CSB327 Lecture 18 Notes – Elastic fibers and tissue resilience (December 3, 2012)
1 – Lecture topics
• Elastin is broadly distributed in tissues.
3 – Artery
• Do not memorize this structure.
• In an artery, they are all lined with endothelium followed with BL then elastin.
• Smooth muscle cells are contractile. If they are disregulated, they can form MVs and
begin hardening of the arteries.
• The precision of the assembly of elastic fibers has to be tightly controlled.
4 – Internal elastic lamina of murine aorta, with associated endothelial cell and smooth muscle
• How is elastin assembled and regulated?
• Elastic fibers are abundant. Elastic fibers are a composite of more than just elastin.
• Where you have high hydrostatic pressure, you will have a lot of elastin. The strength of
the skin is based on the collagen composites.
5 – A network of elastic fibers
• It looks like fibers that are interconnected. If you stretch a rubber band, there is some
cross-linking that enables them to stay as a polymer. What makes the rubber band like
property of elastin?
• Elastin can be purified to a homogenous state. Why is that? Why is elastin easy to
purify? Elastin is hard to study because of its high resilience and stability and amorphous
• The pores allow oxygen and nutrient diffusion, since it is not vascularised.
• They are usually oriented in the direction in which the force is pulling. In this case, it is
6 – TEM of microfibrils and elastin by cultured bovine nuchal ligament fibroblast cultures
• The half life of elastin is 70 years. There are enzymes that can remodel elastin. You can
damage elastin. Exposure to UV light will promote damage to elastin network.
• It is the most stable biomaterial in our body. It does breakdown with age due to
remodelling, oxidation, UV damage, etc.
• Elastin or tropoelastin does not assemble into elastic fibers without the help of other
molecules. The process of tropoelastin to elastic fibers is dependent on the regulation
by other assorted proteins such as fibrillin-rich microfibrils. These set the template onto
which tropoelastin molecules are assembled. however, tropoelastin can self-polymerize.
7 – Light and electron microscopial staining of bovine aorta and ligament
• You can see the individual elastin fibers.
• Elastin has elasticity and tissue resilience. • Collagens prevent overstretching of elastin. Our vascular network does not keep
expanding outward. As a composite material of elastin and collagen, the collagen give
tensile strength and the elastin gives resilience.
8 – Stretching a network of elastin molecules
• Why is it that elastin does not need energy during expansion and recoil? This has to do
with entropy. Elastin fibers are a composite of glycoproteins and elastin.
• Elastin recoiling and extension is driven by entropy.
• How is it cross-linked? What is unique about the cross-linking?
• The stretched state is decreased entropy. The water molecules around elastin molecules
are more ordered. They are less ordered when you have a collapsed network. This
drives the recoiling back to the native state without the use of energy.
• In insects, resilin is structurally similar to elastin.
9 – Inverse temperature solubility
• There is no further processing of tropoelastin except for the removal of the signal
peptide. When it is assembled into a polymer, it is called elastin. The monomer that
makes the elastic fibers is not processed any further than the removal of signal peptide.
• Coacervation is taking a molecule and forming small droplets (e.g., it is undergoing
• Under the right pH and conditions, you get an emulsion. The small spherical tropoelastin
• If I heat up the solution, you will get a gelatinous material. This is called inverted
temperature transition as opposed to solubilising it into the aqueous solution by heating
it up. By heating up, you are forcing it out of solution into spherical hydrophobic
• Tropoelastin is hydrophobic and has a lot of alpha helical regions that are hydrophilic. As
a hydrated polymer, the water molecules are more organized.
• This illustrates the recoiling without energy input because it is driven by entropy. Water
molecules surrounding the tropoelastin in an ordered fashion. When you go through
coacervation and the tropoelastin molecules collapse onto themselves, you have higher
disorder in the water molecule, hence driven by higher entropy.
• This is an inverted temperature transition. This promotes its collapse into a phase
• Elastic recoil can occur without energy.
10 - Tropoelastin
• Tropoelastin is made by a single copy gene. There is no evidence of glycosylation. The
few glycosylation events that you do see are a result of aberrant glycosylation and has
no biological importance.
• Tropoelastin has repeated hydrophobic domains and crosslinking domains. This
molecule can stick to a variety of things. They can interact with fibrillin-1/2.
• Tropoelastin’s appearance is coincident with the formation of a closed circulatory
system. If you look at primitive organisms, they will have molecules that look similar and can have elastic properties. In some cases, even though they look like elastin, they are
purely structural. Often, we classify elastin as part of the non-collagenous structural
11 – Amino acid composition of tropoelastin
• In the tropoelastin molecule, there is no Met residues.
• In the Met cross-linking, the molecule cannot be broken down by CnBr.
• Lys is important in cross-linking by LOX.
12 – Modular organization of tropoelastin
• You have hydrophobic domains. You have a planar arrangement with the R groups
sticking out of the plane.
• The placement of the Lys residues is critical in the design of the molecule. It is a strategy
• If you raise the temperature, it can coacervate and come out of phase.
13 – Hydrophobic repeats
• The hydrophobic domains drive the coacervation process that are coming out of
solution as you increase the temperature or increase the salts.
• These are sequences that are found in many of the vertebrates.
• You have repeated segments of hydrophobic sequences.
• The hydrophobic stretches are important in the assembly of a collagen molecule.
14 – Cross-sectional view of the cross-linking region of elastin
• If you look at the R groups sticking out of an alpha helix, you need the two lysine
residues in proximity on the same side of the helix.
• The Lys residues are separated by Ala residues in the cross-linking domain.
• You never find 1-Ala and 4-Ala in elastin because the Lys residues are too far away.
• Their proximity is critical for cross-linking.
• You can have 2 or 3 Ala residue separating each Lys residue, but not 1 or 4.
15 – Crosslinking between adjacent α-helices
• You set the stage for a special type of cross-linking that gets started with LOX.
• You have a tetrad of Lys molecules. LOX will start forming cross-links that are special to
elastin and extremely stable.
• You have an abundance of cross-linked tropoelastin molecules that form a stable cross-
linked product, using four Lys residues.
16 – Structure of desmosine and isodesmosine tetrafunctional cross-links of elastin
• You start forming tetra-cross-linked desmosine or isodesmosine. This is 4 Lys residues.
This get started by LOX, then you can have spontaneous formation of desmosine and
• These are tetra cross-links. There are also bi and tri cross-links, but not as abundant. • This is extremely stable. This gets started by LOX. This is unique to elastin. It all gets
done because of the Lys residues ordered in a way to promote the formation of
18 – Postulated alignment of elastin peptides during coacervation juxtaposing side chains of
lysine residues for the formation of covalent crosslinks
• You need to align the alpha helix of the Lys residues. You also need to make sure that
adjacent tropoelastin molecules are aligned for LOX to cross-link. (You need to ilne up
the 4 Lys residues promoted by hydrophobic-hydrophobic interaction.)
• This is driven by the hydrophobic residues stacking on top of each other. As a result, you
align the cross-linking repeats so they can be properly cross-linked.
• At a state below 200°C, you have no water, you have no physiological conditions, if you
have pure tropoelastin, then it is brittle. When you hydrate it, you drop down to 30%.
Then you can add salts and coacervation can occur below our body temperature.
o This molecule is useless unless we hydrate it.
19 – Model of the elastin-binding protein (EBP) complex
• Purified tropoelastin at the right salts and right temperature will coacervate. If the cells
are making a lot of tropoelastin, this should be lethal inside our cells. To make sure that
it doesn’t coacervate inside a secretory vesicle, but when you get outside of a cell at
physiological temperature, it can coacervate.
• You keep it from coacervating inside the cell. When the secretory vesicle secretes the
tropoelastin, it is capable of undergoing coacervation and phase separation.
• Inside the secretory vesicle, membrane bound proteins bind to tropoelastin (e.g., 67
kDa). When it is in the bound state, tropoelastin cannot undergo coacervation. There
are two binding sites on 67 kDa. There is no galactose sugar inside secretory vesicles.
Fibrillin is decorated with galactose sugar on the outside of the cell. When the secretory
vesicle is outside, it encounters an environment that is rich in galactose sugars. The
galactose sugar binds, forces a conformational change, which releases tropoelastin.
• You can release tropoelastin by incubating the cells with galactose in vitro.
20 – Model of the elastin-binding protein (EBP) complex
• The hydrophobic domains are key to coacervation. They are key to the alignment of
tropoelastin molecules so they will be cross-linked by LOX extracellularly.
21 – Release of tropoelastin at the plasma membrane
• You get the release of this. The galactose sugars bound to fibrillin releases tropoelastin.
• Fibrillin is not just released tropoelastin. It creates a scaffold onto which tropoelastin
will be deposited to form elastin fibers. Elastic fibers are a composite material of
tropoelastin and glycoprotein, not just tropoelastin forming elastin fibers.
• Using a glycoprotein network as a scaffold to ensure the orientation of the fibers and
ensure coacervation occurs in a rapid and efficient manner. • Coacervation is the falling out of solution and the aggregation of tropoelastin molecules.
The coacervation process is driven by the hydrophobic domain and losing the water
shell. You go to higher disorder with respect to the water. The loss of water organization
undergoes phase separation.
• The tropoelastin is trapped on a receptor complex inside the cell. There are two binding
sites for the receptor