling studies have shown that allysine aldol and dehydrolysinonorleucine will fit on the -helix without distortion with one interesting restriction; allysine aldol can only be accommodated when the precursor lysines are separated by three alanines while dehydrolysinonorleucine can form between lysines separated by either two or three residues(17, 18) .

Analysis of KA sequences in bovine tropoelastin shows four domains with lysine pairs separated by two alanines (-K-A-A-K-) (domains 17, 21, 27, and 31) and an equal number with lysines separated by three alanines (-K-A-A-A-K-) (domains 6, 15, 23, and 29). If intrachain intermediates are required in the biosynthesis of desmosine, the steric restrictions imposed on their formation would predict that domains 6, 15, 23, and 29 (-K-A-A-A-K- sequences) contain allysine aldol intermediates which condense with dehydrolysinonorleucine moieties in domains 17, 21, 27, and 31 (-K-A-A-K- sequences)(17) . These are exactly the domains Baig et al.(19) found to contain desmosine in an earlier study of cross-linked peptides in bovine elastin. It is also important to note that the last lysine in all of the K-A-A-K domains is followed by a bulky hydrophobic residue (Tyr, Phe, Ile, or Leu), whereas three of the four K-A-A-A-K domains have alanines following the last lysine. Several groups have postulated that a large hydrophobic residue next to a lysine might serve to protect the lysyl -amino group from oxidation by lysyl oxidase(6, 14, 17) . This is in agreement with the prediction that K-A-A-K domains contain dehydrolysinonorleucine, which is the condensation of an allysine with an unoxidized lysine side chain.

One of the more interesting findings from the cDNA analysis of tropoelastin sequence was the identification of a second class of cross-link domain. In these sequences, encoded by exons 4, 8, 10, 12, 13, and 35 in bovine elastin, lysine pairs are separated by one or more proline residues (KP domains) and are flanked by prolines and bulky hydrophobic amino acids(20) . Our finding that only one of the lysines in peptide Chy36SZC18 (exon 10) had been oxidized by lysyl oxidase suggests that lysine residues in the KP domains are not equally susceptible to modification. It is important to emphasize that desmosine or isodesmosine have not been found to be associated with these KP domain sequences(19) . This is not unexpected if an intradomain intermediate is required for desmosine formation, since KP domains are unlikely to adopt a helical structure because of steric constraints induced by proline residues. Thus, we can conclude that lysines in KP domains will form cross-links other than desmosine and that any desmosines are most likely, if not exclusively, joining two KA domains.

These assumptions are in agreement with and are supported by the characterization in this study of a cross-linked peptide that contains domains 10, 19, and 25. Our data suggest that domains 19 and 25 are joined by a desmosine and that domain 10 bridges 19 and 25 through two lysinonorleucine cross-links (Fig. 4). Interactions between domains 19 and 25 were expected since these are the only two cross-linking domains in elastin that contain three lysine residues. Thus, it is not unreasonable to predict that these domains must align because of their unique cross-linking requirements.

Based on the conformational restrictions for intrachain cross-link formation, placement of hydrophobic amino acids after lysine residues, and previous sequencing studies(19) , we can predict that domains 19 and 25 are running antiparallel and that the desmosine is formed between the last two lysines in domain 19 and the first two lysines of domain 25. The last two lysines in domain 19 are separated by two alanine residues and are followed by a large hydrophobic amino acid (-K-A-A-K-F-), suggesting a dehydrolysinonorleucine intermediate. The first two lysines of domain 25 are separated by three residues and are followed by an alanine (-K-S-A-A-K-A) and, hence, fit the consensus for allysine aldol formation. Although the second and third lysines of domain 25 also comply with this consensus, Baig et al.(19) identified the sequence -S-A- as being associated with the arm of a desmosine cross-link. The chains must be placed in an antiparallel orientation to position the first lysine of domain 19 and the last lysine of domain 25 on the same side of the desmosine cross-link for the bridging interaction to occur with domain 10.

The importance of domains 10, 19, and 25 to fibrillogenesis is suggested by the uniqueness of these particular domains within the elastin molecule and their conservation among different species. The two triple lysine cross-link domains (exons 19 and 25) are conserved across species, as is the presence of the single KXK motif found in domain 10. Within the KXK motif, X is either an alanine or proline residue. While the presence of a proline in the X position in the human and bovine sequences places steric restrictions on the conformation of this domain, our modeling shows that this constraint still allows domain 10 to bridge lysine residues in domains 19 and 25. Further evidence supporting the importance of domains 10, 19, and 25 in elastin fibrillogenesis is their obvious ability to cross-link under conditions of copper deficiency where the activity of lysyl oxidase is suboptimal. This implies that the lysines within these domains are favored for oxidation.

It is not known what factors are involved in aligning cross-linking domains in tropoelastin molecules. It is likely that an initial alignment is facilitated by interactions between tropoelastin and microfibrillar proteins. Once cross-linking has occurred at one site, however, molecular alignment between tropoelastin molecules may be sufficiently stabilized to allow completion of cross-linking in the absence of microfibrillar proteins. The domain 10-19-25 cross-link isolated in this study is an excellent candidate for nucleating the assembly of up to three different tropoelastin molecules. Because we are limited to working with proteolytic fragments of insoluble elastin, however, it is not possible to determine whether our sequencing is from three distinct molecules, or from amino termini generated by enzymatic cleavage within a single molecule (see Fig. 6). At present there is no easy way of solving this problem, but if three different tropoelastin molecules are joined through this site, then the 10-19-25 cross-link is likely to serve a critical role in directing elastic fiber assembly.


Figure 6: Two explanations of sequence data for Chy62 peptide. In the toppanel, three distinct tropoelastin molecules contribute chains to form the cross-link domain. The bottompanel shows how the same sequence data can be generated from only two molecules where one chain folds back to contribute a second domain to the cross-link. Proteolysis anywhere in the loop would generate the third amino terminus for sequencing. The third possibility (not shown) is where all three chains in the cross-link are contributed through folding of only one molecule.




FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL-26499 and HL-53325. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, Box 8228, 660 South Euclid, St. Louis, MO 63110. Tel.: 314-362-2254; Fax: 314-362-2252; bmecham{at}cellbio.wustl.edu

Domain numbering is based on exon assignment of bovine elastin.

The abbreviations used are: HPLC, high performance liquid chromatography; OPA, o-phthalaldehyde; Des, desmosine.

Peptide nomenclature includes an abbreviation indicating the enzyme used for solubilization (T, trypsin; Chy, chymotrypsin) followed by the fraction number from the first PRP-3 HPLC fractionation. Also indicated, where appropriate, are abbreviations for subsequent purification steps involving sizing (CZ) or C18 reverse phase (C18) chromatography.


ACKNOWLEDGEMENTS

We thank Terese Hall for secretarial assistance.


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