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Tadpole tails disappear in a matter of days, even though they represent
about an eighth of the tadpole’s body weight. They do not wear away; they
do not dissolve. They are simply resorbed into the rest of the tadpole’s
body. In 1962 Jerome Gross, a developmental biologist, and Charles Lapierre,
a dermatologist, both working at the Massachusetts General Hospital in Boston,
decided to find out how. Three decades later, researchers are using their
results as the basis of new drugs for treating arthritis, eye diseases and
cancer.

Gross and Lapierre knew that tadpole tails are made up largely of the
body’s most abundant protein, collagen – a long, rod-shaped molecule made
up of three coils of amino acids wound round each other. They also knew
that collagen is highly resistant to the normal enzymes that break down
proteins. They suspected that the resorbing tail might contain some other
kind of enzyme that could break down collagen.

So Gross purified collagen and coated the bottom of a Petri dish with
it at 37 °C (body temperature). At this temperature, collagen forms
a gel and becomes opaque. Gross and Lapierre then laid tiny amounts of resorbing
tadpole tail on top of the gel and left it overnight at the same temperature.
In the morning there was a clear area around the tail tissue – something
produced by the tail, probably an enzyme, had broken down the collagen.
Gross and Lapierre called it collagenase.

Gross and Lapierre’s work was soon extended to an important source of
collagen in the human body – connective tissue. By 1967 Gross and Yori Nagai
of the Massachusetts Institute of Technology had established that collagenase
was a zinc-containing enzyme that could split collagen at a specific point
in all three of its polypeptide chains, three-quarters of the way from one
end. Gross and another colleague, John Evanson, found the same enzyme in
membrane tissue from rheumatoid joints when they placed it in a nutrient
medium. They began to think that collagenase might be involved in the destruction
of cartilage in arthritis (cartilage is the connective tissue that surrounds
joints).

We now know that any connective tissue, when placed in a nutrient medium,
produces collagenase. From cartilage to cornea and cows to coypus, it seems
that connective tissues in vertebrates all produce an enzyme that can break
them down.

When biochemists began to look more closely at connective tissue, they
found that the collagen ‘matrix’ has other components. In between the fibres
is another protein called GAG (glycosaminoglycan), shaped rather like a
bottle brush. Part of the ‘handle’ binds to a carbohydrate chain called
hyaluronic acid, and the ‘bristles’ are formed from sugar chains which contain
sulphate groups. These huge molecules have negatively charged groups that
bind water. This explains how tissues such as cartilage hold a lot of water,
and so can act as shock absorbers to prevent wear and tear within joints.
People who suffer from arthritis have lost some or all of this cushioning,
and their joint tissue becomes damaged.

Another protein, collagen type IV, does the important job of marking
boundaries between tissues. The molecules form a polymer, rather like a
flat, chain-link fence, to which other proteins bind. Laminin, one such
protein, binds to type IV collagen to form a dense barrier called a basement
membrane. This forms the outermost layer of blood vessels and surrounds
many of the body’s vital organs including the kidney. Other important proteins
in connective tissues include fibronectin, made by cells as a temporary
scaffolding on which collagen fibres are built.

Researchers now know of 13 different types of collagen, all with the
rod-shaped three-coil structure. Types I, II, III, V and XI form fibres,
while types VI, IX and X have short helical regions. They appear mainly
in tissues as cross-linked polymers and sometimes bind specifically to each
other. For example, in cartilage, type IX collagen binds specifically to
type II collagen fibres and limits their size.

These discoveries changed people’s ideas about connective tissues such
as skin, bone, tendons and ligaments. Until then people had considered them
to be static scaffolding for the body, defining boundaries between organs
and filling in spaces. But connective tissue is dynamic. It is constantly
being built up as specialised cells make the different matrix proteins,
and carefully position each one in a precise location outside the cell.
In the cornea, for example, these cells – the corneal fibroblasts – align
each collagen fibre precisely and fill in the spaces between them with just
the right amount and type of GAG. The precision with which they do this
ensures that the tissue is transparent. These ordered structures can also
be dismantled by the same cells: they make collagenase, both in tadpole
tails and in rheumatoid joints.

In the early 1970s, Tony Sellers and John Reynolds at Strangeways Research
Laboratory in Cambridge discovered that connective tissue cells produce
a whole family of enzymes: not only collagenase but also stromelysin and
gelatinase. These enzymes are known as matrix metallo-proteinases or MMPs,
and between them they can break down all the components of the collagen
matrix. Gelatinase breaks down denatured collagens, types IV, V and VII
collagen, and elastin. Stromelysin breaks down GAGs, laminin, fibronectin
and type IV collagen. The enzymes are very powerful and tiny amounts of
them will remove connective tissue. Just 1 milligram of collagenase can
destroy 3 grams of collagen in one hour at body temperature. For this reason,
they are carefully controlled, and cells make them only when needed.

The quantity of MMPs that each cell manufactures is controlled by cytokines
– messenger molecules such as inter-leukin-1 and tumour necrosis factor
that affect the growth and division of cells. Between 12 and 24 hours after
cytokines have bound to the cell membrane, the cell begins to produce the
enzymes. Cytokines and growth factors also stimulate connective tissue cells
to make collagen, GAG and the other matrix proteins, so completing the cycle.

People with inflammatory diseases such as arthritis have large numbers
of cytokines. They are produced by white blood cells called macrophages
when these infiltrate the membrane around joints. They stimulate connective
tissue cells to produce MMPs, which destroy the cartilage. These breakdown
products probably interact with T cells, which then stimulate macrophages
to produce more cytokines. Just a small imbalance between the breakdown
and the synthesis of the matrix can eventually lead to the progressive destruction
of cartilage that signals rheumatoid arthritis (see ‘Confusion in the joints’,
New ÐÓ°ÉÔ­´´, 4 May 1991).

The enzymes are controlled in other ways too. They leave the cell as
‘proenzymes’ which must be activated before they can break down connective
tissue. Proenzymes have an extra peptide sequence of about 80 amino acids.
This contains a sulphur atom that reacts with the zinc atom in the active
site of the enzyme, blocking its action. Disturbing this interaction will
activate the enzyme. There are two ways of doing this: either by proteinases,
such as plasmin, which split off the extra peptide portion, or by chemical
destabilisation, where a molecule binds to sulphur, freeing the active site
of the enzyme which then splits its own peptide. During the 1980s, several
sequencing studies showed that the enzymes all have certain regions of protein
in common. They all contain the zinc-binding region and a sequence of amino
acids at one end of the molecule.

In 1989, Gill Murphy at Strangeways discovered that stromelysins are
important in the activation of procollagenase. Prostromelysins can be activated
by plasmin and can break down GAG molecules in the matrix, leaving the rope-like
fibres of collagen exposed. Procollagenase, which is secreted at the same
time as the stromelysin, is activated and breaks down collagen. We now think
that these two enzymes bring about the rapid, regular removal of connective
tissue matrix around each cell. The type IV collagenases are probably involved
in more drastic removal of connective tissue and in breaching basement membrane
structures. No one knows how the progelatinases are activated.

A further level of control operates within the connective tissues. We
discovered that cells make a specific inhibitor of collagenase, gelatinase
and stromelysin, and called it TIMP (tissue inhibitor of metalloproteinases).
Andy Docherty at Celltech in Slough first deduced its amino acid sequence
in 1985. He showed that it contains six internal sulphur-sulphur bridges
that hold the molecule in a precise three-dimensional arrangement, although
we do not know exactly how it binds to MMPs. The complex formed between
TIMP and an active MMP is tightly bound, preventing the enzyme from breaking
down more matrix protein. TIMP does not bind to proenzymes. In 1989, Bill
Stetler-Stevenson at the National Cancer Institute in Bethesda, Maryland,
discovered a similar inhibitor, TIMP2. Perhaps each enzyme has its own inhibitor.

Designer drugs

We now think that the enzymes are responsible for the destruction of
connective tissues in a wide variety of diseases. These include the slow
but relentless destruction of cartilage in rheumatoid arthritis, its rapid
breakdown in septic arthritis, and the movement of metastatic cells around
the body to form secondary tumours in cancer. These cells move from primary
tumours through connective tissue, secreting MMPs. MMPs can break through
the basement membrane that separates the innermost layer of skin from the
tissues beneath. This enables the cells to punch holes in blood vessels,
by means of gelatinase, so they can move through the blood to another part
of the body. MMPs are also thought to be involved in the breakdown of the
cornea in corneal ulceration and in some types of bone disease.

Pharmaceutical companies, including Smith Kline Beecham, Glaxo, Sterling
and Roche, are developing drugs to block these enzymes. Ian Clark and myself,
with Smith Kline Beecham at Harlow and David Blow at Imperial College in
London, have been attempting to purify enough collagenase to crystallise
it and determine its structure using X-ray crystallography. Last year we
defined the exact conditions needed to make collagenase crystals. We hope
to combine these results with information about the position of amino acids,
obtained by our colleague Mary O’Hare from sequencing studies on pig collagenase.
Together they should identify which chemical groups are on the surface of
the enzyme near its active site, the zinc atom. Then we hope to use molecular
modelling to design chemical inhibitors that bind specifically to the active
site, and so block enzyme activity.

Chemists have already made some inhibitors by copying inhibitors of
another zinc-containing enzyme, angiotensin converting enzyme. They have
also designed inhibitors for thermolysin, a bacterial MMP that has zinc
bound in a similar way. This information formed the basis for designing
MMP inhibitors that couple a chemical group that binds zinc to three amino
acids that copy the region in the proteins where the enzymes cleave.

Do these inhibitors work? We know that they can prevent cancer cells
from moving through basement membrane. Lance Liotta at Bethesda devised
a model system where he separated two chambers with a layer of human base-ment
membrane. In the top chamber he placed invasive tumour cells and in the
bottom a chemical that attracted these cells. The tumour cells moved through
the tissue into the bottom chamber. However, when he added some of our TIMP
to the top chamber, the invasive tumour cells were unable to move through
the basement membrane. Recently this experiment was repeated using the synthetic
inhibitors, with the same result.

We have also used the synthetic inhibitors to investigate the breakdown
of cartilage. Interleukin-1 injected into joints causes the cartilage to
break down GAGs. When we treated cartilage with interleukin-1, both with
and without inhibitors, we found that the inhibitors completely blocked
the release of GAG from the tissue and also prevented the later release
of collagen II.

There is still a lot of work to do before these inhibitors are ready
to be used as drugs. No one knows how stable they will be in the body and
how much of them people would have to take. This is where our crystallisation
studies to determine the three-dimensional structure of the enzymes come
in. They will enable us to design highly specific inhibitors that will selectively
target individual enzymes.

Such specific inhibitors could then be used as drugs in patients suffering
progressive destruction of cartilage and bone. This happens in rheumatoid
and osteoarthritis. In septic arthritis – where cartilage can be removed
completely in a matter of days – it would probably be best to inject the
inhibitor directly into the affected joint.

People suffering from cancer may also benefit from these drugs. Those
at risk of developing secondary tumours could be treated for a short period
to stop tumour cells passing through connective tissue and, perhaps most
importantly, through basement membranes. In the meantime doctors would be
able to remove primary tumour cells by surgery or radiotherapy. Other applications
include eye diseases. In one disease of the cornea, ulcers form and the
normally transparent connective tissue is rapidly broken down. Subsequent
healing leaves a scar which prevents normal sight. Inhibitors could prevent
the initial rapid destruction.

A substance that was first discovered in the tail of the humble tadpole
is now known to be part of a family of enzymes vital to the health of human
connective tissues. An understanding gained from 30 years of research has
put biochemists well on the way towards curing diseases that are caused
by the breakdown of the mechanisms that control the enzyme’s activity.

Tim Cawston is head of the research laboratories at the Rheumatology
Research Unit, Addenbrooke’s Hospital, Cambridge.