2.10 - Extracellular Matrix

Question 1

What is the extracellular matrix?

Answer 1

• the ECM is a complex network of proteins and carbohydrates filling spaces between cells
• comprises both fibrillar (fibre-making) and non-fibrillar (non-fibre making) components
• deposited by cells (notably fibroblasts)
• after being deposited it becomes immobilised outside cells and fills spaces between cells
• like a cellular glue

Question 2

Key functions of ECM

Answer 2

• provides physical support
• determines mechanical (architectural role) and physiological properties of the tissues (influences cell behaviour during development)
• influences the growth, adhesion and differentiation status of the cells and tissues with which it interacts
• essential for development, tissue function and organogenesis

Question 3

Connective tissues

Answer 3

• made of ECM material and the cells, fibroblasts and occasional macrophage
• particularly rich in ECM
• contain a distinct spectrum of collagens, multi-adhesive glycoproteins and proteoglycans (ECM components), together with a cellular component
  Many varieties of ECM component exist:
• collagens: type I, II, III (fibrillar), type IV (basement membrane, non-fibrillar)
• multi-adhesive glycoproteins: fibronectin, fibrinogen, laminins (basement membrane)
• proteoglycans: aggrecan, versican, decorin, perlecan (basement membrane)
• each matrix component is able to interact with the cellular components via specific cell surface receptors

Question 4

The varied properties of connective tissue

Answer 4

• different types and arrangements of collagen, with the presence of absence of different ECM components, gives a wide variety of connective tissues with varied properties
• tendon and skin - tough and flexible
• bone - hard and dense
• cartilage - resilient and shock-absorbing

Question 5

Human disorders resulting from ECM abnormalities

Answer 5

1. gene mutations affecting matrix proteins
   - osteogenesis imperfecta - type 1 collagen
   - Marfan’s syndrome - fibrillin 1
   - Alport’s syndrome - type IV collagen (a5)
   - epidermolysis bullosa - laminin 5 (in all 3 chains)
   - congenital muscular dystrophy - laminin 2 (a2 chain)
2. gene mutations affecting ECM catabolism
   - aka mucopolysaccharidoses (MPSs) - inability to degrade GAGs
   - Hurler’s syndrome - L-a-iduronidase
3. fibrotic disorders due to excessive ECM deposition
   - liver fibrosis - cirrhosis
   - kidney fibrosis - diabetic neuropathy
   - lung fibrosis - idiopathic pulmonary fibrosis (IPF)
4. disorders due to excessive loss of ECM
   - osteoarthritis

Question 6

Collagens

Answer 6

• family of fibrous proteins found in all multicellular organisms
• major proteins in bone, tendon and skin
• most abundant proteins in mammals
  Alignment of collagen fibrils:
• skin - successive layers nearly at right angles to each other (one layer longitudinal, next layer cross-section)
• mature bone and cornea - same arrangement
• these tissues resist tensile force in all directions

Question 7

Molecular arrangements of collagen fibres

Answer 7

• 28 collagen types exist in humans (roman numerals)
• 42 genes encoding collagens in humans
• each collagen molecule comprises 3 alpha chains, forming a triple helix
• type I collagen has chains from two different genes - [a1(I)2][a2(I)] - heterotrimer
• types II and III collagen have only one chain type - [a1(II)]3 and [a1(III)]3 - homotrimer
• triple helix - three alpha chains form a stiff triple helix structure - characteristic gly-x-y repeat
• x is often proline, y is often hydroxyproline
• every third position must be occupied by glycine as it is small enough to occupy the interior (H side chain = simplest AA, allows it to pack closely)
• one alpha chain –> three alpha chains –> collagen fibril –> collagen fibre

Question 8

Collagen biosynthesis (fibrillar)

Answer 8

• all newly synthesised collagen chains have non-collagenous domains at N- and C- termini
• these domains are removed after secretion in the case of fibrillar collagens, but remain part of the collagen in most other types
• procollagen –> collagen –> fibril formation –> cross-linking (increases tensile strength)
• fibrillar collagen biosynthesis: pro-alpha-chains synthesised as longer precursors by ribosomes attached to the ER, then these undergo a series of covalent modifications and fold into triple-helical procollagen molecules, before their release from cells
• then, converted into collagen molecules
• excess collagen production can lead to fibrotic diseases e.g. alcoholic liver cirrhosis, fibrotic lung

Question 9

Covalent cross-links in collagen

Answer 9

• cross-linking provides tensile strength and stability
• both lysine and hydroxy-lysine residues are involved
• the type and extent of cross-links is tissue specific and changes with age
• prolyl and lysyl hydroxylases require Fe2+ and vitamin C - contributes to interchain hydrogen bond formation
• lysine and hydroxylysine are also modified in the formation of covalent cross-linkages - takes place only after collagen has been secreted
• vitamin C deficiency results in under-hydroxylated collagens, with dramatic consequences for tissue stability (scurvy)

Question 10

Ehlers-Danlos syndromes (EDS)

Answer 10

• Ehlers-Danlos syndromes are a group of inherited connective tissue disorders whose symptoms include stretchy skin and loose joints
• several disorders arise due to mutations in collagen, which negatively affect collagen production, collagen structure and collagen processing

Question 11

Non-fibril forming collagens

Answer 11

• type IV collagen is a network-forming collagen and is present in all basement membranes, though its molecular constitution varies from tissue to tissue
• in this network, molecules can associate laterally between triple-helical segments as well as head-to-head & tail-to-tail between the globular domains to give dimers, tetramers and high order complexes
• type IV collagen molecules assemble into a sheet-like network - essential component of basement membranes
• uncleaved N and C terminals interact to form dimers –> tetramers –> network (through rotary shadowing)

Question 12

Basement membranes

Answer 12

• aka basal laminae
• flexible, thin mats of ECM underlying epithelial sheets and tubes
• surround muscle, peripheral nerve and fat cells and underlie most epithelia
• sit above a network of connective tissue
• highly specialised ECM containing a distinct repertoire of collagens, glycoproteins and proteoglycans
• made of type IV collagen and laminin
• apical = top, basal = bottom

Question 13

Diabetic nephropathy

Answer 13

• in kidney - form a key part of filtration unit (glomerular basement membrane)
• in diabetic nephropathy, there is an accumulation of ECM leading to a highly thickened basement membrane
• this restricts renal filtration and can lead to renal failure

Question 14

Alport syndrome

Answer 14

• mutations in collagen IV result in an abnormally split and laminated glomerular basement membrane which is associated with a progressive loss of kidney function (and also hearing loss)

Question 15

Laminins

Answer 15

• laminins are heterotrimeric proteins - alpha chain, beta chain, gamma chain - cross shaped molecule
• large proteins with each chain having a large molecular weight
• multi-adhesive proteins (can bind to various matrix components and cell surface receptors) which can interact with a variety of cell surface receptors including integrins and dystroglycan
• can self-associate as part of the basement membrane matrix, but can also interact with other matrix components e.g. type IV collagen, nidogen and proteoglycans

Question 16

Laminin-a2 deficiency in muscular dystrophy

Answer 16

• LAMA2 gene encodes laminin-a2
• forms a heterotrimer which polymerises with laminin-B1 and laminin-y1
• fails to interact with a7B1 integrin and a-dystroglycan that are required for adhesion and basement membrane assembly
• congenital muscular dystrophy can arise from absence of the a2 chain in laminin 2
• lack of laminin-a2 manifests as progressive muscle weakness and degeneration - symptoms include hypotonia (decreased muscle tension), a generalised weakness and deformities of the joints

Question 17

Elastic fibres and elastin

Answer 17

• elastic fibres provide elasticity to tissues
• found in skin, blood vessels and lungs
• made up of an elastin core and surrounding microfibrils rich in the protein fibrillin
• elastin is a small protein arranged in a random coil, with alternating hydrophilic and hydrophobic domains
• hydrophilic domains contain lysine residues which are cross-linked during the formation of mature elastin

Question 18

Fibrillin-1 mutations - Marfan’s syndrome

Answer 18

• integrity of elastic fibres depends on microfibrils which are made of fibrillin
• Marfan’s syndrome has diverse manifestations, involving primarily the skeletal, ocular and cardiovascular systems
• aberrant thickening of the aortic with fragmentation and disarray of elastic fibres
• individuals can be predisposed to aortic ruptures

Question 19

Fibronectin

Answer 19

• family of closely related glycoproteins of the ECM which are also found in body fluids
• can exist as either an insoluble fibrillar matrix or as a soluble plasma protein
• the basic unit is a 500kD dimer which is disulphide linked
• different domains are discrete sites for binding to different things
• fibronectins interact with cell surface receptors and other matrix molecules
• fibronectins regulate cell adhesion and migration in embryogenesis and tissue repair
• fibronectins important in wound healing, helping promote blood clotting
• form a mechanical continuum with the actin cytoskeleton of many cell types - link ECM and actin
• integrin receptors at the cell surface provide the linkage between the matrix and the actin cytoskeleton - fibronectins bind multiple ligands and cell receptors e.g. collagen fibres, actin filaments

Question 20

Proteoglycans

Answer 20

• proteoglycans are core proteins with attached glycosaminoglycan (GAG) chains
• GAG chains are made of repeating disaccharides
• sulfation / carboxylation can increase negative charges = attract cations –> water is sucked into ECM
  Main GAG varieties:
• hyaluronan (directly from enzyme - other three synthesised and attached to core proteins in ER and Golgi)
• chondroitin sulfate / dermatan sulfate
• heparan sulfate
• keratan sulfate
  Some proteoglycan families are grouped by their structural and functional characteristics:
• basement membrane proteoglycans e.g. perlecan
• aggregating proteoglycans (interact with hyaluronan) e.g. aggrecan
• small leucine-rich proteoglycans e.g. decorin
• cell surface proteoglycans e.g. syndecans 1-4

Question 21

Hyaluronan

Answer 21

• found in ECM of soft connective tissues
• directly from enzyme embedded in plasma membrane
• does not have a core protein, simply an unsulfated carbohydrate chain made of repeating disaccharides
• can undergo a high degree of polymerisation
• can occupy a large volume
• found in highly viscous tissues e.g. vitreous humour of eye, and synovial fluid of joints
• key role in protecting cartilaginous surface from damage

Question 22

Aggrecan

Answer 22

• aggrecan is a major constituent of the cartilage extracellular matrix
• perfectly suited to resist compressive forces
• under compressive load, water is given up, but regained once the load is reduced
• GAGs are highly sulfated and large numbers of carboxyl groups, increasing their negative charge = attracts cations = osmotic effect (large quantities of water retained by environment)

Question 23

Osteoarthritis

Answer 23

• osteoarthritis (OA) is the leading cause of lower extremity disability in older adults
• erosive disease resulting in loss of ECM via degradation
• aggrecan cleavage by aggrecanases and metalloproteinases
• aggrecan filaments lost to synovial fluid
• cushioning properties of cartilage over the end of bones are lost