The Extracellular Matrix

Professor Alfred Cuschieri

Department of Anatomy, University of Malta

Objectives

By the end of this session student should be able to:

· Name the main extra cellular fibers and their locations

· Outline the molecular composition of collagen fibers

· Deduce the major consequences of defects in collagen formation on the functions of the extra cellular matrix

· List the factors that could affect collagen synthesis and affect wound healing

· Name the non-fibrillar components of extra cellular fibers and their functions

· Give examples of how cells interact with the extra cellular matrix

All cells are associated with extra cellular structures. The connective tissues consist predominantly of extra cellular structures, which consist of:

· Fibres - Collagen, elastic and fibrillin

· Matrix (amorphous ground substance)

· Fibroblasts and cells of various types

Collagen

Collagen is a family of extra cellular fibres that are widely represented in the body and have mainly mechanical functions. Collagen fibres provide strength and support.

10 types of collagen are known. They vary in :

a. molecular structure

b. morphology

c. distribution

Molecular Structure

All collagens have a basically similar molecular structure:

They contain large amounts of:

o Glycine (about 30%)

o Hydroxyproline and hydroxylysine (25%).

o (These amino acids are rare in other proteins)

o Oligosaccharide side chains (galactose and glucose)

Collagen is composed of molecules of pro-collagen. A pro-collagen molecule is composed of three polypeptide chains called a-chains, twisted in a helical fashion. The individual a-chains are termed pre-procollagen.

The general chemical structure of pro-collagen molecules is shown in the following diagram. (G – glycine; X – proline or hydroxyproline; Y – lysine or hydroxylysine; A – other amino acids)

– G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A – G – A –

– A – G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A – G –

– A – A – G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A –

There are several different a-chains. They are found in different combinations in the different types of collagen, as shown below.

The following table illustrates the molecular structure, morphology and distribution of the first five types of collagen. The first five types are the most important ones. The other five are less important.

Collagen Type

Morphology

Molecular structure

Distribution

Type I

Fibres with periodicity of 63 nm.

a1-1; a1-1; a1-2

low in carbohydrates

· Ligaments

· Tendons

· Fasciae

· Bones

· Dermis of skin

· Sclera

Type II

Very thin fibrils forming a meshwork

a1-2; a1-2; a1-2

high in hydroxylysine and carbohydrates

· Hyaline cartilage

· Cornea

· Vitreous body

Type III

Loosely-packed network of thin fibrils

a1-3; a1-3; a1-3

low in hydroxylysine

· Associated with type 1

· Blood vessels

· Most internal organs

· Smooth muscle

· Foetal dermis

Type IV

Thin amorphous membrane

a1-4; a1-4; a1-4

Extensive hydroxylysine

Heavy glycosylation

· Basal laminae

Type V

Amorphous

a1-5; a2-5; a3-5;

high hydroxylysine;

low alanine

· Placenta

· Foetal membranes

· Skin

· Blood vessels

Biosynthesis of Collagen

This involves a series of sequential steps occurring in the rough endoplasmic reticulum and the Golgi complex:

1. Synthesis of a chains of pre-procollagen on ribosomes. A signal protein directs them to the RER .

2. Cleavage of signal protein forms procollagen

3. Hydroxylation of lysine and proline

Lysine à Hydroxylysine

Peptidyl lysine hydroxylase

Proline à Hydroxyroline

Peptidyl proline hydroxylase

Ascorbic acid is necessary to activate the hydroxylases.

4. Glycosylation: addition of galactose and glucose to some hydroxylysine residues. The enzymes galactosyl transferase and glycosyl transferase are required for this process.

5. Assembly of three a - chains to form procollagen. This involves the formation of disulphide bonds between parts of the polypeptide chains known as registration peptides, which occur at both ends of the pre-procollagen.

The assembly process aligns the three a - chains relative to one another.

The three alpha chains are wound around one another in the form of a triple helix.

The assembly process occurs in the Golgi apparatus.

6. Secretion of procollagen molecules by exocytosis into the extra cellular space.

7. Cleavage of registration peptides occurs in the extra cellular space, and is catalysed by procollagen peptidases. The resulting molecule is called tropocollagen.

8. Self-assembly or polymerization of tropocollagen molecules form collagen fibrils.

Cross-linkage between adjacent tropocollagen molecules stabilizes the fibrils. This occurs by oxidation – deamination of the amino acid hydroxylysine. It involves the removal of an amino group (NH2), which has a net oxidative effect and the formation of covalent cross-links. It is catalyzed by lysine oxidase (or catalase).

In collagen type 1 the tropocollagen molecules polymerize in a staggered pattern, giving rise to long fibrils that, under high magnification electron microscopy, appear to be striated fibers with a repeat periodicity of 64 nm.

The fibers are usually arranged in bundles that are visible with light microscopy.

Several connective tissue disorders are the result of mutations in the alpha procollagen chains

Osteogensis imperfecta

Ontogenesis imperfecta (OI) is a defect in collagen type I. There are several types of OI, with somewhat different clinical manifestations. Most of them are caused by genetic mutations in the gene for the a 1-1 pre-procollagen.

The main clinical features involve poorly formed bone, which results in:

o Short, curved, and deformed bones

o Multiple fractures, which are present at birth in some cases

o Abnormal dentine

o Deafness due to defects in the small ossicles of the ear

Ehlers Danlos Syndrome

There are several different types of Ehlers Danlos Syndrome. The following are a few common types to illustrate different molecular disorders in the formation of collagen.

Ehlers Danlos Syndrome Type I

This is caused by lack of synthesis or other defect of pro a 2-1, resulting in poorly formed collagen type I. The main clinical features affect:

o The skin is thin, hyperextensible, and easily bruised

o Joint hypermobility

o Anomalies of the heart valves, which are usually incompetent

Ehlers Danlos Syndrome Type IV

This is caused by a defect of collagen type III. It affects mainly the collagen framework in the viscera, and may result in:

o Rupture of viscera e.g. bowel, arteries or uterus

o Thin, translucent (but not hyperextensible) skin

Ehlers Danlos Syndrome Type VI

This is caused by deficiency of lysine hydroxylase, the enzyme responsible for hydroxylation of lysine. This affects the subsequent glycosylation and cross-linking. The main clinical manifestations are:

o Eye abnormalities – abnormalities of sclera and cornea

o Hyperextensible joints

o Scoliosis

o Hyperextensible skin

Ascorbic Acid Deficiency (Scurvy)

This is caused by a dietary deficiency of ascorbic acid. Although it is now rare, it was once common particularly among sailors, and is still found in underdeveloped and undernourished populations. Ascorbic acid is necessary for activating proline and lysine hydroxylases, resulting in poor hydroxylation of lysine and proline. It results in:

o Poor collagen fibre formation

o Poor wound healing

o Blood vessel fragility

o Skin lesions

o diseased gums

Ascorbic acid is present in fresh fruit, and vegetables, and especially in citrus fruit. The old custom of giving oranges when visiting patients in hospital was intended to promote a speedy recovery, but the cheerful colours of flowers appear to have become more popular!

Elastin

Elastin consists of fibers, whose main property is their elasticity. Elastic fibers are composed of protein molecules with the following properties that differ from collagen in many respects. It contains:

o Abundant glycine, about 33% (as in collagen)

o Little hydroxyproline

o No hydroxylysine

o Abundant hydrophobic amino acids

o Helical segments that are responsible for its elasticity

o Desmosine in non-helical segments, which is responsible for cross-linkage of the molecules to form a network that can alter its configuration when stretched.

The following diagrams illustrate the cross-linking of elastin to form a network

(a) in the relaxed state; (b) when stretched.

Fibrillin

Fibrillin is another extra cellular fibril that is often associated with elastic fibres, but distinct from them.

Fibrillin:

o Forms fibrils 10 nm in diameter

o Is rich in cysteine and forms numerous disulphide bonds

o Is present in

o Blood vessels and heart valves

o Suspensory ligament of lens

o Bones, tendons and ligaments

Marfan Syndrome results from a mutation of the fibrillin gene.

The clinical features of Marfan’s syndrome are:

o Skeletal: Tall stature and arachnodactyly

o Cardiac: Aortic aneurysm; Heart valve incompetence

o Ocular: Subluxation of the lens

Proteoglycans are the major components of the amorphous extra cellular matrix

Proteoglycans:

o Are large molecules forming the amorphous matrix of connective tissues.

o Consist of a protein backbone and numerous polysaccharide side chains

o Are rich in anionic radicals - COOH ^- and - SO₄^2-

o The large, branching molecules form complex aggregates that retain water

Properties of proteoglycans

Proteoglycans also give the amorphous matrix the following properties:

o High viscosity

o Water retention

o Form a barrier to free diffusion of substances

o Some proteoglycans are anticoagulants (e.g. heparin and dermatan sulphate)

o Some proteoglycans control cell aggregation and growth during embryogenesis

Examples of proteoglycans

The following examples illustrate the functions of proteoglycans:

Chondroitin sulphate is present in large amounts in the matrix of hyaline cartilage, which is responsible for its hard, rubbery consistency, and a clear (hyaline) appearance. In experiments in which proteoglycans were broken down by enzyme treatment, cartilage lost its rigidity and turgidity.

Keratan sulphate is present in large amounts in the cornea, and is responsible for its transparency.

Other proteoglycans include

Dermatan sulphate is found in skin, blood vessels and heart and

Heparan sulphate is found in basement membranes, lung and arteries.

Hyaluronic acid is a non-sulphated proteoglycan, which is found in synovial fluid, skin and support tissues

Hyaluronidase breaks down hyaluronic acid reducing the viscosity and permeability of the matrix.

o It is produced by some microorganisms to help them penetrate tissues

o It is used therapeutically as an ingredient of ointments such as “Lasonil”, which are applied to the skin to aid diffusion and absorption of haematomas.

Fibronectin and syndecan

Fibronectin and syndecan are proteins that are found in the amorphous matrix of connective tissues, and provide attachment of cells to collagen. Each of these proteins consists of three domains:

o A collagen - binding domain

o Two domains binding to proteoglycans

o A membrane-binding domain that binds to the integral membrane protein integrin

The basal lamina consists of three layers:

o A lamina rara (or lucida), 60 mm thick, in contact with epithelial cells,

o A lamina densa 30-100 nm thick forms an intermediate layer

o A reticular lamina, which merges with the extra cellular matrix

It has four major constituents:

o Collagen type IV - main component of reticular layer

o Heparan sulphate proteoglycan

o Laminin, the main component of the lamina rara, is a glycoprotein that binds the plasma membrane to proteoglycans.

o Entactin, a glycoprotein that binds to collagen type IV and to laminin