Locomotor, ECM & Cartilage Flashcards
What are the different parts that make up the ECM?
ECM – matrix of fibers – protein fibers of various types - predominantly collagen but also elastin
Groudn substance - protein fibers are embedded diffuse matrix of proteoglycans – acidic carbs linked to protein – forms hydrated mesh
Fibroblasts – role to secrete the proteins
What are the mechanical properties of cartilage, tendons, skin and bone?
Cartilage – Elasticity, low friction, resistance to compression
Tendons – strength and flexibility
Skin – Elasticity
Bone – Strength and rigidity
What is the molecular strucutre of collagen?
Collagen – major structural component of bone, tendon, skin, arteries, cartilage, etc
Makes up 25% of dry weight of the body
Class of protein – 28 types known
Collagen – consists of three chain (1000 a.a. long) – 3 helices (left-handed helical structure – 3 a.a. per rotation) that coil around to form a superhelix (twisted to the right)
Superhelix maintained by hydrogen bonds between the three different chains
Helices – gly, pro and hydroxyproline (oxidation of proline – post-translational modification) reason for these amino acids – lack of room for large a.a. side chains
Collagen – defined by unusual triple helical structure
What is the amino acid composition of collagen?
General sequence: [Gly-X-Y]n
X is often proline
Y is often 4-hydroxyproline
Overall composition: 33% glycine
15-20% proline/hydroxyproline
Proline - imino acid – unusual bond angle between carbonyl and amino group – important for maintaining tight helical structure
Collagen is nutritionally poor as hydroxyproline is unusable - deficient in ile, phe, tyr, cys, met
What are the major types of collagen?
Every tissue has at least one major types and some minor types
Types – When it comes to the types of collagen - the helices may be identical (homotrimer) or different (heterotrimer) – note all called alpha chains but they aren’t alpha helices
How many collagen genes have been identified in the genome thus far?
There are at least 40 collagen genes, to encode all the different types of alpha-chain
Each gene has ~ 50 exons; only 20% of the gene encodes protein
Most exons encode 6 glyXY sequences (codes for 18 a.a.), so length is 6 x 3 x 3 = 54 bases
Provide a brief overview of the different parts of the secretory pathway in a cell?
Secretory protein production – all secretory proteins have a signal sequence at their N-terminus (variable length sequence – consistent property – hydrophobic – for collagen 25 aa long)
Secretion pathway
- Cytosol mRNA translated – first the signal sequence translated (5’ end/N-terminal) – recognized by signal recognition particle – directs it to engage with RER channel
- RER – Ribosome becomes bound to RER and protein becomes translated into the lumen of the RER via the channel – co-translational translocation
- Golgi – vesicle with protein is transported to the golgi where it move through the golgi stacks – Cis and trans golgi –buds from the trans golgi
- Different paths – stored in lysosome, regulated (directed) or constitutive (passive) secretion pathways
Outline the different steps in collagen synthesis.
- RER - co-translational translocation - pre-pro-collagen
- Helices form in lumen of RER + signal sequence removed
- Helices need to associate – starts at C-terminus starts by the formation of disulphide bonds + while this happens, we get amino acid modifications (glycosylation and hydroxylation – PTMs)
- Protein winds up into super helix – dependent on the hydroxyproline forming in order to form hydrogen bonds between chains – does not occur - protein is degraded and recycled
- Secrete via golgi apparatus
- End up outside the cell
- Once secreted the non-helical peptides are cleaved – forms tropocollagen
- Types of collagen that form fibrils - fibril formation and crosslinking (modification of lysine and histidine) in the extracellular space
Where are elastin fibres found and what structure do they have?
- Elastin fibers important in lungs, skin and arteries
- Fibers are elastic
- Elastin core surrounded by microfibrils formed from fibrillin
- Elastin - Unusual amino acid composition – small aa – but do not form triple helices – instead helices are cross-linked to-eachother
How are elastin fibres cross-linked - molecular view?
One example - Desmosines
Lysine is oxidized – allows for cross-linking to occur.
Used to generate highly cross-linked structure that is highly elastic
Why is elastin important to understand for emphysema?
- During inflammation neutrophils release proteinases which break down elastin
- Level of proteinase degradation is controlled by a1-antitrypsin, which is able to bind very tightly to proteinases forming an inactive complex – inhibition
- But the process of proteinase inactivation can be inhibited by…
- Mutations in a1-antitrypsin (synthesis and secretion) – increasing predisposition to emphysema
- And inhibition can also occur via oxidization of anti-trypsin by oxidants such as peroxynitrite - which are released from smoking - Both cases – there is elevated levels of proteinase activity which leads to elastin break down and subsequent emphysema
What is the basic structure and characteristics of glycosaminglycans?
Glycosaminglycans - negatively charged polysaccharide units - consist of repeating disaccharide units
Characteristics - polysaccharides, acidic, negatively charged and poly-dispersed (variable MW)
Consists of a unronic sugar and amino sugar
Outline the general structure of proteoglycans and what are it’s properties?
Proteoglycan structure - sugar chains (glycosaminoglycans) attached to a core protein
Sugars attach at consensus sequences
These proteoglycans can then attach to a core protein forming even larger structures.
Examples in cartilage - Aggrecans – can associate non-covalently with hyaluronic acid
Properties - Large structure, high acidic (covered in carboxyl and sulphate groups), negatively charged, heavily hydrated + associates with many cations - acts like a sponge for water
What implication/impact does the negative charge of proteoglycans have?
Negative charge - attracts +ively charged ions
Charged ions draws in water
Faciliates/allow for the property of compressability - movement of water - water drawn in (acting like a cushion) and pushed out
What is mucopolysaccharidoses?
Mucopolysaccharidoses are set of rare genetic conditions where we get accumulation of partially degraded glycosaminoglycan in lysosomes and not in the ECM
This causes skeletal deformities and mental retardation.
What is the chemical composition of bone?
Chemical composition
20% protein (mostly collagen type I) - organic matrix
70% Minerals (inorganic salts – basic form of calcium phosphate - hydroxyapatite) - Other ions like fluoride and magnesium are also present
10% Water
What are the stages of bone deposition?
- Formation of the collagen matrix
- Deposition of CaHPO4 at the ends of collagen fibrils
- Conversion of calcium phosphate to hydroxyapatite
3Ca3(PO4)2.Ca(OH)2 – forms crystalline matrix
What are the causes of poor mineralisation?
- Vitamin D deficiency (affects intestinal absorption of Ca2+)
- Chronic metabolic acidaemia (affects hydroxyapatite formation)
Name some nutritional and genetic conditions associated with ECM?
Nutritional – associated with vitamin C (Scurvy) and D (e.g. rickets)
Hereditary conditions –most effecting collagen but not all
- Osteogenesis imperfecta type I (decreased) and II (abnormal) - brittle bone disease
- Alport syndrome – rare condition that effects type 4 collagen – mainly effects the kidney
- Elhers Danlos syndrome - heterogenous collection of diseases – classification runs to 14 types – effecting collagen synthesis, processing and assembly
Most common type – hypermobile EDS – cause unknown – hypereleastic joints + risk of dislocations
Most dangerous type – decreased amount of type III collagen
What goes wrong in collagen synthesis when vitamin C is deficient - mechanism?
Oxidation/hydroxylation of proline requires ascorbic acid as a co-factor – required to maintain enzyme in active state
This keeps iron in prolyl hydroxylase in it’s ferrous form (Fe2+) instead of the ferric form (3+) - ascorbic acid acts as a reducing agent
Prolyl hydroxylase - responsible for catalysis
What are some typical symptoms associated with scurvy?
- Poor wound healing
- Hair and tooth loss
- Capillary weakness
- Stunted growth
What roles does vitamin D play in terms of calcium metabolism, how is it generated and what happens when it is not present?
- Promotes Calcium uptake in the gut
- Promotes mineral deposition in the skeleton
- Inhibits calcium and phosphate excretion
Vitamin D – not taken up in diet – UV dependent reaction – Cholesterol is the precursor for vitamin D – followed by hydroxylation in the liver and kidney before forming active form
In the absence of vitamin D – poor bone mineralization – rickets
If we have a heterozygous individuals with a mutation in alpha1 chain of collagen - what proportion of collagen will carry 0, 1 or 2 copies of the alpha1 point mutation?
Example using type 1 collagen
Heterozygous mutation in genes encoding a1 chain–
- 50% of type 1 collagen chains will carry mutated alpha 1 strand - 50% of chains produced will carry this mutation
- 25% of type 1 collagen chains will 2 strands with mutations
- 25% of type 1 collagen chains wil be completely normal
Total of 75% of collagen will be effected
Does the position of mutations matter in terms of their impact of collagen triple helix formation?
Effect of mutations on the triple helical structure
Yes, mutations do exhibit polarity
Tendency for severe mutations to be located at the C-terminus - Why? Assembly starts at the C-terminal end – will effect winding up of helix from the start –> mutations here are less permissable
What will we be focusing in the locomoter module?
Introduction to fundamental biomedical and clinical aspects of the bones, cartilage and joints of the musculoskeletal system
Look at the basic science of connective tissue all the way to how disorders of the connective tissue manifest at the clinical level
What are the different groups/categories of connective tissue?
What are chondrocytes?
Cells responsible for cartilage formation - synthesis and breakdown of ECM
Can synthesise the full range of ECM proteins (collagens/ proteoglycans, degradative enzymes and inhibitors of enzymes).
Normally synthesise cartilage-specific ECM components
Cell metabolism highly sensitive to the physico-chemical environment.
Specialised matrix surrounds cells (lacuna or chondron)
No cell division in healthy tissue – life-span years/decades?
What are fibroblasts?
A fibroblast is a type of cell that contributes to the formation of connective tissue
Fibroblasts and fibrocytes are two states of the same cells, the former being the activated state, the latter the less active state.
Involved in the synthesis of fibrous matrix proteins, particularly collagens (mainly type I collagen) - forming rope-like network.
Cell metabolism is highly sensitive to the physico-chemical environment.
Critical role in wound healing.
The lifespan of fibroblasts is months
What is hyaline cartilage? What are its properties/characteristics?
Hyaline cartilage - otherwise known as articular cartilage - present on surfaces of moveable joints
Glassy/shiny appearance & low friction surface
- Withstands compressive and tensile forces – load-bearing but NOT elastic
- Pliable - spreads loads over ends of bones – no focal points of pressure
- working with synovial fluid, provides a low friction surface for articulating joints.
Mainly made up of type II colagen and some type IX (9) collagen
Connects to the underlying bone at the osteochondrial junction
Cells - Chondrocytes - surrounded by lacuna
What are the resident cells of fibrocartilage?
Fibroblasts
Mainly produce type I collagen – not a basket structured matrix but rather a rope-like matrix
Produce small proteoglycans rather than large
Where is elastic cartilage found? What are its properties?
Elastic Cartilage - auricle of ear, epiglottis, etc.
Histologically very similar to hyaline
BUT - Contains elastin - highly & reversibly deformable.
Ideal for a flexible skeleton
Fibroblasts synthesise elastin, collagens, small PGs, and degradative enzymes (and their inhibitors)
What are the role of tendons and ligaments?
Tendons transmit load from muscle to bone
Ligaments transmit load/give stability from bone to bone (joins bones together)
Structures are adaptive - Cells adapt to prevailing mechanical forces by modifying ECM synthesis.
For example - depending on the location of the tendon and the different compressive forces we can see varying levels of proteoglycans and collagen
What factor determines whether we can synthesis or breakdown of cartilage?
Matrix synthesis/breakdown is totally controlled by chondrocytes
But this is dependent on the balance of loading
Normal dynamic loading; synthesis = breakdown
Greater loading; synthesis > breakdown
Cartilage degeneration; breakdown > > synthesis –> can cause permanent cartilage damage
Concept is illustrated by attached diagram
Do we expect high levels of dye penetration in a mobile joint?
Yes, mobile joints show higher levels of dye penetration when compared to immobile joints
How do tendons and ligaments differ in terms of elasticity, toughness, fibroblast organisation, Collagen/proteoglycan/elastin content?
