Degradation of tropoelastin by matrix metalloproteinases –
cleavage site specificities and release of matrikines
Andrea Heinz
1
, Michael C. Jung
1
, Laurent Duca
2
, Wolfgang Sippl
1
, Samuel Taddese
1
,
Christian Ihling
1
, Anthony Rusciani
2
,Gu
¨
nther Jahreis
3
, Anthony S. Weiss
4
, Reinhard H. H. Neubert
1
and Christian E. H. Schmelzer
1
1 Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany
2 Faculte
´
des Sciences, Laboratoire de Biochimie, Reims, France

and Val, at P
1
¢. MMP-7 shows a strong preference for Leu at P
1
¢, which is
also well accepted by MMP-9 and MMP-12. Of all three MMPs, MMP-12
best tolerates bulky charged and aromatic amino acids at P
1
¢. All three
MMPs showed a clear preference for Pro at P
3
that could be structurally
explained by molecular modeling. Analysis of the generated peptides
revealed that all three MMPs show a similar ability to release bioactive
sequences, with MMP-12 producing the highest number of these peptides.
Furthermore, the generated peptides YTTGKLPYGYGPGG,
YGARPGVGVGGIP, and PGFGAVPGA, containing GxxPG motifs that
have not yet been proven to be bioactive, were identified as new matrikines
upon biological activity testing.
Structured digital abstract
l
MINT-7709630: MMP-7 (uniprotkb:P09237) cleaves (MI:0194) Tropoelastin (uniprotkb:
P15502)byprotease assay (MI:0435)
l
MINT-7709668: MMP-9 (uniprotkb:P14780) cleaves (MI:0194) Tropoelastin (uniprotkb:
P15502)byprotease assay ( MI:0435)
l
MINT-7709289: MMP-12 (uniprotkb:P39900) cleaves (MI:0194) Tropoelastin (uniprotkb:
P15502)byprotease assay ( MI:0435)
Abbreviations

various malignant cells, ras-transformed murine cells,
and chemically stimulated fibroblasts. It has, for
instance, been shown to cleave native type IV and VII
collagens, gelatin, laminin, and plasminogen [2].
MMP-12 (macrophage elastase) is expressed mainly by
macrophages. The enzyme cleaves a variety of sub-
strates, including collagens, gelatin, laminin, pro-tumor
necrosis factor-a, and plasminogen [2]. Natural sub-
strates that are known to be degraded by MMP-7,
MMP-9, MMP-12, and a further member of the
MMP family, MMP-2, are the connective tissue pro-
tein elastin and its monomeric precursor tropoelastin
[6–11].
The biopolymer elastin, which provides elasticity
and resilience to several tissues, including lungs, arter-
ies, and skin, shows a unique chemical composition
characterized by the presence of large amounts of the
four hydrophobic amino acids Gly, Val, Ala, and Pro.
The protein consists of molecules of its soluble precur-
sor tropoelastin that are cross-linked at Lys residues.
Owing to its hydrophobicity and extensive cross-link-
ing, elastin is insoluble and highly resistant to proteo-
lytic degradation. Moreover, elastin does not undergo
substantial turnover in healthy tissue [12–16].
In the last two decades, studies have revealed that
elastin is not only a structural protein influencing the
architecture and biomechanical properties of the ECM
but also plays an active role in various physiological
processes [16]. Some elastin-derived peptides (EDPs),
which may occur upon proteolytic degradation of elas-

tous skin diseases [34]. MMP-7 is strongly expressed in
tumors of almost every organ in the body and seems
to play a vital role in tumor progression and angiogen-
esis [35,36]. Taken together, these examples show that
it is of utmost importance to understand and charac-
terize elastin-degrading processes, including the cleav-
age behavior of elastinolytic MMPs and the nature of
the peptides released on degradation. This approach
may aid in the development of directed therapies to
treat pathologies related to elastin degradation, the
overexpression of MMPs, and the consequent release
of bioactive peptides.
Few studies have investigated the enzymatic degra-
dation of elastin or its precursor tropoelastin [10,37,38]
and the release of bioactive peptides upon enzymatic
degradation of elastin, tropoelastin or synthesized
domains derived from tropoelastin [39–41]. The aim of
the present study was to obtain detailed information
Tropoelastin degradation by elastinolytic MMPs A. Heinz et al.
1940 FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS
on the cleavage site specificities of MMP-7, MMP-9,
and MMP-12 in tropoelastin using complementary MS
techniques and to characterize and compare the cleav-
age behavior of the three enzymes using molecular
modeling. Tropoelastin was chosen as substrate in this
study because of its biological relevance during elastin
turnover and matrix remodeling and to increase the
number of identifiable peptides resulting from proteo-
lytic digestion. Mature elastin, in contrast, shows only
limited suitability for characterization of the cleavage

ment with previous studies on bovine and human elas-
tin [10,37]. Altogether, for MMP-12, 89 cleavage sites
and 132 peptides were identified in almost all domains
of tropoelastin with the exception of domains 8, 9, and
11. In contrast, for MMP-7 and MMP-9, only 58 (84
peptides) and 63 (74 peptides) cleavage sites could be
determined, respectively. For MMP-7, no cleavages
were observed in domains 8–11, 17, 19–21, 23, and 36.
MMP-9 showed a similar cleavage behavior and did
not degrade domains 8–11, 16–20, and 36. Altogether,
23 cleavage sites and 20 peptides were found that were
common for all three MMPs. It is worth mentioning
that in MALDI-TOF experiments several unidentified
higher-mass peptides of between 10 kDa and 20 kDa
were observed for the three MMPs, underlining the
finding that some domains resisted proteolysis (data
not shown).
Aliphatic and
⁄
or hydrophobic residues favored at
P
1
¢
The P
1
¢–S
1
¢ interaction has been identified as the main
determinant of the cleavage position of MMPs in pep-
tide substrates [5,10,42,43]. The results of this work

Leu. Figure 2A and Table 1 show that MMP-7 has a
strong preference for Leu at P
1
¢, which has also been
described previously [44,45]. MMP-7 cut at 29 of
40 (73%) possible cleavage sites with Leu, whereas
MMP-9 and MMP-12 only cleaved at 14 (35%) and
19 (48%) sites, respectively. The preference of MMP-7
is also shown in Fig. 3, where the interaction of the
hexapeptide substrate PQGLAG containing Leu at P
1
¢
is modeled. Small differences in the cleavage site speci-
ficities of the three MMPs were observed at cleavage
sites with bulky aromatic amino acids such as Tyr and
Phe at P
1
¢, which were cut, in particular, by MMP-12.
While MMP-12 hydrolyzed 53% (8 of 15) of the possi-
ble x-Tyr peptide bonds, MMP-7 and MMP-9 showed
similar cleavage behavior and only cut at 3 and 4 of
15 possible x-Tyr cleavage sites, respectively. x-Phe
bonds were found to be hydrolyzed by MMP-7 (2 of
16) and MMP-12 (4 of 16) but not by MMP-9.
The cleavage behavior of MMP-7, MMP-9, and
MMP-12 at x-Pro, x-Gly, x-Ile, and x-Val peptide
bonds is very similar. All three MMPs similarly toler-
ate amino acids with relatively small aliphatic and ⁄ or
hydrophobic residues at P
1

1
-P
4
and P
1
¢-P
4
¢ after digestion with MMP-7, MMP-9, and MMP-12.
Values are based on the total amounts of each of the amino acids in tropoelastin (isoform 2).
A. Heinz et al. Tropoelastin degradation by elastinolytic MMPs
FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS 1943
the order Leu (48% of all identified cleavage
sites) >> Val ⁄ Gly (each 12%) > Pro (10%) > Tyr
(5%), with Leu being clearly preferred over other
aliphatic and ⁄ or hydrophobic amino acids (Table 2).
The cleavage site specificity of MMP-9 follows the
order Leu (22% of all identified cleavage sites) > Ala
(19%) > Gly (14%) > Lys (11%) > Val (9%).
MMP-12, which is the most active of the three
enzymes, shows a cleavage site specificity according to
the order Ala (26% of all identified cleavage site-
s) > Leu (20%) > Lys (12%) > Val ⁄ Tyr (each
9%) > Gly (7%).
With respect to the charged or polar amino acids
Arg, Gln, Ser, Asp, Cys, and Glu, which together con-
stitute only 3.6% of the tropoelastin sequence, this
study revealed that hardly any cleavage occurred
N-terminal to these amino acids upon digestion with
MMP-7, MMP-9, and MMP-12. An exception is a sin-
gle cleavage between Ala and Cys, which was found

1
(Table 2).
A
B
C
Fig. 3. Interaction of hexapeptide substrates with the binding
sites of MMP-7, MMP-9, and MMP-12. For clarity, only non-
conserved residues of the S
1
¢ pocket within the three
studied MMPs are shown. The zinc ion at the catalytic site is
shown as a yellow ball. (A) Interaction of the peptide substrate
PQGLAG containing a P
1
¢ Leu with the MMP-7 binding site. (B)
Interaction of the peptide substrate PQGKAG containing a P
1
¢
Lys with the MMP-9 binding site. (C) Interaction of the peptide
substrate PQGKAG containing a P
1
¢ Lys with the MMP-12
binding site.
Tropoelastin degradation by elastinolytic MMPs A. Heinz et al.
1944 FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS
Mainly Gly and Ala are found at P
2
–P
4
and P

3
¢ better than MMP-9 and
MMP-12.
Peptides with bioactive sequences released upon
proteolytic digestion of tropoelastin with MMP-7,
MMP-9 or MMP-12
Table 3 and Fig. 1 show that some of the 42 bioactive
sequences [17,19,46–65] partly overlap in tropoelastin.
Altogether, 35 of these sequences were found: 15
sequences, of which 11 were nonrepetitive, were found
in 27 peptides of different lengths released by MMP-7;
22 sequences, of which 11 were nonrepetitive,
were found in 23 peptides released by MMP-9; and 20
sequences, of which 13 were nonrepetitive, were
found in 41 peptides released by MMP-12 (Tables 3
and 4).
Table 2. Occurrence of different amino acids at the substrate positions P
1
-P
4
and P
1
¢ -P
4
¢ after digestion with MMP-7, MMP-9, and
MMP-12. Values are based on the number of cleavage sites identified on MS analysis of the digests.
P3‘
P4‘
A B
Fig. 4. Interaction of the natural substrate LPYGYGPG (residues 226–233 from tropoelastin isoform 2; see Fig. 1) and the MMP-12 active

MMP-12 and contained 9 partly overlapping bioactive
sequences (Tables 3 and 4).
It is worth mentioning that domain 24, which can be
considered as one huge matrikine encompassing 15 par-
tially overlapping bioactive peptides, remained intact
upon proteolytic digestion with MMP-7, MMP-9, and
MMP-12 (Fig. 1). An interesting finding is a peptide
that was released after digestion with MMP-9 and
spans the sequence 442–523 and so contains all the 15
bioactive sequences within domain 24 (Table 4). This
includes GLVPG and repeats of VGVAPG and
GVAPGV, which have been reported to show biologi-
cal effects [46–60], as well as further GxxPG sequences
(GIGPG and GLAPG). Because none of the three
MMPs was capable of cleaving within domain 24, it is
also likely that treatment with MMP-7 and MMP-12
resulted in additional peptides comprising the whole of
domain 24; however, these could not be identified upon
MS analysis.
Among the different peptides released by MMP-7,
MMP-9, and MMP-12, three contain GxxPG motifs
for which no biological activity has yet been described:
YTTGKLPYGYGPGG (residues 221-234, released by
MMP-7, MMP-9, and MMP-12), YGARPGVGVG-
GIP (residues 383–395, released by MMP-12), and
Table 4. (continued)
Fig. 5. Zymography analysis of pro-MMP-2
secretion. Cells were stimulated for 24 h
with or without elastin peptides, and cell
culture media were subjected to gelatin

has been reported to contain pockets of hydrophobic
clusters [72] that may be only partially solvent-accessi-
ble and hence restrict cutting in this area.
Cleavage site specificity
To gain a better understanding of the cleavage site
specificities of the three MMPs, the active sites were
modeled in complex with peptide substrates (Figs 3
and 4). Previous studies of the crystal structures of dif-
ferent MMPs have indicated that, in general, S
1
¢ pock-
ets are relatively large, although they differ in size and
shape among various MMPs depending on the amino
acids constituting the S
1
¢ loop which varies in second-
ary structure and length [2,5,73–76]. In the case of
MMP-7, Tyr214 has been found to extend into the
opening of the S
1
¢ pocket, restricting the size of the
binding site (Fig. 3A) [5]. Thus, the S
1
¢ pocket of
MMP-7 is less accessible to large polar or aromatic
residues such as Ser, Lys, Arg, Phe, and Tyr, whereas
it easily accommodates medium-sized hydrophobic res-
idues, including Leu and the even bulkier Ile
[5,44,45,76,77], as confirmed in this study (Tables 1
and 2; Fig. 2A). Both MMP-9 and MMP-12 contain

¢ pocket is restricted by the
bulky and flexible Tyr393, whereas in MMP-12 the
smaller Thr210 is observed at the same position, indi-
cating that MMP-12 can accept larger residues.
Overall, the MS findings (Tables 1 and 2; Fig. 2A)
and the modeled structures (Fig. 3) show that all three
MMPs can accommodate medium-sized aliphatic
and ⁄ or hydrophobic amino acids at P
1
¢, including Ala,
Gly, Pro, Val, Leu, and Ile, which is in agreement with
previous studies on the cleavage site specificities of
MMPs [10,37,38,43]. The differences in the cleavage
behavior of the three MMPs at charged or bulky aro-
matic amino acids such as Lys, Tyr, and Phe (Tables 1
and 2; Fig. 2A) result from differences in the second-
ary structures and amino acid compositions of the
active sites of the three MMPs (Fig. 3). As described
above, MMP-12 has a more polar and larger active
site than MMP-7 and MMP-9 and hence tolerates such
residues better. The low number or lack of cleavages
by all three MMPs N-terminal to large and polar
amino acids, including Arg, Gln, Ser, Asp, Cys, and
Glu, is most likely a result of unfavorable interactions
and may also be due to the fact that these amino acids
only exist in small numbers in tropoelastin and consti-
tute only 3.6% of the tropoelastin sequence.
A difference between the present and three previous
studies on elastin [10,37,38] concerns the amino acid
Lys. While Lys was identified as a relatively well

¢, which is
consistent with previous studies [10,38,44]. It has been
reported that Ala and Gly are found at P
1
for 75% of
the time when Leu or Ile is in P
1
¢ [10]. Furthermore, it
was found that the amino acids experimentally identi-
fied at P
1
¢ are, in most cases, preceded by Gly or Ala
[10]. Thus, the current and previous studies indicate
that the occurrence of a specific amino acid at P
1
might correlate with the total number of molecules of
the respective amino acid in tropoelastin and may also
be influenced by the amino acid at P
1
¢. As Ala and
Gly constitute more than 50% of the tropoelastin
sequence, it seems likely that the nature of the amino
acid at P
1
is governed not only by the amino acid pref-
erences of the elastinolytic enzyme at this position, but
also by the tropoelastin sequence itself. Furthermore,
the connection between P
1
¢ and P

2
pocket, as shown for Tyr (Fig. 4B, C) at P
2
, and can
also be seen from the experimental data (Tables 1
and 2).
Release of peptides containing bioactive
sequences
All three MMPs released peptides containing the
motifs GLGVGAGVP, PGAIPG, VPGVG, and
VGVPG, which have been reported to act as matrikin-
es by stimulating cell proliferation and showing
chemotactic activity on different cell types (Table 4)
[47,55,59,61–63]. Moreover, the bioactive sequence
VVPQ [64] was found in peptides after digestion with
MMP-7 and MMP-12. MMP-9 released a peptide con-
taining VGVAPG [46–60], GVAPGV [59] and their
repeats, which exhibit a range of biological activities,
in addition to a peptide containing bioactive GLVPG
[19,46]. Considering the fact that biological effects of
the above mentioned sequences have already been
reported, it is likely that the peptides containing these
sequences display bioactivity, provided that they are
liberated in vivo.
It has been suggested that the ability of EDPs to
exhibit biological activities is associated with their
structural conformation. In particular, GxxP sequences
in which x „ Gly are able to adopt a type VIII b-turn
conformation that enables the peptides to bind to the
EBP and thus show biological activities [19,61]. Most

significant increase in pro-MMP-2 secretion (Fig. 5). It
is important to note that all three peptides increased
pro-MMP-2 expression to the same extent, as no signif-
icant difference in bioactivity was observed between the
peptides. Overall, these results show that YTTGKL-
PYGYGPGG, YGARPGVGVGGIP and PGFGAV-
PGA can be considered as new matrikines that are
likely to be released by the action of elastinolytic
Tropoelastin degradation by elastinolytic MMPs A. Heinz et al.
1950 FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS
MMPs. The results of the bioactivity study confirm the
assumption that a variety of amino acids (in this case
Tyr, Ala, Gly, Arg, or Val) may occur in the x position
of active GxxPG motifs [19]. Interestingly, the peptide
YTTGKLPYGYGPGG, containing GYGPG, pos-
sesses a Gly in the x position and is nevertheless
biologically active. This could be explained by the
fact that the GYGPG motif is preceded by eight
amino acids that could facilitate the adoption of an
active conformation and its stabilization. This has
also been suggested by Moroy et al. [19] who showed
that a residue located before the GxxP motif increases
the stability of the bioactive type VIII b-turn confor-
mation.
With regard to the long peptide spanning more than
the entire length of domain 24 that was released by
MMP-9 and contains a variety of bioactive sequences,
it is likely that it has biological effects. As has previ-
ously been stated, the presence of multiple parts of a
peptide in the type VIII b-turn conformation increases

lacks domains encoded by the frequently spliced exons 22,
24A, and 26A (isoform 2) [83]. Recombinant MMP-7 and
MMP-12 expressed in Escherichia coli and full-length
human MMP-9 purified from human neutrophils were
obtained from Biomol (Plymouth Meeting, PA, USA). The
catalytic domains of MMP-7 and MMP-12 were used
because these are the biologically relevant forms of the pro-
teases. MMP-7, as the shortest member of the MMP
family, does not contain a hemopexin-like domain and acts
only with its catalytic domain, and MMP-12 is special in
that it autocatalytically loses its hemopexin-like domain
soon after activation without loss of its elastin-degrading
capacity [84]. Gelatin was purchased from Sigma (Saint-
Quentin Fallavier, France). All reagents for cell culture
were obtained from Gibco BRL (Invitrogen, Cergy
Pontoise, France). HPLC-grade acetonitrile (ACN) (VWR
Prolabo, Leuven, Belgium) and water purified by means of
a Millipore (Milford, MA, USA) filtration device (Milli-Q
grade) were used. Analytical-grade Tris, formic acid,
trifluoroacetic acid (TFA), sodium chloride, and calcium
chloride dihydrate were purchased from Merck (Darmstadt,
Germany).
Synthesis of peptides
The peptides YTTGKLPYGYGPGG, YGARPGVGVG-
GIP, and PGFGAVPGA were produced by solid-phase pep-
tide synthesis using 0.15 mm of preloaded Fmoc amino acid
Wang resins (NovaBiochem, La
¨
ufelfingen, Switzerland) on
a SYRO II multiple peptide synthesizer (MultiSynTech,

Proteolysis of tropoelastin
For proteolysis using the catalytic domains of either MMP-7
or MMP-12, recombinant tropoelastin was dissolved at a
concentration of 1 mgÆmL
)1
in 50 mm Tris containing
200 mm NaCl and 10 mm CaCl
2
at pH 7.5. The digestions
were performed for 4 h at 37 °C at an enzyme-to-substrate
ratio of 1 : 500 (m ⁄ m). Prior to proteolysis using MMP-9,
the enzyme was activated through incubation with 1.76 mm
aminophenylmercuric acetate for 1 h at 37 °C. Recombi-
nant tropoelastin was dissolved at a concentration of
1mgÆmL
)1
in 50 mm Tris containing 100 mm NaCl and
5mm CaCl
2
at pH 7.5, and subsequently digested with
activated MMP-9 at an enzyme-to-substrate ratio of
1 : 500 (m ⁄ m) for 4 h at 37 °C. Before MS analysis, all
samples were stored at )30 °C.
MALDI-TOF
⁄
TOF MS analysis
Analysis of the digests was conducted by offline nano-
HPLC ⁄ MALDI-TOF ⁄ TOF MS, using a nanoHPLC sys-
tem (Ultimate 3000; Dionex, Idstein, Germany) and an
Ultraflex III MALDI-TOF ⁄ TOF mass spectrometer

214 nm and 280 nm. Between 8 min and 75 min of the run,
191 fractions of the effluent (each of 21 s) were spotted,
together with a solution of MALDI matrix (1.1 lL per spot
in total; 0.71 mgÆmL
)1
a-cyano-4-hydroxy-cinnamic acid in
90% ACN ⁄ 10% H
2
O containing 0.1% TFA and 1 mm
NH
4
H
2
PO
4
), onto an AnchorChip MALDI target (384
spots; 800 lm anchors; Bruker Daltonik) using a Protein-
eerFC HPLC ⁄ MALDI fraction collector (Bruker Daltonik).
Mass spectra in the m ⁄ z range 740–5000 were acquired in
positive ionization mode and reflectron mode by accumulat-
ing data from 1800 laser shots per spot. Data acquisition was
performed by flex control 3.0.101.1, which was controlled
by warplc 1.1 (Bruker Daltonik). Ion signals with signal-to-
noise ratio higher than 10 were automatically subjected to
MALDI-LIFT-TOF ⁄ TOF MS ⁄ MS experiments by applying
2000 laser shots.
NanoHPLC–nanoESI–qTOF MS analysis
On-line nanoHPLC, nanoESI MS and MS ⁄ MS experiments
were conducted using an Ultimate nanoHPLC system (LC
Packings ⁄ Dionex) coupled to a quadrupole TOF (qTOF)

The tandem mass spectra obtained from qTOF experiments
were processed by masslynx (version 4.0; Waters ⁄ Micro-
mass) with the add-on Maximum Entropy 3 (maxent3). The
fragment ion spectra obtained in MALDI-TOF ⁄ TOF
MS experiments were analyzed using flex analysis 3.0.54
(Bruker Daltonik). All fragment ion peak lists were submit-
ted to a local mascot server (version 2.2.1; Matrix Science,
London, UK) [85]. The searches were taxonomically
restricted to ‘Homo sapiens’, the enzyme was set to ‘none’,
and the mass error tolerances for precursor and fragment
ions were typically set to 0.15 u for qTOF MS ⁄ MS and
50 p.p.m. and 1 u, respectively, for MALDI-TOF ⁄ TOF MS
data. The formation of pyroglutamic acid moieties from Glu
and Gln were considered as variable modifications.
Moreover, automated de novo sequencing of the nano-
ESI–qTOF and MALDI-TOF ⁄ TOF MS data followed by
database matching (peaks protein id and spider) [86] was
Tropoelastin degradation by elastinolytic MMPs A. Heinz et al.
1952 FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS
performed using the in-chorus search of the software
peaks studio (version 4.5; Bioinformatics Solutions,
Waterloo, Canada) [87] with precursor and fragment mass
error tolerances as described for mascot searches. The
enzyme entry was set to ‘unknown’, and the same post-
translational modifications as used for the mascot searches
were considered. For searching purposes, swissprot
extended by the splice variant of the used tropoelastin was
implemented.
Molecular modeling
All calculations were performed on a Pentium IV 2.6 GHz-

grown as monolayer cultures in Dulbecco’s modified Eagle’s
medium (DMEM) containing 1 gÆL
)1
glucose, glutamax I,
and pyruvate, supplemented with 10% fetal bovine serum in
the presence of 5% CO
2
. Cells at subcultures 4–8 were used.
For experiments, fibroblasts were grown to subconfluence in
the medium containing 10% fetal bovine serum. Before
stimulation, the cells were incubated for 18 h in DMEM
supplemented with 0.5% fetal bovine serum, washed twice
with NaCl ⁄ P
i
, and incubated in serum-free DMEM with or
without elastin peptides (200 lgÆmL
)1
) for 24 h. The cell
media were then harvested and centrifuged at 400 g to
eliminate cellular debris.
Gelatin zymography
To analyze pro-MMP-2 secretion from the conditioned
media of fibroblasts, equal amounts of proteins were ana-
lyzed using SDS ⁄ PAGE with 0.1% gelatin. After electro-
phoresis, the gels were soaked in 2.5% (v ⁄ v) Triton X-100
solution for 1 h to remove SDS. The gels were then incu-
bated in 50 mm Tris ⁄ HCl (pH 7.6) containing 5 mm CaCl
2
and 200 mm NaCl at 37 °C for 24 h. The gels were stained
with 0.1% (m ⁄ v) G250 Coomassie Brilliant Blue in a

3 Sternlicht MD & Werb Z (2001) How matrix metallo-
proteases regulate cell behavior. Annu Rev Cell Dev Biol
17, 463–516.
4 Nagase H & Woessner JF Jr (1999) Matrix metallopro-
teinases. J Biol Chem 274, 21491–21494.
5 Bode W (2003) Structural basis of matrix metallopro-
teinase function. Biochem Soc Symp 70, 1–14.
6 Verma RP & Hansch C (2007) Matrix metalloproteinas-
es (MMPs): chemical–biological functions and
(Q)SARs. Bioorg Med Chem 15 , 2223–2268.
7 Shipley JM, Doyle GAR, Fliszar CJ, Ye Q-Z, Johnson
LL, Shapiro SD, Welgus HG & Senior RM (1996)
A. Heinz et al. Tropoelastin degradation by elastinolytic MMPs
FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS 1953
The structural basis for the elastolytic activity of the
92-kDa and 72-kDa gelatinases. J Biol Chem 271,
4335–4341.
8 Filippov S, Caras I, Murray R, Matrisian LM,
Chapman HA Jr, Shapiro S & Weiss SJ (2003) Matrily-
sin-dependent elastolysis by human macrophages. J Exp
Med 198, 925–935.
9 Murphy G, Cockett MI, Ward RV & Docherty AJ
(1991) Matrix metalloproteinase degradation of elastin,
type IV collagen and proteoglycan. A quantitative com-
parison of the activities of 95 kDa and 72 kDa gelatin-
ases, stromelysins-1 and -2 and punctuated
metalloproteinase (PUMP). Biochem J 277, 277–279.
10 Mecham RP, Broekelmann TJ, Fliszar CJ, Shapiro SD,
Welgus HG & Senior RM (1997) Elastin degradation
by matrix metalloproteinases. Cleavage site specificity

Hematol 49, 199–202.
19 Moroy G, Alix AJP & He
´
ry-Huynh S (2005) Structural
characterization of human elastin derived peptides con-
taining the GXXP sequence. Biopolymers 78, 206–220.
20 Jung S, Rutka JT & Hinek A (1998) Tropoelastin and
elastin degradation products promote proliferation of
human astrocytoma cell lines. J Neuropathol Exp Neurol
57, 439–448.
21 Antonicelli F, Bellon G, Debelle L & Hornebeck W
(2007) Elastin-elastases and inflamm-aging. Curr Top
Dev Biol 79, 99–155.
22 Duca L, Blanchevoye C, Cantarelli B, Ghoneim C,
Dedieu S, Delacoux F, Hornebeck W, Hinek A, Marti-
ny L & Debelle L (2007) The elastin receptor complex
transduces signals through the catalytic activity of its
Neu-1 subunit. J Biol Chem 282, 12484–12491.
23 Hinek A, Rabinovitch M, Keeley F, Okamura-Oho Y
& Callahan J (1993) The 67-kD elastin ⁄ laminin-binding
protein is related to an enzymatically inactive, alterna-
tively spliced form of beta-galactosidase. J Clin Invest
91, 1198–1205.
24 Privitera S, Prody CA, Callahan JW & Hinek A (1998)
The 67-kDa enzymatically inactive alternatively spliced
variant of beta-galactosidase is identical to the elas-
tin ⁄ laminin-binding protein. J Biol Chem 273, 6319–
6326.
25 Mecham RP, Hinek A, Entwistle R, Wrenn DS, Griffin
GL & Senior RM (1989) Elastin binds to a multifunc-

32 Hautamaki RD, Kobayashi DK, Senior RM & Shapiro
SD (1997) Requirement for macrophage elastase for cig-
arette smoke-induced emphysema in mice. Science 277,
2002–2004.
33 Chung JH, Seo JY, Lee MK, Eun HC, Lee JH, Kang
S, Fisher GJ & Voorhees JJ (2002) Ultraviolet modula-
tion of human macrophage metalloelastase in human
skin in vivo. J Invest Dermatol 119, 507–512.
34 Vaalamo M, Kariniemi A-L, Shapiro SD & Saarialho-
Kere U (1999) Enhanced expression of human metallo-
elastase (MMP-12) in cutaneous granulomas and mac-
rophage migration. J Invest Dermatol 112, 499–505.
Tropoelastin degradation by elastinolytic MMPs A. Heinz et al.
1954 FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS
35 Wielockx B, Libert C & Wilson C (2004) Matrilysin
(matrix metalloproteinase-7): a new promising drug
target in cancer and inflammation? Cytokine Growth
Factor Rev 15, 111–115.
36 Nagashima Y, Hasegawa S, Koshikawa N, Taki A,
Ichikawa Y, Kitamura H, Misugi K, Kihira Y, Matuo
Y, Yasumitsu H et al. (1997) Expression of matrilysin
in vascular endothelial cells adjacent to matrilysin-pro-
ducing tumors. Int J Cancer 72, 441–445.
37 Taddese S, Weiss AS, Neubert RHH & Schmelzer CEH
(2008) Mapping of macrophage elastase cleavage sites
in insoluble human skin elastin. Matrix Biol 27, 420–
428.
38 Barroso B, Abello N & Bischoff R (2006) Study of
human lung elastin degradation by different elastases
using high-performance liquid chromatography ⁄ mass

Traniello S, Spisani S & Tamburro AM (1994) Migra-
tion of monocytes in the presence of elastolytic
fragments of elastin and in synthetic derivates.
Structure–activity relationships. Int J Pept Protein Res
44, 332–341.
47 Castiglione Morelli MA, Bisaccia F, Spisani S, De Biasi
M, Traniello S & Tamburro AM (1997) Structure–activ-
ity relationships for some elastin-derived peptide chemo-
attractants. J Pept Res 49, 492–499.
48 Hance KA, Tataria M, Ziporin SJ, Lee JK &
Thompson RW (2002) Monocyte chemotactic activity
in human abdominal aortic aneurysms: role of elastin
degradation peptides and the 67-kD cell surface elastin
receptor. J Vasc Surg 35, 254–261.
49 Senior R, Griffin G, Mecham R, Wrenn D, Prasad K &
Urry D (1984) Val-Gly-Val-Ala-Pro-Gly, a repeating
peptide in elastin, is chemotactic for fibroblasts and
monocytes. J Cell Biol 99, 870–874.
50 Kamoun A, Landeau J-M, Godeau G, Wallach J,
Duchesnay A, Pellat B & Hornebeck W (1995) Growth
stimulation of human skin fibroblasts by elastin-derived
peptides. Cell Commun Adhes 3, 273–281.
51 Tajima S, Wachi H, Uemura Y & Okamoto K (1997)
Modulation by elastin peptide VGVAPG of cell prolif-
eration and elastin expression in human skin fibroblasts.
Arch Dermatol Res
289, 489–492.
52 Mochizuki S, Brassart B & Hinek A (2002) Signaling
pathways transduced through the elastin receptor facili-
tate proliferation of arterial smooth muscle cells. J Biol

Hornebeck W et al. (2001) Conformational dependence
of collagenase (matrix metalloproteinase-1) up-regula-
tion by elastin peptides in cultured fibroblasts. J Biol
Chem 276, 5222–5227.
60 Faury G, Garnier S, Weiss AS, Wallach J, Fu
¨
lo
¨
pT,
Jacob M-P, Mecham RP, Robert L & Verdetti J (1998)
Action of tropoelastin and synthetic elastin sequences
A. Heinz et al. Tropoelastin degradation by elastinolytic MMPs
FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS 1955
on vascular tone and on free Ca
2+
level in human vas-
cular endothelial cells. Circ Res 82, 328–336.
61 Fuchs P, Debelle L & Alix AJP (2001) Structural study
of some specific elastin hexapeptides activating MMP1.
J Mol Struct 565–566, 335–339.
62 Lin S-Y, Hsieh T-F & Wei Y-S (2005) pH- and ther-
mal-dependent conformational transition of PGAIPG, a
repeated hexapeptide sequence from tropoelastin.
Peptides 26, 543–549.
63 Wachi H, Seyama Y, Yamashita S, Suganami H, Uemura
Y, Okamoto K, Yamada H & Tajima S (1995) Stimula-
tion of cell proliferation and autoregulation of elastin
expression by elastin peptide VPGVG in cultured chick
vascular smooth muscle cells. FEBS Lett 368, 215–219.
64 Spezzacatena C, Pepe A, Green LM, Sandberg LB,

72 Muiznieks LD & Weiss AS (2007) Flexibility in the
solution structure of human tropoelastin. Biochemistry
46, 8196–8205.
73 Terp GE, Cruciani G, Christensen IT & Jorgensen FS
(2002) Structural differences of matrix metalloproteinas-
es with potential implications for inhibitor selectivity
examined by the GRID ⁄ CPCA approach. J Med Chem
45, 2675–2684.
74 Rowsell S, Hawtin P, Minshull CA, Jepson H, Brock-
bank SMV, Barratt DG, Slater AM, McPheat WL,
Waterson D, Henney AM et al. (2002) Crystal structure
of human MMP9 in complex with a reverse hydroxa-
mate inhibitor. J Mol Biol 319, 173–181.
75 Massova I, Kotra LP, Fridman R & Mobashery S
(1998) Matrix metalloproteinases: structures, evolution,
and diversification. FASEB J 12, 1075–1095.
76 Browner MF, Smith WW & Castelhano AL (1995)
Crystal structures of matrilysin–inhibitor complexes.
Biochemistry 34, 6602–6610.
77 Wilson CL & Matrisian LM (1996) Matrilysin: an epi-
thelial matrix metalloproteinase with potentially novel
functions. Int J Biochem Cell Biol 28, 123–136.
78 Kozel BA, Wachi H, Davis EC & Mecham RP
(2003) Domains in tropoelastin that mediate elastin
deposition in vitro and in vivo. J Biol Chem 278,
18491–18498.
79 Deng SJ, Bickett DM, Mitchell JL, Lambert MH,
Blackburn RK, Carter HL, Neugebauer J, Pahel G,
Weiner MP & Moss ML (2000) Substrate specificity of
human collagenase 3 assessed using a phage-displayed

Kirby A & Lajoie G (2003) PEAKS: powerful software
for peptide de novo sequencing by tandem mass spec-
trometry. Rapid Commun Mass Spectrom 17, 2337–2342.
88 Case DA, Cheatham TE III, Darden T, Gohlke H, Luo
R, Merz KM Jr, Onufriev A, Simmerling C, Wang B &
Woods RJ (2005) The Amber biomolecular simulation
programs. J Comput Chem 26, 1668–1688.
Tropoelastin degradation by elastinolytic MMPs A. Heinz et al.
1956 FEBS Journal 277 (2010) 1939–1956 ª 2010 The Authors Journal compilation ª 2010 FEBS