The extracellular matix (ECM)
Three types of molecules are abundant in the extracellular matrix of all tissues:
1. proteoglycan: a glycoproteins, high viscosity, it can bound variety of ECMs
2. Collagen fibers: provide mechanical strength and resilience.
3. Soluble multiadhesive matrix proteins: bind to and cross-link cell-surface adhesion receptors and other ECM components
Adhesion receptor (molecule) can bind to three types
The ECM of eipthelial sheets In animals, ECM:
1. Organize cells into tissue
2. Regulated the cell function via signal transduction pathway 3. Migration (development)
connective tissue → ECM is plentiful (充足)
• cells sparsely distributed within it
epithelial tissue → ECM is scant (不足)
• cells bound tightly together in sheets
• most of volume is occupied by cells
TEM
Thin section of cell
Connective tissue
quick-freeze deep etc of skeletal muscule
The basal lamina provides a foundation for epithelial sheets
Basal lamina has other function:
1.Helps four and eight-celled embryos adhere together 2.Development of neurons migrate
3.Tissue repair
Most of ECM components in the basal lamina are synthesized by the cells that rest. About four types:
1. typeIV collagen: trimeric molecules (rodlike & globular), form 2D
network
2. Laminins: form 2D network with collagen, also can bind to integrins 3. Entactin: cross-link collagenIV and
laminin, and helps incorporate other components into the ECM; a proteoglycan
4. Perlecan: a proteoglycan, can binds to and ECM and cell surface
molecules (cell surface receptor)
Interstitial Connective Tissues
Interstitial ECM’s have the same pattern of
organization as basement membrane ECMS
fibrillar
fibrillar proteins proteins glycoproteins
glycoproteins proteoglycans proteoglycans
Some examples of Interstitial Connective Tissues:
Bone, cartilage, tendons, ligaments, fascia,
lamina propria, submucosa, vitreous humor
Laminin, a multiadhesive matrix protein helps cross-link components of the basal lamina
LAMININ: a heterotrimeric protein found in all basal lamina
It binds to cell surface receptors as well as various matrix components
b: left, intact laminin molecule, characteristic cross appearance
right, carbohydrate binding LG domains Multiadhesive matrix proteins
Long and flexible with multiple domains
Bind collagen, other matrix proteins, polysacc, cell-surface adhesion receptors and extra-cell
ligands
Function in organization of extracell matrix, regulating cell-matrix adhesion, cell
migration, and cell shape
Laminin, principale multiadhesive matrix protein in basal
Heterotrimeric 820,000 daltons
Columnar and epithelia is a foundation on one surface of the cells rests Muscle or fat the basal lamina surrounds each cell
Laminin, a multiadhesive matrix protein, helps cross-link
components of the basal lamina
Sheet-forming type IV collagen is a major structural component in basal lamina (基底層)
20 types of collagen participate in the formation of ECM
All collagen are trimeric protein made from three polypeptide called collagen a chain; May homotrimeric or
heterotrimeric
Has triple helical structure, because of an unusual abundance of three amino
acids: glycine, proline, and
hydroxyproline (modified from proline)
The unique properties of each type of collagen by difference:
1.The number and lengths of the triple- helical segment
2.The segment effect 3-D structure 3.Covalent modification
glycine
Motif: Gly-X-Y, X and Y are any, but often are pro and (OH-)-pro repeats of gly-pro-(OH-)pro
Very narrow
纖維
細纖維
The triple helix is interrupted by non- helical segments
A lateral association of triple helices combined with C-terminal
associations results in sheet formation
Type IV collagen assembly
•EM of in vitro formed network
•thin arrows- side-to-side binding
•thick arrows- C-term domain binding
亞伯氏症候群(Alport's syndrome)
Mutation of C-terminal globular domain of IVα chain
Sensorineural hearing loss, blood-filled capillaries in kidney Goodpastures syndrome 古德巴斯德症候群
Autoimmune disease → auto antibody→ self attacking → α3 chains of type IV collage→ glomerular and lung basement membrane→
cellular damage → renal failure or pulmonary hemorrhage dysfunction of basal lamina
1. Autoimmune disease
2. Ab against α3 chains of type IV collagen of kidney and lungs
3. Cellular damage, progressive renal failure and pulmonary hemorrhage
The ECM II: connective and other tissue
Fibrillar collagens are the major fibrous proteins in the
ECM of connective tissue
Characterizations of COLLAGEN
The various isoforms are the most abundant proteins in the animal kingdom There are at least 16 types (or 24 types)
Types I, II and III are the most abundant and form fibrils Type IV forms sheets (found in the basal lamina)
They form triple helices
They have unique segments that interrupt the triple helix and are responsible for the unique properties of individual collagen
They contain a three residue repeat of: glycine, proline, X They are rich in hydroxyproline
There are three amino acids per turn of the helix, with pyrrolidone rings on the outside of the helix
The helix is stabilized by hydrogen bonds
The fibrous backbone of the extracellular matrix
Formation of collagen fibrils(細纖維) begins in the endoplasmic reticulum and is completed outside the cell
1. Synthesis of procollagen a on ribosomes (ER)
2. Formed trimers and
glycosylation (modification) 3. Facilitate zipperlike (拉錬)
formation and stabilization of triple helices, and binding by chaperone Hsp47. it
procollagen
4. Transport to golgi complex 5. folded precollagens
6. Secretion
7. N- and C- terminal propeptides removed
8. Trimers assemble into fibrils and are covalently the corss- link
PROCOLLAGEN:
Transfers to the Golgi
• There is a further addition of oligo-saccharides
• There is further processing to remove disulfide-containing regions and insertion into transport vesicles
• Exocytosis results in the removal of termini by extracellular enzymes
and assembly of cross-linked fibers
Synthesized by fibroblasts in connective tissue Made by osteoblasts in bone
Secreted by cells as “procollagen” →collagenase cuts off terminal domains at each end → assembly only after molecules emerge into extracellular space
Propeptides function to:
• guide intracellular formation of triple-strand structure
• prevent intracellular formation of large collagen fibrils
Posttranslational modifications
Critical for collagen molecule formation And assembly into fibrils
Scurvy (壞血病)
vitC deficiency- cofactor for hydroxylases adding -OH to pro and lys
pro-α chains not modified triple-helix not formed at RT
procollagen does not assemble into fibrils ->No collagen
Blood vessels, tendons and skin become fragile
Bruck and one form Ehler-Danlos Syndromes 結遞組織疾病 Lysyl hydroxylase deficiency
connective-tissue defects
Pro-a chain → post-translational modification → hydroxylase
→adding hydroxy group to proline → assembly → fibrils→ strong
scurvy
壞血病是一種缺乏維生素C所引起的疾病
VitC cofactor
Support the formation of normal collagen 1/3 Gly, 1/5 Pro or Hyp
Triplet Gly-X-Pro (or Gly-X-Hyp) repeats
Supertwisted coiled coil is right-handed, made of 3
left-handed a-chains
Hydroxylysine and hydroxyproline residues. These modified amino acids are common in collagen; they are formed by enzymes that act after the lysine and proline are incorporated into procollagen molecules
The covalent intramolecular and intermolecular cross-links formed
between modified lysine side chains within a collagen fibril. The cross- links are formed in several steps. First, certain lysine and hydroxylysine
residues are deaminated by the extracellular enzyme lysyl oxidase to yield highly reactive aldehyde groups. The aldehydes then react spontaneously to form covalent bonds with each other or with other lysine or hydroxylysine residues. Most of the cross-links form between the short nonhelical segments at each end of the collagen molecules.
Collagen → collagen fibril
Interaction of fibrous collagens with nonfibrous associated collagens Type I and II collagens from diverse structure and associate with
different non-fibrillar (非纖維) collagens
Includes Types VI and IX
Type IX cannot form fibrils due to interruptions in the helical structure, but it can associate with fibrils of other collagen types
Type VI is bound to the sides of Type I fibrils, linking them together Non-helical regions anchor Types VI and IX to proteoglycans/other ECM components
Strong
Bone, tendons cartilage
Ehlers-Danlos 先天結締組織異常
Joint hypermobility skin hyperextensibility
skin tends to split with minor trauma
nodules
tendency to bruise
Mutation in lysyl hydroxylase gene
成骨不全症(Osteogenesis Imperfecta),簡稱OI
Type I collagen, every third position in a collagen α chain must glycine→ mutation of glycine site → unstable helix.
Tendency of bones to fracture
Collagen found in all multicellular animals, mammals; approx 25 different genes Are main proteins in bone, tendon and skin → approx. 25% of total protein
Connective Tissue = mainly types I, II, III, V and XI, type-1 by far most common Rope-like super-helix with 3 collagen polypeptide chains wound around each
another
Packed together in ordered fashion → collagen fibrils = thin cables, 10-300 nm diameter → these pack together → thicker collagen fibres
Synthesized by fibroblasts in connective tissue Made by osteoblasts in bone
Secreted by cells as “procollagen” → collagenase cuts off terminal domains at each end → assembly only after molecules emerge into extracellular space
Propeptides function to:
guide intracellular formation of triple-strand structure prevent intracellular formation of large collagen fibrils
Characterization and functions of collagen
All 16 collagen types contain a repeating gly-pro-X sequence and form triple helices
Collagens vary in their associations to form sheets, fibrils and cross- linkages
Most collagen is fibrillar - made of Type I molecules The basal lamina contains Type IV collagen
Fibrous collagen molecules (I,II & III) form fibrils stabilized by aldol cross-links
Procollagen chains are assembled into triple helices in the RER, aligned by disulfide bonds among propeptides (which are
subsequently removed)
Fibrous collagen is subject to mutations which exhibit a dominant phenotype
Summary - Collagen
Secreted and cell surface proteoglycan are expressed by many cell type Proteoglycans and their constituent GAGs play diverse roles in ECM
Viscous proteins and glycoprotein, covalently linked to charged
glycosaminoglycan also called GAG (specialized polysaccharide chains) polysaccharides; protein + GAGs = proteoglycan
Found in all connective tissues, extracellular matrices and on the surface of many cells
A core protein is attached to one or more polysaccharides called
glycosaminoglycans* (repeating polymers of disaccharides with sulfate residues
Four classes: hyaluron, chondroitin sulfate, heparan sulfate, keratan sulfate Proteoglycans is very diversity
Modifications in GAC chains can determine proteoglycan functions (Fig 6-19)
Dense, compact connective tissues (tendon, bone)
→ proportion of GAGs is small → very little water → matrix consists almost entirely of collagen
Other extreme = jelly-like substance in interior of eye → mainly one type of GAG → mostly water, → very little collagen.
GAGs in general;
strongly hydrophilic
adopt highly extended conformations huge volume relative to their mass.
form gels at very low concentrations
multiple -ve charges attract cations → osmotically active → large amounts of water adsorbed into matrix
Create swelling pressure that is counterbalanced by tension in the collagen fibres and interwoven with the PGs.
Gels of Polysaccharide and Protein Fill Spaces and Resist
Compression
The repeating disaccharides of glycosaminoglycans (GAGs), the polysaccharide components of proteoglycans
non sulfated GAG
Localization
1. Cell surface receptors 2. Extracellular
Function
1. Bind & present growth factors 2. Extracellular matric
Glycosaminoglycan (GAG)
Biosynthesis of heparan and chondroitin sulfate chains in proteoglycans
Glycosaminoglycans (heparan or chondroitin sulfate) are covalently linked to serine residues in the core protein via linking sugars (three); keratan sulfate attached to asparagine residues, N-linked oligosaccharides
Core protein synthesis at ER; GAG chains assembled in Golgi complex Addition of keratan sulfate chains are oligosaccharide chains attached to
asparagine residues: N-linked oligosaccharides
GAG + protein = proteoglycan
GLYCOPROTEINS VERSUS PROTEOGLYCANS
Glycoproteins are vast in number & structurally very diverse
Proteoglycans are few and share a simple structure
Core protein
Repeating sugar pair
Core protein
O O O
X S S S S S S S S S S
Conserved attachment
}
Two main types of linkage: O & N N
S
S S
O
S S S S S
S
Xyl Gal G
S - Sugar in chain
{
proteoglycan = protein + GAG
GLYCOPROTEINS VERSUS PROTEOGLYCANS
Two main types of linkage: O &
N & several core attachment structures
CORE PROTEIN
N
S
S S
O
S S S S S
S
Conserved attachment
CORE PROTEIN
Repeating sugar pair
O O
X S S S S } S S Asparagine
Serine Threonine
Asparagine Serine
Serine Threonine
PGs - Only O linkage
*
GLYCOPROTEINS VERSUS PROTEOGLYCANS
Sugars varied, not all hexose
Sugar chains short (sometimes very short, or a single sugar)
Less negative charge
Sugar chains can branch
Characteristic core proteins
Sugar chains are all glycose- aminoglycans (GAGs)
Sugar chains are long
GAGs often sulfated Large negative charge
Sugar chains do not branch Sugars - small repertoire
Own core proteins
GAG can be independent of
protein or have PGs attached, eg., hyaluronan
Red (sulfate group) are essential for heparin function Blue may be present but are not essential.
Modifications in GAG chains can determine proteoglycan functions
Pentasaccharide GAG sequence that regulates the activity of antithrombin III;
heparin bind to ATIII and activated for inhibited blood clotting
ECM can regulated many functions
Heparin side chain: longer GAG
Hyaluronan resists compression and facilitates cell migration
Also called hyaluronic acid (HA), is a nonsulfated GAG.
A long, negatively charged polysaccharide that forms hydrated gels. It synthesis by a plasma membrane bound enzyme (HA synthase) and is directly secreted into
extracellulat space.
It is not covalently linked to a protein
It imparts stiffness (硬), resilience (彈 性) and lubricating (潤滑) qualities to connective tissues
Behaves as a random coil in solution
Takes up water (1000-fold its own weight) in the ECM
Binds via the CD44 receptor to the surface of migrating cells – keeping them apart Degraded by the action of hyaluronidase, an extracellular enzyme
Structure of proteoglycan aggregate from cartilage
Hyaluronan resists compression, facilitates cell migration, and gives cartilage its gel like properties
Proteoglycans form large aggregates
– proteglycans attached to a hyaluronate backbone
– can be as long as 4000 nm and a diameter of 500 nm
Function of aggregation:
– increased water retention – increased stiffness
– regulate collagen fibril deposition
Aggregated proteoglycans
Aggrecan aggregate
Proteoglycans form large aggregates
Aggrecan monomer:
– a protein backbone of 210- 250 kDa
– both chondroitin sulphate and keratan sulphate chains
attached to backbone
– chondroitin sulphate chains (100 - 150 per monomer), being located in the C terminal 90%
– the keratan sulphate (30 - 60 per monomer) is
preferentially located towards the N terminal
Hyaluronan is a glycosaminoglycan enriched in connective tissues
Hyaluronan is a glycosaminoglycan.
– It forms enormous complexes with proteoglycans in the extracellular matrix.
These complexes are especially abundant in cartilage.
– There, hyaluronan is associated with the proteoglycan aggrecan, via a linker protein.
Hyaluronan is highly negatively charged.
– It binds to cations and water in the extracellular space.
• This increases the stiffness
硬
of the extracellular matrix .• This provides a water cushion (墊子) between cells that absorbs compressive forces.
Unlike other glycosaminoglycans, hyaluronans chains are:
– synthesized on the cytosolic surface of the plasma membrane – translocated out of the cell
Cells bind to hyaluronan via a family of receptors known as hyladherins.
– Hyladherins initiate signaling pathways that control:
• cell migration
• assembly of the cytoskeleton
Glycosaminoglycans
GAG Localization
Hyaluronate synovial fluid, vitreous humor, ECM of loose connective tissue
Chondroitin sulfate cartilage, bone, heart valves
Heparan sulfate basement membranes,
components of cell surfaces
Heparin mast cells lining the arteries of the lungs, liver and skin
Dermatan sulfate skin, blood vessels, heart valves
Keratan sulfate cornea, bone, cartilage aggregated with chondroitin sulfates