Section 11: Extracellular Macromolecules

Section 11: Extracellular Macromolecules

• Fibrous proteins: keratin, collagen and elastin

• Fibrous proteins: keratin, collagen and elastin

02/21/06

Selected Extracellular and Cytoskeletal Proteins

• Connective Tissue Fibrous Proteins– collagen– elastin– keratin – fibronectin

• Other Fibrous Proteins– fibrin– myosin (partially)

• Cytoskeleton Proteins– actin– keratin – intermediate filaments– microtubules

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Cell Adhesion

• Receptors and Integrin are transmembrane proteins

Fibronectin

FibronectinReceptor

Epithelial Cell

Fibroblast

Proteoglycans

Laminin:Entactin

Laminin Receptor

Integrin

ExtracellularMatrix:

Collagen IV

2

Cell adhesion proteinsCell adhesion proteins

Laminin FibronectinLaminin Fibronectin

© 2000 by Geoffrey M. Cooper

Integrin

3

A chain

Collagenbinding

B2 chainB1 chain

Cellbinding Cell

binding

Enactinbinding

Proteoglycan binding

Collagen bindingCell binding

Proteoglycan binding

Matrix binding

Actin

Integrin

Extracellular matrix

Plasma membrane

Association Between Cell and

Extracellular Matrix

• Some of the fibrous proteins are transmembane and connect (and communicate) to the cytoskeleton (actin, keratin, microtubule, tailin, vinculin).

Fig. 11-22, Lehninger.4

Elastin• Elastin (64-66kD) is rich in prolines and non-polar side

chains, and one third of its amino acids are glycine.• As a result, its has uncommon secondary structure

(more random structure than found in other proteins). It does not have a stable tertiary structure.

• Elastin is very resilient. It can be stretched to lengths many times greater than in its relaxed state. It can also be compressed.

• Elastin is common in many connective tissues, along with collagen, especially if the tissue undergoes physical stress. It surrounds arteries, is in the lung and in ligaments.

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Elastin Structure and Function• Elastin interconverts between a number of conformations, both

disordered (upper two on left) and -spiral (bottom left).• After cross-linking, when elastin is stretched (or compressed) it

is less stable and it returns to the disordered conformations.

6 (Fig. 4-28, Rawn) (Fig. 4-30, Rawn)

Elastin Cross-linking

• Some lysine residues in elastin are deaminated and oxidized to the aldehyde level.

• They combine with each other and with other lysines to form lysinonorleucine and desmosine cross-links

NH

CH

O

CH2 CH2 CH2 CH2NH CH2

CH2CH2

NH

CH

O

CH2

lysinonorleucine

+CHNH

CH

CH2

CH2

CH2 CH

CH2

CH2

CH2

CH2

CH

CH2CH2CH

NH

NH

NH

OO

O

CH2

CHNH

O

CH2

desmosine

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Keratin• Keratin is rich in cysteines.• Its secondary structure is mostly -helical.• The helices form coiled coils (on right).• The coiled coils pack into higher order

elongated structures.• Keratin properties depend strongly on the

degree of disulfide cross-linking.– With low levels of cross-linking, it is flexible (hair,

skin).– It can be made very hard with additional cross-linking

(claws, horns).

• Extracellular via whithering of keratin-filled cells.• Intracellular: cytoskeletal intermediate filaments.

Fig. 3.34

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2 nm

Keratin Supramolecular Structure• Two coiled coils bind together to

form a protofibril (below).• Protofibrils assemble into

various microfibrils (on the right).

Fig. 4-5Fig. 4-6Rawn

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Keratin Cross-linking

• The structure of keratin is strengthened by disulfide cross-links from one helix to another.

CH CH2 S

NH

O

CHCH2

NH

S

O

CH CH2 SH

NH

O

disulfidecross-link

CHCH2

NH

SH

O

two cysteines

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Collagen Types Collagen Types Fibrils – long triple helicesI. Skin, tendon, bone, dentin II. Cartilage and vitreous humor III. Skin, muscles, blood vessels (frequently found with type I)V. Fetal tissues, placenta, interstitial tissues XI. Cartilage

Fibril associated – interrupted triple helicesIX. Cartilage, vitreous, humor XII. Embryonic skin and tendons XIV. Fetal skin and tendons

Fibril associated -- beadedVI. Most interstitial tissues

SheetsIV. All basal laminae VIII. Endothelial cells, X. Cartilage growth plate

Fibrils – long triple helicesI. Skin, tendon, bone, dentin II. Cartilage and vitreous humor III. Skin, muscles, blood vessels (frequently found with type I)V. Fetal tissues, placenta, interstitial tissues XI. Cartilage

Fibril associated – interrupted triple helicesIX. Cartilage, vitreous, humor XII. Embryonic skin and tendons XIV. Fetal skin and tendons

Fibril associated -- beadedVI. Most interstitial tissues

SheetsIV. All basal laminae VIII. Endothelial cells, X. Cartilage growth plate

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Collagen• Collagen has glycine in every third position, is rich in

proline, and contains hydroxyproline and hydroxylysine residues.

• Collagen does not have secondary structure, but three highly extended strands interact to form a triple helix.

• Collagen triple helices form a rod-like fibril or sheet aggregate that is somewhat flexible, not extensible, and can be very strong.

• Cross-linking increases its strength.• It is common to connective tissue and is present in

bone, dentin and cementum.• Collagen is the most abundant protein in the

biosphere.

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Collagen Triple Helix• Prolines, especially hydroxylated prolines,

keep the individual chains extended, and increase Tm (keep it above body temperature).

• The small size of the glycine sidechain in every third position allows the three strands to come close together.

• There are interstrand hydrogen bonds.• It is not very extensible because it is

already extended (3.1 per residue vs 1.5 for -helix).

• Glycosylation of hydroxylysines appear to modulate fibril or sheet formation by the triple helices.

Fig. 11-5Stryer 3rd

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Collagen Amino Acid Modifications

• Hydroxyprolines form interstrand hydrogen bonds.• These post-translational modifications require ascorbate to

reverse the prolyl hydroxylase active site Fe(III)-O- .

N CH

CH2CH2

CH2

O

N CH

CH2CH2

CH

O

OH

NH3

+

CH2

CH2

CH2

CH2

CHNHO

NH3

+

CH2

CH

CH2

CH2

CHNHO

OH

-ketogutarate succinate

-ketogutarate succinate

prolyl hydroxylase

lysyl hydroxylase

(ascorbate)

(ascorbate)

O2 + CO2 +

O2 + CO2 +

proline4-hydroxyproline

lysine 5-hydroxylysine

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Glycosylation of Hydroxylysine

• The hydroxylysines are modified by sequential glycosylations, giving lysyl-gal-(12)-glc

• Activated sugar complexes are usually UDP-sugars.• Higher levels of glycosylation favor formation of

sheet structures by the collagen triple helices.

galactosyl transferase

glucosyltransferase

UDP-galactose UDP-glucoseNH3

+

CH2

CH

CH2

CH2

CHNHO

OH

O

CH2

O

OHOH

OH

OH

H

H

H H

H

O

CH2O

O

OH

OH

OH

H

HH

H

H

CH

CH2

CH2

CH2

CH

NH3

+

NH

O

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Procollagen and

Tropocollagen Formation

• The three strands of procollagen are cross-linked by disulfide bonds near the C-terminal end, initiating triple helix formation.

• Procollagen is soluble, but tropocollagen, formed by hydrolyzing away peptide fragments at both ends, is not.

Fig. 4-24Rawn

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Aggregation and Cross-linking• Tropocollagen

spontaneously aggregates into elongated staggered arrays, shown in two dimensions at right.

• Hydroxylysine glycosylation determines fibril or sheet formation.

• Cross-linking strengthens the structure (lower).

• In bone, dentin and cementum, biomineralization begins in the gaps (hole zones) between the individual tropocollagens (type I).

Fig. 4-24, Rawn

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Collagen Cross-linking

• Lysines and hydroxylysines are used in cross-linking.• Other than oxidation to the aldehyde level, the reactions

appear to be non-enzymatic.

Aldol Cross-link

Aldehyde Derivatives

Lysines

lysyl oxidase

NH

CH

O

CH2 CH2 CH2 CH2NH3

+

NH

CH

O

CH2 CH2 CH2

H

O

NH

CH

O

CH2 CH2 CH2

H

C CH2

NH

CH

O

CH2

O O

NH3

+CH2 CH2

CH2

NH

CH

O

CH2

CH2CH2

NH

CH

O

CH2

O

Hhydroxypyridimiumcross-link(from 3 lysines)

H

H

CHN

CH

CH2

CH

CH2

CHNH

O

O

CH2

CH2

CH

NH

O

CH2

CH NH

O

CH2

O

+

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Fibroblast to Mature Collagen Fiber

• Procollagen in vesicles is transported to the cell membrane in vesicles, and then secreted via exocytosis.

• Proteolysis, assembly, and lysine oxidation leading to cross-linking occurs outside the cell.

1. Polypeptide synthesis

2. Hydroxylation and glycosylation

3. Triple helix formation

4. Secretion into extracellular matrix

Procollagen bundles

Tropocollagen bundles

Collagen fiber

Maturecollagen fiber

5. Hydrolysis of peptide bonds

6. Assembly near the cell surface

7. Cross-link formation

fibroblast cell

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Collagen Degradation

• Collagenase cuts the 1000 aa triple helix into 250 and 750 aa fragments which melt and are proteolyzed.

• Some animal tissues (for example tadpole tails) have collagenases that are used to degrade collagen during growth and remodeling.

• The collagenase of Clostridium histolyticum destroys host connective tissue, helping to make it a highly invasive bacterium.

• In periodontal disease, host collagenases help break down periodontal ligament (collagens type I and III).

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Next topic:

Section 12: Mineralized tissues.

Calcium and phosphate metabolism