The International Journal of Biochemistry & Cell Biology 41 (2009) 494497

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The International Journal of Biochemistry
& Cell Biology
j our nal homepage: www. el sevi er . com/ l ocat e/ bi ocel
Molecules in focus
Tropoelastin
Steven G. Wise, Anthony S. Weiss

School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia
a r t i c l e i n f o
Article history:
Received 27 February 2008
Received in revised form 25 March 2008
Accepted 26 March 2008
Available online 1 April 2008
Keywords:
Tropoelastin
Elastin
Extracellular matrix
Biomaterial
a b s t r a c t
Tropoelastin is a 6072kDa alternatively spliced extracellular matrix protein and a key
component of elastic bres. It is found in all vertebrates except for cyclostomes. Secreted
tropoelastin is tethered to the cell surface, where it aggregates into organised spheres for
cross-linking and incorporation into growing elastic bres. Tropoelastin is characterised by
alternating hydrophobic and hydrophilic domains and is highly exible. The conserved C-
terminus is an area of the molecule of particular biological importance in that it is required
for both incorporation into elastin and for cellular interactions. Mature cross-linked tropoe-
lastin gives elastin, which confers resilience and elasticity on a diverse range of tissues.
Elastin gene disruptions in disease states and knockout mice emphasise the importance
of proper tropoelastin production and assembly, particularly in vascular tissue. Tropoe-
lastin constructs hold promise as biomaterials as they mimic many of elastins physical and
biological properties with the capacity to replace damaged elastin-rich tissue.
2008 Elsevier Ltd. All rights reserved.
1. Introduction
Elastic bres are major extracellular matrix assemblies
that provide elasticity and resilience to a range of tis-
sues. They consist of two main protein components, elastin
and microbrils. Elastin, in turn is formed following the
assembly and cross-linking of its soluble precursor, tropoe-
lastin. The complex nature of elastic bre assembly has
been reviewed more broadly elsewhere (Wagenseil and
Mecham, 2007).
Historically, elastin and tropoelastin have been difcult
to isolate for study. Both were harvested from a variety
of animal sources particularly bovine, porcine, human and
equine elastin-rich tissues such as ligament and aorta.
Elastin was rst puried and characterised in 1952 from
humanaorta and horse ligamentumnuchae. Elastinextrac-
tion techniques were reviewed by Daamen et al. (2001).
Obtaining the tropoelastin monomer from animal sources
requires the perturbation of the natural cross-linking pro-

Corresponding author. Tel.: +61 2 9351 3464; fax: +61 2 9351 5858.
E-mail address: a.weiss@mmb.usyd.edu.au (A.S. Weiss).
cess, mediated by the family of copper-requiring lysyl
oxidase enzymes (Lucero andKagan, 2006) andso achieved
using animals with a copper decient diet or treated with
a chemical inhibitor, though both means were found to be
highly inefcient.
Given the difculty in obtaining large quantities of
tropoelastin, researchers have studied hydrolysed, soluble
elastin; recombinant tropoelastin fragments; and elastin-
like peptides as discussed by Daamen et al. (2007). These
systems provide an approximation of the structure and
assembly of tropoelastin. However, the expression of full-
length tropoelastin, and its various isoforms in a bacterial
system have made it possible to access gram quantities
of puried, uncross-linked material, allowing for signi-
cant increase in tropoelastin research (Mithieux and Weiss,
2005).
2. Structure
A comprehensive review of the tropoelastin gene and
amino acid sequence across several species was described
by Chung et al. (2006). Human tropoelastin is encoded by a
single gene that possesses 34 exons and gives rise to mul-
1357-2725/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocel.2008.03.017
S.G. Wise, A.S. Weiss / The International Journal of Biochemistry & Cell Biology 41 (2009) 494497 495
Fig. 1. Domain map of human tropoelastin containing all possible exons. Hydrophilic cross-linking domains are further divided into KP- and KA-rich
regions. Alternative splicing is a hallmark of tropoelastin biosynthesis. Exons subject to alternative splicing are marked in bold outline. Domain 36 is
assigned differently because of its unique structural features.
tiple isoforms. The mRNA encodes a 72kDa polypeptide
depending on the splicing pattern and removal of a signal
peptide which leaves a mature protein with a molecular
weight of at least 60kDa. At least 11 human tropoelastin
splice forms have been characterised, resulting fromdevel-
opmentally regulated alternative splicing of domains 22,
23, 24, 26A, 32 and 33 (Fig. 1).
The tropoelastin amino acid sequence is divided into
two major domain types: hydrophilic and hydropho-
bic domains. The hydrophobic domains are rich in the
non-polar residues glycine, valine and proline, typically
occurring in repeating motifs. Alternating with these
domains arehydrophilic regions characterisedbytheir high
lysine and alanine content and their involvement in cross-
linking. At the amino acid level tropoelastin has greater
than 70% similarity when sequences are globally aligned
across a range of vertebrate mammals (Piontkivska et al.,
2004). Subtle evolutionary substitutions of similar amino
acids can be seen that would not be expected to dras-
tically alter the structure of the protein and to maintain
overall hydrophobicity. Sequences of important structural
features such as splice junctions and cross-linking regions
are strictly preserved, while repeating hydrophobic motifs
have more exibility and variation.
One of the most important and well-conserved regions
in tropoelastin is the C-terminus. It contains two char-
acteristic features: the only two cysteine residues in the
molecule are closely spaced in the nal domain 36 and
form a disulphide bond, while the protein terminates with
a positively charged RKRK sequence. The disruption of the
disulphide bond dramatically reduces the ability of tropoe-
lastin to assemble and be included in developing bres,
as does the removal of the C-terminal region (Kozel et al.,
2003).
The tertiary structure of the expressed protein has not
yet been denitively elucidated, given that no crystal struc-
ture information has been obtained for the full-length
monomer. However, there have been signicant investiga-
tions into the secondary structure of tropoelastin and its
derivatives (Pepe et al., 2007). In solution, it can exist in
either a globular or elongated form and has a high degree
of exibility. Analysis of individual domains of tropoe-
lastin reveal secondary structure features that include
poly-proline II, compact -turns and disordered structure,
in agreement with the view of a highly exible elastic pro-
tein (Muiznieks and Weiss, 2007).
3. Expression, activation and turnover
Tropoelastin is secreted fromelastogenic cell types, pri-
marilyvascular smoothmusclecells anddiversebroblasts.
The protein is proposed to be chaperoned to the cell sur-
face by a 67kDa protein, termed the elastin binding protein
(EBP), proposed to be important in elastic bre production
(Hinek et al., 2004). Tropoelastin self assembles into con-
sistent globules of several microns in diameter that remain
tethered to the cell surface. This early microassembly stage
is likely to be the rst commitment in elastic bre forma-
tion, followed by further, microbril directed aggregation
(Kozel et al., 2006).
After deposition, tropoelastin production is substan-
tiallyreducedandthereis verylittleturnover of themature,
cross-linked form of the eventual elastin. The majority of
elastin formation in mammals occurs during late fetal and
in early neonatal periods, and at maturity the production
of new elastin ceases (Swee et al., 1995). In the event
of injury, the production of tropoelastin can be quickly
restarted and is inuenced by diverse exogenous factors
such as tumour necrosis factor-, interleukin 1, insulin-
like growth factor-1 and strongly by transforming growth
factor (Pierce et al., 2006).
A very specic set of proteases, broadly grouped under
the name elastases are responsible for elastin remodelling.
The major classes of elastases, their mechanism of action
and their pathogenesis were reviewed by Antonicelli et al.
(2007). The matrix metalloproteinases (MMPs) are partic-
ularly important in elastin breakdown, with MMP-2, -3,
-9 and -12 explicitly shown to degrade elastin. A detailed
discussion of MMP activity, regulation and substrates was
presented by Ra and Parks (2007).
4. Biological function
4.1. Coacervation and assembly
Tropoelastin undergoes rapid ordered assembly and
cross-linkingintoelastinfollowingexcretionintothe extra-
cellular space. Tropoelastin monomers assemble in a self
496 S.G. Wise, A.S. Weiss / The International Journal of Biochemistry & Cell Biology 41 (2009) 494497
Fig. 2. Schematic representation of elastogenesis. (A) Secreted tropoelastin is tethered to the cell surface creating sites for further aggregation. (B) Discreet
packages of tropoelastin are released for further microbril directed assembly, and undergo lysine oxidation which facilitates cross-linking. (C) Tropoelastin
is incorporated into growing elastic bres. The diagram is not drawn to scale. Adapted from Mithieux and Weiss (2005).
association process called coacervation, aligning and con-
centrating the protein into quantized spheres, prior to
cross-linking. Between31and32

C, the15nmmonomers
coalesce intopackages that are spherically26mindiam-
eter (Clarke et al., 2006). Coacervation is optimal at 37

C,
in a pH range of 78 and in physiological salt conditions.
Following coacervation, tropoelastinplays animportant
role in generating the elastic bre. The coacervate package
remains tethered to the cell surface, collecting tropoelastin
until its release to the nascent elastic bre. The specic
binding interaction between tropoelastin and cell surface
has not been fully illuminated, though possible binding
partners include integrins and glycosaminoglycans on the
cell surface and possibly additional non-integrin proteins
(Broekelmann et al., 2005). The delivered packages of accu-
mulated tropoelastin are xed in place by cross-linking,
initiated through the action of a lysyl oxidase, deposited
ontomicrobrils andthenincorporatedintomaturing elas-
tic bres (Sato et al., 2007) (Fig. 2).
4.2. Cross-linked tropoelastin
The cross-linked protein as elastin is responsible for the
elasticityof multipletissues impartingresilienceandrecoil.
It is prevalent in several tissues, constituting up to 57% of
the aorta, 50% of elastic ligaments, 32% of major vascular
vessels, 7%of lungand5%of thedryweight of skin(Vrhovski
and Weiss, 1998). Elastin is an extremely durable and insol-
uble biopolymer that does not turn over appreciably in
healthy tissue.
Mice with a disrupted elastin gene provide an impor-
tant insight into the function of the protein. Heterozygous
(ELN +/) mice demonstrated a decreased level of arte-
rial compliance and an increase in hypertension, but had
a normal life expectancy. Arterial remodelling, in response
to the reduced amount of elastin was suggested to explain
the reduced physical performance of these vessels. ELN+/
micealsodisplayedalteredagingprocesses intheaorta, sig-
nalling that early elastin arrangement determines the way
the vessel evolves (Pezet et al., 2008). The total absence of
elastin (ELN /) results in the early death of mice from
uncontrolled proliferation of smooth muscle cells causing
arterial obstruction (Li et al., 1998). Together these stud-
ies demonstrate the dual roles of elastin: mechanical and
physical properties coupled with cell regulation. The dra-
matic effects on vasculature in both instances emphasise
the importance of elastin to these tissues in particular.
5. Possible medical applications
Abnormalities in the elastin gene are known to be
responsible for several genetic disorders, disrupting the
architecture and function of elastin-rich tissues. Mutations
and truncations of the C-terminal end of tropoelastin are
particularly severe, resulting in serious disease states par-
ticularly supravalvular aortic stenosis (SVAS) and cutis laxa
(CL). SVAS is characterised by arterial narrowing, a reduc-
tion in vascular elastin content and order, while CL patients
have loose, sagging skin (Milewicz et al., 2000).
Additionally, uncontrolled protease activity causing
destruction of elastin has been implicated in the devel-
opment of several disease states including emphysema,
rheumatoid arthritis, and cystic brosis. It is also symp-
tomatic in about approximately half of acquired CL cases,
frequently leading to systemic elastolysis and causing aor-
tic rupture(Huet al., 2006). Proteaseinhibitor development
S.G. Wise, A.S. Weiss / The International Journal of Biochemistry & Cell Biology 41 (2009) 494497 497
to combat these conditions has been the subject of consid-
erable research (Leung et al., 2000).
Currently, the most promising commercial application
of tropoelastin is in its use as a biomaterial. Derivatives of
natural elastin, tropoelastin fragments and recombinantly
produced variants have been engineered to potentially
replace elastin-rich tissues such as skin dermis and vascu-
lature. Potential skin substitutes seek to take advantage of
the increased elasticity, improved cellular interactions and
enhanced tissue regeneration observed for tropoelastin-
based biomaterials. As a vascular substitute elastinbenets
from being able to support new matrix synthesis and to be
endothelialised, while providing the physical strength and
recoil required of these vessels (Daamen et al., 2007).
References
Antonicelli F, Bellon G, Debelle L, Hornebeck W. Elastin-elastases and
inamm-aging. Curr Top Dev Biol 2007;79:99155.
Broekelmann TJ, Kozel BA, Ishibashi H, Werneck CC, Keeley FW, Zhang L,
Mecham RP. Tropoelastin interacts with cell-surface glycosaminogly-
cans via its C-terminal domain. J Biol Chem 2005;280:4093947.
Chung MI, Miao M, Stahl RJ, Chan E, Parkinson J, Keeley FW. Sequences
and domain structures of mammalian, avian, amphibian and teleost
tropoelastins: clues to the evolutionary history of elastins. Matrix Biol
2006;25:492504.
Clarke AW, Arnspang EC, Mithieux SM, Korkmaz E, Braet F, Weiss AS.
Tropoelastin massively associates during coacervation to form quan-
tized protein spheres. Biochemistry 2006;45:998996.
Daamen WF, Hafmans T, Veerkamp JH, Van Kuppevelt TH. Comparison of
ve procedures for the purication of insoluble elastin. Biomaterials
2001;22:19972005.
Daamen WF, Veerkamp JH, van Hest JC, van Kuppevelt TH. Elastin as a
biomaterial for tissue engineering. Biomaterials 2007;28:437898.
Hinek A, Braun KR, Liu K, Wang Y, Wight TN. Retrovirally mediated
overexpression of versican v3 reverses impaired elastogenesis and
heightened proliferation exhibited by broblasts from Costello syn-
drome and Hurler disease patients. Am J Pathol 2004;164:11931.
HuQ, ReymondJL, Pinel N, Zabot MT, UrbanZ. Inammatory destructionof
elastic bers in acquired cutis laxa is associated with missense alleles
in the elastin and bulin-5 genes. J Invest Dermatol 2006;126:28390
(erratum appears in J Invest Dermatol 2006;126(Jun(6)):1426).
Kozel BA, Rongish BJ, Czirok A, Zach J, Little CD, Davis EC, Knutsen RH,
Wagenseil JE, Levy MA, MechamRP. Elastic ber formation: a dynamic
view of extracellular matrix assembly using timer reporters. J Cell
Physiol 2006;207:8796.
Kozel BA, Wachi H, Davis EC, Mecham RP. Domains in tropoelastin
that mediate elastin deposition in vitro and in vivo. J Biol Chem
2003;278:184918.
Leung D, Abbenante G, Fairlie DP. Protease inhibitors: current status and
future prospects. J Med Chem 2000;43:30541.
Li DY, Brooke B, Davis EC, Mecham R, Sorenson LK, Boak BB, Eichwald E,
Keating MT. Elastin is an essential determinant of arterial morphogen-
esis. Nature 1998;393:27680.
Lucero HA, Kagan HM. Lysyl oxidase: an oxidative enzyme and effector of
cell function. Cell Mol Life Sci 2006;63:230416.
Milewicz DM, UrbanZ, BoydC. Genetic disorders of theelastic ber system.
Matrix Biol 2000;19:47180.
Mithieux SM, Weiss AS. Elastin. Adv Protein Chem 2005;70:43761.
Muiznieks LD, Weiss AS. Flexibility in the solution structure of human
tropoelastin. Biochemistry 2007;46:8196205.
Pepe A, Bochicchio B, Tamburro AM. Supramolecular organiza-
tion of elastin and elastin-related nanostructured biopolymers.
Nanomedicine 2007;2:20318.
Pezet M, Jacob MP, Escoubet B, Gheduzzi D, Tillet E, Perret P, Huber P,
Quaglino D, Vranckx R, Li DY, Starcher B, Boyle WA, MechamRP, Faury
G. Elastin haploinsufciency induces alternative aging processes in
the aorta. Rejuvenation Res 2008;11:97112.
Pierce RA, Moore CH, Arikan MC. Positive transcriptional regulatory ele-
ment located within exon 1 of elastin gene. AmJ Physiol Lung Cell Mol
Physiol 2006;291:L3919.
Piontkivska H, Zhang Y, Green ED, Elnitski L. Multi-species sequence
comparison reveals dynamic evolution of the elastin gene that has
involved purifying selection and lineage-specic insertions/deletions.
BMC Genomics 2004;5:31.
Ra HJ, Parks WC. Control of matrix metalloproteinase catalytic activity.
Matrix Biol 2007;26:58796.
Sato F, Wachi H, Ishida M, Nonaka R, Onoue S, Urban Z, Starcher BC,
Seyama Y. Distinct steps of cross-linking, self-association, and mat-
uration of tropoelastin are necessary for elastic ber formation. J Mol
Biol 2007;369:84151.
Swee MH, Parks WC, Pierce RA. Developmental regulation of elastin
production. Expression of tropoelastin pre-mRNA persists after
down-regulation of steady-state mRNA levels. J Biol Chem
1995;270:14899906.
Vrhovski B, Weiss A. Biochemistry of tropoelastin. Eur J Biochem
1998;258:118.
Wagenseil JE, Mecham RP. New insights into elastic ber assembly. Birth
Defects Res (Part C) 2007;81:22940.