THE JOURNAL OF Bmmorcal, CHEMISTRY Vol. 249, No. 19, Issue of October 10, pp. 6191-6196, 1974

Printed in U.S.A.

Isolation and Characterization of Cross-

Linked Peptides from Elastin*

(Received for publication, February 15, 1974)

JUDITH ANN FOSTER,~, 9 LISA RUBIN, HERBERT M. KAGAN, AND CARL FRANZBLAU

From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

EVELINE BRUENGER AND LAWRENCE 13. SANDBERGS

From the Department of Surgery, University of Utah, Salt Lake City, Utah 84112

SUMMARY

Two peptides containing the desmosine cross-link were isolated and purified from a subtilisin digest of oxalic acid- solubilized elastin. Molecular weights averaged approxi- mately 6000. Automated sequence data suggest that the desmosines cross-link two polypeptide chains and that both desmosine and isodesmosine are present in the same primary sequence approximately equally substituted. The primary sequence of the peptides is in good agreement with sequence data obtained on the soluble elastin precursor tropoelastin.

Investigations into the primary structure of elastin have re- cently advanced considerably with the isolation of an elast’in-like soluble component from the aortas of copper-deficient swine (1, 2). Sandberg et al. (3, 4), while examining peptides from enzymatic digests of the soluble elastin (tropoelastin), have ob- tained evidence for a striking clustering of alanine and lysine residues. Since lysine has been shown to be the precursor to the elastin cross-links (5-7), it might be expected that areas of cross- link formation are rich in alanine residues. This, in fact, has been verified by several investigators who have isolated alanine- enriched cross-linked peptides from mature elastin (8-11). However, as yet, little or no primary sequence data have been published on the mature elastin peptides.

It was the aim of the present investigation to characterize the area of mature elastin containing the desmosine cross-links (12). Since the pyridinium ring of the desmosines may serve to link 4, 3, or 2 polypeptide chains (13), we were particularly interested in defining which of these situations act,ually exists in mature elastin. It was also hoped that sequence data obtained would yield insights into the specific areas of tropoelastin destined for desmosine formation.

* This work was supported by Grant 1161 from the Greater Boston Chapter of the Massachusetts Heart Association, National Institutes of Health Grants HL-15964, AM-07697, HE-11963, and AM-51051, and the American Heart Association.

$ To whom correspondence should be addressed. $ Established Investigator of the American Heart Association.

MATERIALS AKD METHODS

Preparation of Soluble Elastin-Elastin was isolated from bovine ligamentum nuchae according to Partridge et al. (14) and solubi- lized with oxalic acid as described below. Ten grams of elastin were refluxed with 200 ml of 0.25 M oxalic acid until the mixture became clear. After cooling, the solut,ion was dialyzed against water at 4’ and subsequently lyophilized. The lyophilized elas- tin, 2.2 grams (by weight), was suspended in 44 ml of 0.1 M ammo- nium acetate buffer, pH 8.6. Subtilisin (purchased from Nutri- tional Biochemicals) was added to give an enzyme to substrate ratio of 1: 100 and the mixture was allowed to stir at room tempera- ture for 2 hours. The subtilisin addit,ion was then repeated, giving a final enzyme to substrate ratio of 1:50, and the digestion was continued for 2 more hours. At the end of this time, the reaction was terminated by lowering the pH to 3.0 with acetic acid, and the mixture, which was at this point a clear solution, was lyophilized.

Initial Separation of Elastin Peptides-The lyophilized sub- tilisin digest was not completely soluble when taken up in water at a concentration of 50 mg per ml (with respect to the original elastin starting material). The insoluble material was removed by centrifugation at 10,000 X 9 for 15 min. It should be noted here that this water-insoluble material represented less than 5yc of the starting elastin sample and contained a high percentage of polar amino acids. The clear supernatsnt was chromatographed on an ion exchange resin (Technicon peptide resin, 4y0 cross-link) in 10 successive runs of 2 ml each. The column (0.9 X 52 cm, 60”, 50 ml per hour) was eluted with a continuous pyridine-acetate gradient as specified in Table I. Following the gradient elution, the column was washed with 10 ml of 8.5 M pyridine and equili- brated with starting buffer. The elution pattern was established by ninhydrin analyses preceded by alkaline hydrolysis (15).

Amino acid analyses were performed on a Beckman 121 amino acid analyzer.

Pvrijkation of Peptide Fractions-Chromatography on Sepha- dex G-50 (fine, 1.3 X 200 cm) was used for further enrichment of the cross-linked fractions, employing 507, acet,ic acid as the eluant. These cross-linked peptide fractions were then purified on the same ion exchange column mentioned above utilizing a different pyridine-acetate gradient described by Schroeder (16).

Purity of the isolated peptides was tested by the dansyl’ tech- nique for NHz-terminal amino acid determinations (17) and the resulting dansyl amino acids were identified by thin layer chro- matography on polyamide sheets (18). Free desmosine was in- cluded as a standard. Both the ethyl acetate and the aqueous layers of each dansyl determination were examined because of the possibility of the desmosines being NH*-terminal.

Sequential Analyses of Peptides-Automated sequence analyses were performed on a Beckman model 890 C Sequencer according to

1 The abbreviations used are: dansyl, 5-dimethylaminonaph- thalene-1-sulfonyl; PTH, phenylthiohydantoin derivatives of amino acids.

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TABLE I Composition of pyridine-acetate buffers used for the elution of

elastin peptides on a Technicon peptide resin

A continuous gradient of pyridine-acetate was generated with a Technicon Varigrad containing 120 ml of buffer per chamber.

Chamber PH Normality of pyridine

1 2.7 0.025 2 3.2 0.050 3 3.5 0.100 4 3.8 0.500 5 4.2 1.000 6 4.9 2.000

Edman and Begg (19). Because of the extreme nonpolar character of the elastin peptides, a peptide program utilizing dimethylal- lylamine buffer was used for all analyses.

PTH-norleucine was added to all tubes in the machine and served as an internal standard. The sequenator fractions were converted to PTH derivative in the usual manner (19) and ex- tracted into ethyl acetate. The aqueous layer of each of the sequenator runs containing the desmosine cross-linked peptides was examined for ultraviolet absorption at 275 nm in order to detect the desmosines (12).

The majority of PTH derivatives were identified on a Beckman model 65 gas chromatograph according to Pisano and Bronzert (20). Silylation of PTH derivatives was performed with N,O- bis(trimethylsilyl)acetamide by direct injection of sample and reagent onto the column (21). In some cases, PTH derivatives (including both ethyl acetate and aqueous extracts) were hydro- lyzed to free amino acids in 6 N HCl at 120’ for 24 hours. The resulting amino acids were analyzed on a Jeolco 6AH amino acid analyzer. For standardization, free desmosine was subjected to two cycles in the sequenator and both the ethyl acetate and aque- ous layers were examined by gas chromatography and amino acid analysis.

In some cases the residual peptide material remaining in the sequenator cup after completion of analysis was extracted with 50% acetic acid and the amino acid composition determined.

RESULTS

Initial Separation of Elastin Peptides from Oxalic Acid-soluble Elastin-The chromatographic distribution of the subtilisin digest on the Technicon peptide resin is given in Fig. 1. Accord- ing to the ninhydrin pattern a total of 23 contiguous fractions were pooled. Corresponding fractions of 10 columns were com- bined and concentrated by flash evaporation. Based upon amino acid analyses of the soluble digest, desmosines and lysino- norleucine recoveries in the various fractions were 72 and 59%, respectively. Since we were particularly interested in the desmosine cross-linked area, we focused our attention on Frac- tions 19, 20, and 21, which contained 50% of the desmosines applied to the column and 70% of the total recovered desmosines.

Purification of Desmosine-enriched Fractions-Each of the three desmosine-enriched areas (Fractions 19, 20, and 21) were first chromatographed on the Sephadex G-50 column described above. Components eluted from the gel column were indi- vidually pooled and rechromatographed separately on the same column. We attempted to sequence several of the peptides after this purification step. However, there was a high back- ground of glycine, valine, and proline. Although these analyses provided some information concerning the primary sequence, further purification was necessary.

Fractions containing the highest content of desmosines were pooled and rehcromatographed on an ion exchange column em- ploying a steeper gradient (16) of pyridine-acetate than used for the original separation (see Table I). The compositions of those

I.0

0.8

2

g 0.6 d 0:

0.4

8.5M Pyridine

20 40 60 80 100 16C Tube number

FIG. 1. Elution pattern of the subtilisin digest from a Technicon peptide resin (0.9 X 52 cm, 60”, 50 ml per hour). A continuous gradient of pyridine acetate was used as specified elsewhere. Peptide material was located by ninhydrin preceded by alkaline hydrolysis. Peptide fractions were pooled as designated, i.e. 1 to 23.

peptides appearing pure as judged by the dansyl technique in conjunction with amino acid analyses and molecular weight estimated by gel filtration (Sephadex G-50 column), are given in Table II. Also included in the table are the amino acid composi- tions of the peptides during the various stages of purification.

Sequence Analyses of Desmosine Peptides-Tables III and IV contain data obtained on peptides S19 and S20. Table V pre- sents our interpretation of the sequencing data. It should be noted that the assignment of a particular amino acid residue to one or the other chain is tenuous when two chains are sequenced simultaneously. Our interpretation is based upon sequences ob- tained from the soluble precursor (15) (see “Discussion”). Each of the two peptides were sequenced twice; thus, there are dupli- cate sets of sequence data given for each peptide. Amino acid analyses of the residual peptide remaining in the sequenator cup after completion of 12 cycles were in good agreement with the sequencing data. The residue from both peptides S19 and S20 contained no tyrosine, glutamic acid, or desmosines and the majority of alanines had been removed by the 12th cycle.

The difficulty in interpreting the sequencing data of the desmosine peptides is a-fold. First, the two chains which are sequenced simultaneously are covalently linked in four different locations. Therefore, three of the four loci of cross-linking may not be detectable as a PTH derivative until the fourth arm of the cross-link is cleaved. Second, if the actual area of the cross-link is comprised of a two-stranded polyalanine structure, as proposed (see Table V), the interpretation of the sequencing data will be complicated by the degree of carryover and the concomitant loss of peptide through the extraction cycles (19). An illustration of a hypothetical desmosine peptide is given in Fig. 2 in order to clarify and justify our interpretation of the sequencing data ob- tained. Since both Chains a and b are sequenced simultaneously, Steps 1 and 2 of the Edman degradation will yield only alanine in an amount approximately equal to the amount of total desmo- sines. Step 3 would yield an increased amount of alanine repre- sentative of 2 residues. Step 4 would release Chain b from the desmosine; however, the only PTH derivative detectable will be

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TABLE II Amino acid compositions of purified desmosine peptides

Values are based on the average of at least two analyses. A space indicates less than 0.2 residue.

Amino acids Elastic starting materiala

Lysine ...................... 0.9 Histidine ....................... 0.3 Arginine ........................ 5.4 Hydroxyproline. .............. 10.6 Aspartic acid. ................... 1.9 Threonine ....................... 2.1 Serine. ......................... 2.4 Glutamic acid ................. 4.7 Proline ........................ 38.6 Glycine ..................... 78.2 Alanine. ....................... 61.4 Valine ........................ 41.9 Isoleucine ....................... 7.7 Leucine ......................... 19.1 Tyrosine ...................... 2.1 Phenylalanine. ............... 9.8 Lysinonorleucine .............. 0.2 Desmosine plus isodesmosine ... 1.0

NHz-terminal amino acidsc. .....

-

Gly, Ala, Val, Leu NDd

sl9*

Original isolation

1.0

1.2 0.7 0.6 0.8 1.2 1.6

12.0 20.0 25.8

6.7 1.6 4.1 1.4 4.2 0.2 1.0

0.9

0.9 0.3 0.6 0.5 0.8 1.1 6.8

12.4 19.5

4.7 1.2 2.8 0.8 3.2

1.0

Ala, Gly, Val

IOU exchange

Original isolation

0.2 0.9

0.3 0.2

0.7 1.0 7.2

12.9 20.8

4.3 1.2 2.7 0.7 2.8

1.0

0.9 1.1 0.5 0.9 0.9 2.1

14.0 27.4 45.6

8.4 2.1 5.3 1.7 4.9 0.2 1.0

Ala NDd

I- s20*

IOIl exchange

iephadex G-50 column

-

0.5

0.5 0.7 0.4 0.7 1.0 1.5

11.4 23.6 38.7

8.0 1.3 4.0 0.8 3.4

1.0

Ala, Gly, Val

0.6 0.3

1.1 1.3

11.1 20.4 31.2

7.6 1.4 4.0 1.1 3.8

1.0

Ala

a Reported on a residue basis derived from the division of the individual amino acid values per 1000 residues by the combined amount of desmosine plus isodesmosine.

* Results are expressed as residues per peptide. The three compositions listed correspond to various purification steps (see text for details).

c Determined as dansyl amino acids. d ND, not done.

TABLE III

Sequential degradation of peptide S19

Step No. Deduced residuesa

1 Ala + Ala Ala 127 Ala 212 2 Lysc + Ala Ala 85 Ala 130 3 Ala + Lys Ala 61 Ala 89 4 Ala + Ala Ala 81 Ala 89 5 Lys + Ala Ala 34 Ala 64 6 Tyr + Ala Ala + (Tyr)d 44 + 9 Ala + Tyrd 76 + 34 7 Ala + Des Ala + (Des), 24 + 21 Ala + Des 31 + 31 8 (Ala)f + Ala Ala 34 Ala 31 9 (Pro) + Ala Ala + (Pro) 20 + 6 Ala + (Pro) 12 + 9

10 Gly + (Glu) GUY 5 Gly + (G~u)~ 5.2 + 3.1 11 Xf + Phe Phe 4 Phe 2.8 12 X + WY) WY) 2 GUY) 0.8

Run 52

SP4OOb nmoles -

Run 60

SP400 IlIIlOkS

a Each sequenator step is assigned two amino acids since two chains are sequenced simultaneously (see text for explanation). b Packing material present in the columns employed in gas chromatography. c Every Lys represents a modified lysine residue which is involved in desmosine formation. They are not detected as a PTH de-

rivative by itself since each of these modified residues has been incorporated into the pyridinium ring. d Silylated derivative. c Des is used to abbreviate the combined content of desmosine plus isodesmosine which has been determined by amino acid analyses. f X signifies an unknown amino acid; parentheses indicate a suspected residue.

1 alanine residue from Chain a. Step 5 will yield an alanine the detachment of Chain b would be expected to result in a drop residue plus a tyrosine residue. Since the positively charged in yields of subsequent steps. At Step 6 the last arm of the pyridinium ring of the desmosines enhances the hydrophilicity of desmosine would be cleaved, producing a tetrathiazolinone the peptide, rendering it less extractable in the organic solvents, derivative plus an alanine residue. At this point, Chain a would

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TABLE IV

Sequential degradation of peptide S20

For explanation of abbreviations see footnotes to Table III.

step No

1 2 3 4 5 6 7 8 9

10 11 12

Deduced residues -

Ala + Ala Ala + Ala Ala + Ala Lys + Ala Ala + Lys Ala + Ala Ala + Ala

Des Ala + Tyr Ala + Gly

(Glu) + X Phe + X

SP400 I

nmoles

Ala 230 Ala 236 Ala 184 Ala 102 Ala 83 Ala 96 Ala 104

(Ala) + Des 29 + 20 Ala + Tyr 9 + 11 Ala + Gly 6th

(Ala) 1.9 Phe 1.8

Run 50

S19 arid 5’20

Tentative assignments arc given in parentheses, i.e. (Ala) means an alnnine is susprcted at this position, bllt not definitely proven. 1,~s connected to the abbreviation I>es is llscd to denote the arms of the desmosinc cross-link which are not detected as PTH derivatives (see text for dct nils), but, originally represent modified lgsine residues involved in the formation of the pyri- dinium ring of the desmosines. (X) signifies that the residue is unknown.

S 19 >Des<

A~~t.Ala-I,~~-Ala-Ala-Ala-Lys-Ala-Aia-Aia-~Gl~~~-Phe

( -.- Ala Ala Ala Ala Lys Ala Ala 1,~s Tyr Gly (X) (X) (X)

s 20

1 2 3 4 5 6

(a)

(b) a 1 2 34 5 6

FIG. 2. An illustration of a hypothetical desmosine peptide (see text for details). A, alanine; Y, tyrosine; d, desmosine plus isodesmosine; X, a modified lysine residue which constitutes an arm of the pyridinium nucleus.

also be freed from the cross-link. It should be noted that if the NHz-terminal residue on Chain b were alanine, the first sequen- ator step would yield an amount of alanine approximately equal to twice the total desmosine content.

Run 53

SP400

Ala 323 Ala 373 Ala 343 Ala 128 Ala 117 Ala 161 Ala 174

(Ala) + Des 35 + 30 Ala + ‘I’yr 21 + 21 Ala + Gly 10 + 6 (Glu) 2.1

Phe 3.8

TABLE VI

NHz-terminal analyses of desmosine peptides

Peptide Sequence run

19 it-60 1.8 19 it-52 1.5 20 R-50 1.7 20 IL-53 1.6

a Expressed as the micromoles of NHz-terminal amino acid recovered per Kmole of desmosine plus isodesmosine applied to the sequencer.

DISCUSSION

The original separation of the elastin peptides (see Fig. 1) re- sulted in the enrichment of dcsmosine peptide fractions which contained a concomitant enrichment of alanine residues. Purifi- cation of these fractions revealed several unique features in the amino acid compositions of the desmosine peptides. On a molar basis each of the peptides contained 1 serine, 1 glutamic acid, 1 tyrosine, and 2 to 3 residues of phenylalanine per peptide. The combined amount of the above mentioned amino acid residues in the total ligament elastin is relatively small (22) which might sug- gest some clustering of these residues within the area of the desmosine cross-link. Evidence for the association of tyrosine, glutamic acid, and phenylalanine within the sites destined for elastin cross-links is seen in the soluble elastin precursor. Se- quencing data obtained on a tryptic digest of tropoelastin (3, 15) have shown: (a) that of the 7 tyrosine residues already placed, 5 occupy a position adjacent to the carboxyl group of lysine, and (b) the sequence Ala-Ala-Glx-Phe follows a lysine residue at a minimum 2 to 3 times within the molecule.

The sequencing data indicate that the desmosines cross-link two chains based on: (a) micromoles of NHz-terminal amino acids recovered per pmole of peptide (see Table VI), and (b) the finding of two peptide sequences comparable to those obtained from the soluble elastin precursor (15). It should be noted that the molecular weights and residue numbers of the cross-linked pep- tides were based on the assumption that the amount of total desmosines (desmosine plus isodesmosine) be equal to 1 residue. This implies that the two isomers, desmosine and isodesmosine, are in the same primary sequence approximately equally substi-

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FIG. 3. Schematic illustration of the formation of either desmosine or isodesmosine via the reaction of ally- sine aldol with dehydrolysinonorleu- tine. As drawn, the direction of the condensation of two allysine residues (a) could result in two different reac- tion pathways (5) to form the desmo- sine isomers (c) within the same pri- mary sequence. Several carbons are numbered for clarification. (See text for further details.)

tuted. Sequence analyses offer evidence that this assumption is valid, since only one primary structure was seen and both desmosine and isodesmosine were cleaved at the same sequenator step (see Tables III and IV). A possible explanation for the existence of the two isomers within the same peptide is illustrated in Fig. 3. As diagrammed, the prime determinant in the forma- tion of either desmosine or isodesmosine would be the direction of the condensation of the 2 allysine residues. Specifically, the aldol condensation can result in two products, chemically indis- tinguishable, depending upon which of the allysine residues re- tains its aldehyde group. The remaining half of the desmosine moiety, whether it be in the form of dehydrolysinonorleucine as shown, or as an unreacted allysine and lysinc residue, is limited to one reactive direction.

We have drawn the pathway for desmosine synthesis via the reaction of the allysine aldol with dehydrolysinonorleucine for illustrative purposes. There is no direct evidence as yet for the formation of the pyridinium nucleus via this particular pathway. It is interesting to note that previously Piez (23) and more recently Gray el al.* (24) have postulated that the desmosines are in fact formed from allysine aldol and dchydrolysinonorlcucine. The latter authors, using CPK space-filling atomic models (25) have observed that lysines separated by 2 or 3 alanine residues in a LY helical conformation protrude on the same side of the helix. Hence, both dehydrolysinonorleucine and allysinc aldol could independently form within the same polypeptide chain. The sequence Lys-Ala-Ala-Lys allows for the formation of dehy- drolysinonorleucine, whereas the sequence Lys-Ala-Ala-Ala-Lys accommodates either allysine aldol or dehydrolysinonorleucine. The condensation of these two intramolecular cross-links would result in the formation of the carbon and nitrogen skeleton of the intermolecular linkage, resulting ultimately in desmosine or iso- desmosine-like moieties (26).

Several investigators have postulated that the pyridinium nucleus of the desmosine ring is actually formed from the reac- tion of merodesmosine with an allysine residue (27, 28). Our sequencing data do not contradict this postulate, the only restric- tion being that the allysine aldol is donated from one of the elastin chains and that the second chain contributes a lysine and an allysine residue either free or in the Schiff base form.

In the initial portion under “Discussion,” we have mentioned the clustering of tyrosine, phenylalanine, and glutamic acid in the desmosine area. Further evidence to support this finding

2 W. Et. Gray, L. B. Sandberg, and J. A. Foster, to be submitted shortly.

and also to substantiate the sequencing data presented here has come from a chymotryptic digest of tropoelastin.3 Of the chy- motryptic peptides already characterized, the sequences Ala-Lys- Ala-Ala-Lys-Tyr and Ala-Ala-Lys-Ala-Ala-Ala-Lys-Ala-Ala-Glx- Phe have been determined. Both of these sequences strongly support our interpretation of the data on the environment of the desmosine cross-links and for the involvement of only two poly- peptide chains, each of which can independently form an intra- molecular cross-link.

As stated above, we have determined that tyrosine frequently occupies a position adjacent to a lysine residue (5 of 7 times) in the primary sequence of tropoclastin The finding of tyrosine residues positioned next to the pyridinium nucleus may be signifi- cant in terms of assigning a probable role of tyrosine in cross-link formation. Since I of the 4 lysinc residues involved in the syn- thesis of the desmosine ring must retain its e amino group, it is conceivable that a neighboring tyrosine residue inhibits enzy- matic deamination either sterically or through hydrogen bonding.

It is interesting to note at this point that the tyrosine content varies considerably between different clastin samples (29). For example, ligamentum elastin contains 6 tyrosine residues per 1000 residues, whereas aortic elastin contains 18 tyrosine residues per 1000 residues. Significantly, aortic clastin is reported to possess almost twice the amount of dcsmosines as that present in liga- ment (30).

The source of the peptide material in this study was bovine ligamentum nuchae elastin, whereas the soluble elastin precursor, to which we are comparing our sequences, was isolated from porcine aorta. Interestingly, despite these differences in animals and tissues, we find some similarity in primary sequence in those areas destined for cross-link formation. We are presently trying to extend the sequence information toward the COOH-terminal area of the peptides to further compare the primary structure of the two elastin samples.

Acknowledgments-We are grateful to Dr. William R. Gray for his helpful discussions during the course of this work.

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and Lawrence B. SandbergJudith Ann Foster, Lisa Rubin, Herbert M. Kagan, Carl Franzblau, Eveline Bruenger

Isolation and Characterization of CrossLinked Peptides from Elastin

1974, 249:6191-6196.J. Biol. Chem. 

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