Biochemistry: Collagen And Connective Tissue

Question 1

Connective tissues

Answer 1

Cartilage Bone Tendon Skin

§ highly specialised tissues
§ provide mechanical support and assist in movement
§ matrix around cells regulates their behaviour
§ may contain blood vessels, nerves
§ arena for fighting infection
§ diverse structure and function, but composed of same building blocks

Provide mechanical support to the body
Matrix around the cell affects behavior

Question 2

Connective tissue components

Answer 2

§ cells – dynamically synthesise and break down the connective tissue
§ fibroblasts in skin, tendon ligament
§ chondrocytes in cartilage
§ osteocytes, osteoblasts, osteoclasts in bone

§ extracellular matrix

(a) structural components – give the tissue its mechanical properties
§ ‘ground substance’ – mostly proteoglycans (e. g. aggrecan) § elastic fibres
§ collagen fibres

(b) matricellular proteins – have regulatory, non-structural roles

Question 3

Proteoglycans

Answer 3

Important for mechanical properties of connective tissue

consist of a protein core, decorated with sugar (called “glycans” or
“glycosaminoglycans” or “GAGs”) chains

there are various kinds of GAG chains, built from different building blocks. Most are sulfated - this is what makes it negatively charged

Sugars are negatively charged so bring water into the cell

Question 4

Proteoglycans 2

Answer 4

§ variable in size - small (~40kDa) to large (100 kDa+)
§ hold water in tissues
§ confer viscoelastic properties
§ interact with cells, cytokines and collagen

Question 5

Aggrecan

Answer 5

Forms huge multimeric aggregates in cartilage

§ Link protein stabilises binding to hyaluronan (HA) ‘backbone’
§ huge complexes of sulfated chondroitin & keratan sulfate are highly
hydrophilic
§ draws water into tissue and so enables cartilage to resist compression

Interaction between collagen and aggrecan gives collagen it’s properties
They’re highly hydrophilic

Question 6

In osteoarthritis: degradation of aggrecan and type II collagen impairs mechanical properties of cartilage

Answer 6

§ in osteoarthritis, altered joint mechanics causes breakdown of aggrecan and also collagen by proteolytic enzymes
§ this impairs cartilage functions, causing pain in affected joints.

Question 7

Elastic fibres (10 nm microfibrils)

Answer 7

§ consist of multiple components:
§ elastin
§ fibrillin-1, -2, and -3
§ fibulins (esp 4 and 5)
§ matrix-associated glycoproteins

§ structural role :
§ enable stretching of blood vessel
walls, alveoli, bladder, tendons

§ regulatory role :
§ regulate targeting and activation of
growth factors (especially TGFb)

§ form long microfibrils that sit between collagen fibre bundles

Question 8

Collagens are a diverse and varied ‘family’

Answer 8

27 collagens, 42 genes – many roles and functions

No need to know all of the names

Question 9

Collagen fibrils (collagen type I, II and III)

Answer 9

the main structural component of connective tissues

§ very plentiful protein - makes up 12-17% of whole-body protein content

§ collagen fibrils are made of many collagen molecules, with each molecule consisting of 3 “alpha” polypeptide chains arranged in a triple helix :
§ each chain is a single gene product
§ either 3 identical a chains (homotrimer) e.g. type 2 collagen (cartilage) is a13 (3 is subscript)
§ or two or more different a chains (heterotrimer) e.g. type I collagen (skin, bone) is a12a21

§ very stable and slowly turned over – has been extracted from dinosaur bones!

Question 10

Collagen structures (triple helix)

Answer 10

Each molecule of fibrillar collagen is made up of 3 intertwined (triple helix) polypeptides

§ unique amino acid composition of Gly-X-Y repeats
(X= often proline, Y = often hydroxyproline)

§ tight packing of triple helix depends on glycine being every 3rd residue - § glycine is the smallest amino acid

§ proline & hydroxyproline provide rigidity and stability -
§ hydroxyproline is an unusual amino acid, mostly found in collagen

§ stabilised by hydrogen bonds between chains

Question 11

Collagen fibril synthesis

(Only need to know first steps)

Answer 11

processing is essential for stability

post-translational modification
§ Hydroxylation (NB for chain stability!) § glycosylation

assembly of three alfa chains
disulfide bond formation
assembly of triple helix

Question 12

What vitamin is hydroxylation dependent on

(& scurvy)

Answer 12

Vitamin c

Vitamin C deficiency causes reduced collagen hydroxylation, and causes scurvy

reduced hydroxylation of proline weakens collagen triple helix

weaker collagen fibrils leads to scurvy:

§ bleeding gums
§ loss of teeth
§ skin lesions, bruises § poor wound healing
§ joint pain & weakness

Question 13

Connective tissues have different collagen composition and organisation

Answer 13

Skin
§ 60% type I collagen
§ 30% type III collagen
§ meshwork of fibres

Tendon/Ligament
§ 90+% type I collagen
§ 5% type III collagen
§ parallel fibres

Bone
§ 90% type I collagen
§ 3% type V collagen
§ sheets (lamella)

Cartilage
§ 95% type II collagen
§ meshwork

No need to learn percentages

Question 14

In bone: type I collagen fibrils form ‘lamellae’

Answer 14

§ type I collagen is principal protein component
§ type I collagen fibers are arranged into ‘lamellae’ (concentric circles)
§ hydroxyapatite crystal deposits harden bone

Question 15

Collagen In tendon:

Answer 15

tensile strength is provided by type I collagen fibrils arranged in parallel bundles

Question 16

In tendon:

Answer 16

the insertion (‘enthesis’) has a different matrix composition & organisation

Uncalcified fibrocartilage:
As for tendon plus:
type II collagen: 2-5%
proteoglycan (aggrecan): 3%

Tendon:
type I collagen: 90+%
type III collagen: ~5%
proteoglycan: 0.5%

No need to learn %

enthesis is a common site of tendon pathology
§ is a region where mechanical stress is concentrated
§ common site of microdamage and ‘overuse’ injury
§ e.g. tennis elbow, golfer’s elbow

Changes associated with tendinopathy:
§ loss of collagen fibril organisation and reduced fibril diameter
§ changes in collagen content
§ increased type III collagen
§ increased turnover
§ vascular and nerve infiltration (painful tendons only)
§ failure to repair

Question 17

Collagen in Skin and blood vessels:

Answer 17

are rich in type III collagen

§ type III collagen forms mixed fibrils with type I collagen
§ deficiencies in type III collagen (COL3A1 gene) that affect fibril formation lead to fragility of skin and blood vessels (e. g. Ehlers Danlos Syndrome Type IV)

Question 18

examples of acquired connective tissue diseases are:

Answer 18

Scurvy – a disease of impaired collagen synthesis
§ Vitamin C deficiency causes poor proline hydroxylation
§ structure of fibril-forming collagens (types I, II and III) is weakened
§ skin/bone/gums affected

Diseases of altered mechanics
§ abnormal mechanics lead to altered connective tissue structure, which
impairs the tissue function

§ e. g. tendinopathy
§ often affects the enthesis
§ altered collagen content and organisation

§ e. g. osteoarthritis
§ increased breakdown of collagen (type II) and aggrecan in cartilage

Question 19

Genetic conditions of connective tissue

Answer 19

§ the conditions reflect the function of the protein and where it is expressed
§ phenotypic effects of mutations are often highly variable depending on the specific
mutation, and so therapy is often guided by genotype
§ rarer forms of these diseases can be caused by mutation of other genes

Question 20

Mafan syndrome

Answer 20

Protein affected: fibrillin 1
Connective tissue component: elastic fibers
Genes: FBN1
Tissue affected: CV system, eyes, lungs, tendons, ligament

§ affected individuals are tall, with long hands/feet and cardiovascular effects
§ mutation in fibrillin-1 (>1800 different mutations have been reported, often missense)

Question 21

Osteogenesis imperfecta

Answer 21

Protein affected: type 1 collagen
Connective tissue component: collagen fibers
Genes: COL1A1 , COL1A2
Tissue affected: Bone

§ type I collagen is abundant in bone, so this mutation causes ‘brittle bone disease”
§ position of mutation determines severity of phenotype
§ most common mutations cause glycine to be replaced with larger amino acid (e. g. cysteine) that distorts packing of the collagen helix

TYPE I Osteogenesis imperfecta
§ mildest and most common form
§ mutations cause a quantitative defect, with ~half the amount of type I collagen produced (called haploinsufficiency)
§ bones fracture easily
§ sclerae can appear blue – thin sclerae allow the pigmented coat of the choroid to be visible

TYPE II Osteogenesis imperfecta
§ most severe form of OI – perinatal lethal
§ mutations cause a qualitative defect, with
abnormal type I collagen produced
§ very fragile bones – intracranial fractures, severe breathing difficulties
e.g. lack of skull mineralisation, severe osteopenia, multiple fractures, long bone and rib deformities

Question 22

Vascular Ehlers Danlos Syndrome (EDS)

Answer 22

Protein affected: type III collagen
Connective tissue component: collagen fibers
Genes: COL3A1
Tissue affected: blood vessels, skin

Question 23

Alport syndrome:

Answer 23

Protein affected: type IV collagen
Connective tissue component: collagen fibers
Genes: COL4A3, COL4A4, COL4A5
Tissue affected: Basement membrane of many tissues (kidneys, ears, eyes)

Question 24

Classical EDS

Answer 24

Protein affected: type V collagen
Connective tissue component: collagen fibers
Genes: COL5A1, COL5A2
Tissue affected: skin, joints

Question 25

Genetic diseases affecting collagen

collagenopathies

Answer 25

§ 1000’s of mutations, affecting a range of different tissues
§ impact depends on where the collagen gene is expressed
§ most common mutations are single base substitutions that lead to an altered amino acid (missense mutations)
§ less common mutations introduce a premature stop codon – leads to ‘haploinsufficiency’

Question 26

COL2A1 mutations — defective type II collagen

e. g. Skeletal development abnormalities

Answer 26

§ type II collagen is abundant in cartilage
§ bone is formed on a cartilage scaffold (by
“endochondral ossification”)

So mutation in COL2A1 leads to:
§ short stature
§ enlarged joints
§ spinal curvature
§ early arthritis
§ vision & hearing defects § cleft palate

For example:
§ Type 2 Achondrogenesis
§ Hypochondrogenesis
§ Kniest dysplasia
§ Spondyloepimetaphyseal dysplasia
(Strudwick type)
§ some cases of Stickler syndrome

Question 27

COL4A3, COL4A4, COL4A5 mutations — defective typeIVcollagen

e.g.Alportsyndrome

Answer 27

§ type IV collagen is abundant in basement membranes
e.g. kidney, ears, eyes

§ so mutation in these genes causes:
§ chronic kidney disease
§ hearing loss
§ eye abnormalities

§ Alport syndrome has an incidence of 1 in 5000 to 10000

§ most common form (~85%) is due to COL4A5 mutations
§ gene is on X-chromosome so inheritance is X-linked
§ affected boys get more severe disease (renal failure)

Question 28

Some of the inherited connective tissue diseases are caused by mutation in one of several genes

Answer 28

§ Stickler Syndrome (mutation of COL2A1, COL11A1, COL11A2, COL9A1, or COL9A2)

§ Ehlers Danlos Syndrome (EDS) (mutation of 1 of at least 19 genes, including COL3A1,COL5A1, COL5A2

Question 29

Stickler Syndrome

Answer 29

§ flattened facial features § eye and ear problems § joints also affected

§ at least 5 variants:
§ type I - COL2A1 mutations (75% of cases) § type II - COL11A1
§ type III - COL11A2 (non-ocular)
§ type IV - COL9A1 (recessive)
§ type V - COL9A2 (recessive)

§ collagens IX and XI are essential for type II collagen fibril formation

Question 30

Ehlers Danlos Syndrome (EDS)

Answer 30

§ at least 13 types of EDS, caused by different mutations in at least 19 genes § overlapping symptoms and variable presentation, even within families

Hypermobile EDS (type III)
§ incidence of 1 in 10000 to 15000
§ gene defect not known – active area of research! § “Benign hypermobility”

Classic EDS (type I/II)
§ incidence 1 in 20 000
§ due to COL5A1/COL5A2 null allele - expressed in
skin and other tissues that express type I collagen
§ stretchy skin that bruises and scars easily
§ hypermobile joints

Vascular EDS (type IV)
§ incidence 1 in 200 000
§ due to COL3A1 mutations – expressed in blood
vessels and skin
§ fragile skin and blood vessels - bruising, varicose
veins, arterial rupture
§ organ rupture e. g. bowel

Question 31

Vascular EDS

Answer 31

Molecular understanding of the genetic cause of inherited connective tissue diseases can guide appropriate surveillance and prophylactic intervention.

type III collagen is a homotrimer i.e. a triple helix made of 3 COL3A1 polypeptides
§ >700 COL3A1 mutations causing vascular EDS have been described - these can be divided into 2 molecular types:

Minimal type

Haploinsufficiency (HI) type