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Three types of molecules are abundant in the extracellular matrix of all tissues:

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(1)

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

(2)

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

(3)

TEM

Thin section of cell

Connective tissue

quick-freeze deep etc of skeletal muscule

(4)
(5)

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)

(6)

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

(7)

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

(8)

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

(9)

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

(10)

纖維

細纖維

(11)
(12)

The triple helix is interrupted by non- helical segments

A lateral association of triple helices combined with C-terminal

associations results in sheet formation

(13)

Type IV collagen assembly

•EM of in vitro formed network

•thin arrows- side-to-side binding

•thick arrows- C-term domain binding

(14)

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

(15)

The ECM II: connective and other tissue

Fibrillar collagens are the major fibrous proteins in the

ECM of connective tissue

(16)

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

(17)

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

(18)

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

(19)
(20)

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

(21)

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

(22)

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

(23)

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

(24)

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

(25)

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

(26)

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

(27)

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

(28)

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

(29)

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

(30)

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)

(31)

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

(32)

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)

(33)

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

(34)
(35)

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

(36)

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

*

(37)

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

(38)

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

(39)

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

(40)

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

(41)

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

(42)

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

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

(43)

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

Dermatan Sulphate:

absent in cartilage

identified in meniscus, tendon,

skin and joint capsule

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