WE ARE all used to the idea that structures like buildings and boats are made
of materials strengthened by fibres—take reinforced concrete and
fibreglass for example. But it’s easy to forget that nature had the idea first,
and without fibres life itself would literally be a complete flop.
While plants rely on cellulose—a fibrous carbohydrate—to prop
themselves up and keep their shape, most multicellular animals use a protein
called collagen as their structural scaffolding. Collagen is an ingredient of
many connective tissues, including bone, tendons, cartilage, and skin. It is
also a key constituent of specialised membranes that surround and separate
groups of cells and organs.
A collagen fibre, like a piece of rope, is built up from smaller strands
wound together. There are four structural layers
(three are shown in Figure 1).
A molecule of collagen consists of three protein chains wrapped around each
other, and each of these is made up of many smaller units called amino acids
(see Box 1). Collagen molecules join together side by side and head to tail to
form larger cylinders (fibrils), which in turn link up into bigger fibres.
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Collagen mixes with a range of substances and can assemble in many different
ways. This enables particular tissues to have unique properties. For example in
bone, collagen mingles with calcium crystals to give a rigid structure. Tendons
possess great tensile strength because of the parallel arrangement and
cross-linking of collagen fibrils. In cartilage, a connective tissue found in
joints, collagen combines with a gel to create a completely different
effect—shock absorption and resistance to compression. Fibrils in skin are
woven together like wickerwork and interspersed with the stretchy protein
elastin to allow stretching without breaking. The window of the eye (the cornea)
is transparent mainly as a result of collagen’s arrangement into a regular open
network. Similarly, in the kidneys collagen forms the basis of a filter between
the blood and urine.
Much of the deterioration that comes with old age is due to the decreasing
amount and quality of collagen in tissues. As we get older, the plump skin of
youth starts to sag and wrinkle, mainly because collagen fibres weaken and break
like old bed springs. The skin also becomes tough and sinewy due to increased
cross-linking of fibrils. Arthritis occurs when the joints start to produce
unusually high levels of enzymes that break down collagen. The structure of
bones also deteriorates, making them more prone to fractures.
Because of its role in ageing, collagen has become the centre of a
multimillion-dollar industry. A combination of human vanity and a better
understanding of skin has enabled collagen replacement therapy to become popular
in recent years (see Box 2). Medically, the protein plays an important role in
applications as varied as post-surgical stitches and corneal implants.
And collagen’s uses don’t stop there. Gelatin, used as a setting agent in
many foods, is merely a solution of collagen and water heated up so that its
strands fall apart and form a gel on cooling. Some natural glues are made with
collagen, and it is worth remembering that leather—tanned animal
hide—gets its toughness from collagen.
Three in one
The triple helix
A molecule of collagen consists of three protein chains (alpha chains), each
curled into a left-handed helix. In turn these chains are intertwined to form a
right-handed cable or triple helix
(see Figure 1). In other words, three protein
chains, each about 1000 amino acids (protein building blocks) long, make up a
single molecule of collagen. At each end of the molecule are a few extra amino
acids that are not part of the helix. They are responsible for forming strong
links with nearby molecules, mainly collagen.FIG-mg21358201.JPG
Alpha chains have a very regular structure. The amino acid glycine (gly) is
repeated at intervals of three, so the sequence goes gly-A-B-gly-A-B (A and B
are any other amino acids, except those containing sulphur). Glycine is the
smallest amino acid, so it takes up the least space in the chain. This
one-in-three arrangement means glycine is always positioned at the cramped
centre of the triple helix, enabling the molecule to pack tightly together.
Osteogenesis imperfecta, a rare genetic disease that weakens bones,
illustrates the need for the recurring glycine triplet. A genetic mutation
replaces a single glycine with cysteine, preventing the normal intertwining and
dense packing of the chains.
As well as containing one-third glycine, another 30 per cent of the alpha
chain is proline (pro) and hydroxyproline (hyp). There may also be hydroxylysine
(hyl) and other amino acids. The sequence gly-pro-hyp is very common. Other
structural proteins like fibroin, a key ingredient of silk, and elastin, which
gives skin and other organs elasticity, also have regular repeating series of
amino acids. Globular proteins (which are “glob”-shaped), such as the enzymes
which speed up biological reactions, usually have a much more irregular sequence
of amino acids.
Hyp and hyl rarely feature elsewhere in biological systems. Each in its own
way provides strength and solidity. Bulky hyp and pro residues stabilise the
helix by chemically repelling one another—a phenomenon called steric
repulsion. Hyl and lysine form strong bonds called aldol links. Glycine, hyp and
hyl also form thousands of weak, but nevertheless crucial, hydrogen bonds
between alpha chains. Hydrogen bonding occurs when a positively charged hydrogen
in one part of a molecule is attracted to a negatively charged part of another
(or the same) molecule. This attraction holds the molecules in shape.
Collagen molecules spontaneously assemble into fibrils. The molecules are
united by various links, including a very strong three-way bond between two hyls
and a lysine, formed with the help of an enzyme. The amount of cross-linking
depends on the type of tissue and increases with age. Collagen in the Achilles
tendon, which connects the calf muscles to the heelbone, has far more
cross-linking than collagen in the ear, reflecting the need for extra resistance
to tensile stress in the lower leg.
When stained and viewed with an electron microscope, fibrils show regularly
spaced light and dark bands. This is because the parallel molecules within are
staggered by one quarter of their length, every 67 nanometres (67 thousand
millionths of a metre). Small gaps exist at these intervals
(see Figure 1),
where other molecules may attach such as sugars or minerals, depending on the
type of tissue. As the gaps absorb more of the metallic stain used in creating
the electron micrograph, they show as dark bands. This uniform but staggered
array safeguards against weak points that could buckle under stress. Fibrils in
turn associate into giant fibres—the final level of complexity.FIG-mg21358201.JPG
A huge family
Mysterious roles
Over the past 30 years, discoveries of new roles and configurations for
collagen have kept biochemists very busy. Researchers have observed
approximately 25 variations of the alpha chains that make up the triple helix.
Each has a different amino acid sequence and is coded by a separate gene. In
theory, this could make nearly 16 000 (253) combinations of triple helix, or
collagen types. In reality, only about 15 types have been identified, and in
many cases their roles have yet to be elucidated.
There are two main groups in the collagen family: fibrillar and
non-fibrillar. Fibrillar collagens are more widespread and are hardier and more
rigid than non-fibrillar types, because they must bear the stresses in tissues
such as skin, bone, cartilage, tooth dentine and tendons. They form the long
fibres described above.
The non-fibrillar collagens are more diverse, both in terms of their assembly
and function. For example, the “fibril-associated collagens” exist alongside
their fibrillar cousins. They aid the orientation of hefty fibres and link
fibrils to each other and to other important substances.
Other non-fibrillar collagens form networks and sheets (types IV and VII).
Type IV is a crucial ingredient of basement membranes, thin but sturdy sheets
that separate the layers of cells (epithelia) that line tubes, cavities and
organs. This type is also a vital part of connective tissue, the extracellular
matter that connects and supports organs. The triple helices of the type IV
chains form a two-dimensional mesh like chicken wire
(see Figure 3). The strands
contain numerous non-helical segments or kinks, which add flexibility. This open
meshwork is the perfect base or other cells to attach to. In the kidney, the
basement membrane lies between two cellular sheets, where it acts as a filter
between the blood and urine through which only molecules small enough may
pass.
Different collagen types also intermingle to form unique structures. For
example, in the cornea of the eye, extremely fine and regularly organised fibres
of types I and V are essential for transparency. However, most of the roles and
combinations of collagen types remain a mystery.
Well connected
A sturdy framework
Collagen molecules are far too large to fit inside cells and instead are
found in the surrounding region or “extracellular matrix”. The matrix is a
tangle of proteins and carbohydrates that occurs in varying amounts in different
parts of the body.
Connective tissue is a combination of matrix and cells. There are many
varieties, but as the name suggests, they all have one thing in
common—they provide a framework for the body and “connect” organs. There
are two categories of connective tissue, “dense” (for example that found in bone
and skin, which is fairly cell-free and mostly matrix), and the “loose” type,
such as spinal cord, which is packed with cells.
Collagen is a key element in many connective tissues because of its all-round
robustness. Apart from a few cells and blood vessels, the dense fibrous
connective tissue of tendons (where muscles attach to bones) and ligaments
(where bones attach to each other at joints) is almost exclusively made of type
I collagen fibrils. This setup allows an impressive resistance to force. The
downside is that the poor blood supply to these tissues hinders speedy healing.
This is why sprains, in which the connective tissue surrounding a joint is
stretched or overstrained causing fluid to enter the area, take such a long time
to heal.
Another well-known connective tissue is cartilage. This versatile matrix
reduces friction and acts as a shock absorber at the joints, provides an
architectural framework for the nose and ears, protects tissues and serves as a
precursor to the skeleton in the embryo—to name just of few of its many
uses.
As with tendons and ligaments, collagen is also a key feature of cartilage,
but the tissue is given very distinct properties by another ingredient of
extracellular matrix with which it works closely—proteoglycans. Molecules
of proteoglycans have long rod-like protein backbones, off which branch many
carbohydrate strands (carbohydrates—which include sugars and
starch—are a family of molecules containing only carbon, hydrogen and
oxygen). This arrangement is akin to a toilet brush, with the central protein as
the handle and the carbohydrate chains the brush filaments.
Carbohydrate chains in proteoglycans are highly negatively charged, and so
attract positive ions (cations). This dense cloud of cations, such as sodium
(Na+), causes lots of water to be sucked into the matrix by a process known as
osmosis, creating a gel-like environment that is superb for withstanding
compression. Collagen fibres nestle in this watery environment and provide
resistance to stretching forces. In this way, joints can support extremely high
stresses over an entire lifetime. Cells responsible for synthesising matrix
constituents are also present. They occupy holes, called lacunae, that speckle
the matrix at intervals. Cartilage also has a poor blood supply, so repair after
an injury is slow.
Cartilage in the external ear needs to be more flexible than that at the knee
joint. Elastic fibres made of the protein elastin give it this extra
springiness. Elastin is present in many tissues and organs that need to be
flexible, such as the arteries and skin.
A review of connective tissue would be incomplete without a glance at bone.
As well as giving mechanical support, the skeleton provides attachment sites for
muscles, allowing movement. The centre or marrow of bones acts as a factory for
producing blood cells and a depot for inorganic salts. Once again, collagen
interacts with another molecule to confer unique properties, in this case the
rigidity of bone. The salt calcium hydroxyapatite sits in the gaps in the fibril
network, binding to the nearest molecules.
Although we know quite a lot about their structure, the roles and
biochemistry of the more recently discovered collagen types have yet to be
determined. Matters were further complicated by the discovery a few years ago of
a related group, the curiously named non-collagen collagens. They also possess
the trademark triple helix motif, but have globular, non-helical segments that
perform special roles, mainly in binding other molecules. They are common as
membrane receptors and as binding sites in enzymes and recognition markers on
cells.
So even though all collagens share the triple helix motif, as a group they
have enormous diversity, in their assembly, composition and usage. Whether
they’re bracing our tendons and ligaments, reinforcing our bones, keeping our
skin firm, or acting as part of a filter in the kidneys, we owe a great deal to
these tough, sinewy molecules. As we have seen, without them life really would
be a flop.
LIKE all proteins, the synthesis of collagen begins in the nucleus of cells
(1). Most collagen is manufactured by cells called fibroblasts, which are found
in the extracellular matrix of connective tissue. First the DNA code for
collagen is copied onto a template molecule in the nucleus. The templates are
used to make pro-alpha chains (precursors to the individual alpha chains) out of
amino acid building blocks. The assembly process takes place on the
ribosomes—the cell’s protein factories (2). Pro-alpha chains differ from
alpha chains in having an extra peptide group, or pro-peptide, attached at each
end. These act as “destination labels” during subsequent stages of synthesis.
They also stop fibrils forming inside the cell and clogging it.
The pro-alpha chains then worm their way through a network of tubes known as
the endoplasmic reticulum (ER), guided by the pro-peptides. In the ER, enzymes
selectively add sugars and hydroxyl groups (OH) to amino acids in the chains
(3). Helped by the pro-peptides, which link the three chains together, a triple
helix forms (4). The newly formed “pro-collagen” molecule then exits the cell.
On secretion, enzymes chop off the pro-peptides (5), and the final product is
now ready to form into fibrils and fibres.
Diseases that affect collagen can be traced to each stage of the synthetic
journey. Scurvy occurs when stage 3 is prevented. Our daily requirement for
vitamin C (ascorbic acid) reflects the importance of hydrogen bonds in holding
the collagen cable together. Ascorbic acid keeps active the enzyme responsible
for adding a hydroxyl group (OH) to proline to make hydroxyproline. So when we
are deficient in vitamin C, this leads to decreased proline hydroxylation and
hydrogen bonding. Without this crucial molecular glue, the helix falls apart
with disastrous results: wounds that won’t heal, spongy gums unable to grip
teeth and fragile blood vessels prone to rupturing.
Ehlers-Danlos, a genetic disease, is caused by an inability to synthesise an
enzyme that catalyses the linkage of collagen molecules and fibrils at stage 5.
Patients have stretchy skin that is unable to resist pulling, as the intact
elastin fibres have no collagen fibrils to keep them in check.
FED UP with those sagging cheeks, crow’s-feet around your eyes and wrinkles
around your mouth? Would expanding your lower lip give you added sex appeal? And
now that you’re looking, what about that unsightly chickenpox scar you have had
since you were 11? All these imperfections can be treated with collagen therapy.
Film and TV stars have paid as much as ÂŁ600 a go to enhance their lips and
cheeks. Although cosmetic uses for the protein have emerged only recently,
collagen has been used for stitching up post-surgical gashes since the end of
the 19th century.
Surgical collagen originates from the hide of cows, and less frequently
pigs. Chemically, the bovine version is very similar to the human form. This is
crucial as the human immune system will reject anything that deviates too much
from its own proteins. The amino acid sequences at each end of the alpha chains
vary the most between species, making them the more “allergenic” parts—in
other words, the most likely to provoke an immune response. In order to reduce
sensitivity, chemists lop these bits off using the enzyme pepsin (also found in
the stomach, where it breaks down dietary protein).
A treatment for wrinkles is spread over about six sessions. The surgeon uses
a fine needle to squirt collagen into the dermis, the lower layer of skin
housing cells, hair follicles and collagen fibres (see Inside Science No. 78).
The injected molecules fill the space weakened by age or disease. They mingle
with other fibres, but do not combine. Eventually the implant degrades or falls
prey to immune cells, so injections must restart a few months later.
You may have attended a dinner where wine was served in ornate crystal
glasses. If so, perhaps you noticed the pretty reflections on the tablecloth
formed by light shining through the facets cut into the sides of the glass.
Imagine trying to guess the pattern cut into the glass by looking solely at the
reflections on the tablecloth, and you have some idea of how biochemists use
X-ray crystallography to analyse the structure of a protein crystal.
In X-ray analysis of a protein, an X-ray beam is fired at a protein crystal.
The atoms in the protein crystal scatter the X-ray in different directions,
creating an X-ray diffraction pattern which is captured on a photographic plate
behind the crystal. The crystal is rotated slowly and the process repeated until
there are X-ray diffraction patterns of the protein from several angles. The
three-dimensional structure is interpreted by studying these diffraction
patterns. As most proteins, including collagen, contain thousands of atoms, this
is no mean feat.
The advent of X-ray crystallography forced a rethink about the structure of
collagen. It was previously thought that the gaps between the molecules that
make up a fibril of collagen
(see Figure 1) were smaller than they actually are.
The discrepancy arose because the main technique then used to analyse collagen,
electron microscopy (which uses electron beams instead of light), required
mixing the samples with harsh metallic stains, causing them to shrink. X-ray
analysis uses tissue in its natural state, and bypasses this problem.FIG-mg21358201.JPG
1: A recipe for collagen
2: The perfect pout
3: Proteins made crystal-clear
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Further reading:
Biochemistry
(John Wiley & Sons, 1997), pp 156-161 -
Biochemistry: molecules, cells and the body
(Addison-Wesley, 1996), pp 467-477