(Received for publication, February 27, 1995; and in revised form, May 8, 1995)
From the
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. 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 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 NaB
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
Table 1shows the
radioactivity released by each successive digest of
[
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
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.
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.
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.
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 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 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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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) .
(
)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.
H
(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
NaB
H
was removed by sequential washes of the
reduced protein with 0.01 M HCl, ethanol, and diethyl
ether(10) .
. 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.
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.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.
(
)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.
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).
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.
-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) .
-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.
We thank Terese Hall for secretarial assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.