Chapter 8

Fibrous proteins

Major fibrous protein of epithelial tissues is keratin

Major fibrous proteins of connective tissue are:

Collagen

Elastin

Dr. Stephen C. Hardies 7-3735 437D

hardies@uthscsa.edu

The fundamental building block of a keratin fiber is a coiled coil dimer of a type I and type II keratin polypeptide.

A.

coiled coil

B.

C. heptad repeat: hxxhxxx, where "h" means hydrophobic

and "x" means any residue.

-helix

-helical coiled coil domain

-Keratin

Where: Major protein of hair, skin, nails, some oral mucosa; small amounts in all epithelial cells.

Cellular location: Intracellular; not found in connective tissues

Structure: fibrous bundle of coil-coiled -helixes.

Crosslinks: disulfide bonds

Multigene family: family members differ by:

tissue structure Hard Soft

amt. Cys More Less

amt. Crosslinks More Less

Function: Provide insoluble structural protein for body surfaces

True or False?

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a) Proteins are linear chains of amino acids with polarity. Polarity means that there is directionality caused by adjacent amino acids being joined from COOH group to NH2 group. One end of a polypeptide will be termed the N-terminus and the other the C-terminus.

b) Disulfide bonds are crosslinks between sulfur atoms of eithercysteine residues or methionine residues.

c) An alpha helix can accommidate any sequence of amino acids.

Collagen

Where: 30% of total protein. Major protein of connective tissues: bones, tendons, ligaments, basement membranes, dentin, cementum, (not enamel).

Cellular location: extracellular matrix.

Structure: triple helix (tropocollagen). Subsequent to secretion, tropocollagen is assembled and crosslinked to make insoluble collagen fibers.

Function: Provides tensile strength to soft connective tissues. Tissues that must be elastic but exhibit tensile strength (e.g. ligaments) have a mixture of collagen and elastin. Collagen fibers in bone reinforce against fracture.

True or False?

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a) In woven bone, collagen fibers are laid down in a disorganized array, whereas in lamellar bone they arelaid down in a more organized parallel fashion.

b) Woven bone is stronger than lamellar bone.

c) Laminar bone is made by remodeling woven bone.

Features of collagen primary structure

-Gly-Pro-Met-Gly-Pro-Ser-Gly-Pro-Arg-

-Gly-Leu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp-

-Gly-Pro-Gln-Gly-Phe-Gln-Gly-Pro-Hyp-

Repetitive character of sequence:

Collagen 3 stranded helix

The collagen triple helix is stabilized by an interchain hydrogen bonding network involving the hydroxyl group of hydroxyproline, the glycine carbonyl group, and water molecules.

True or False?

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a) Hydroxyproline and hydroxylysine are among the 20 amino acids inserted into polypeptides by ribosomes.

b) Glycine is NH2-CH2-COOH.

c) Glycine is the only amino acid that doesn’t have a D and an L isomer.

Type I collagen fibril

EM

670 angstrom D period

tropocollagen

Key assembly

interaction

Gap

gap

gap

gapgap

Ca10(OH)2(PO4)6

True or False?

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a) Ca10(OH)2(PO4)6 is the same mineral (hydroxyapatite) that forms enamel.

b) Whereas bone is reinforced by collagen fibers, enamel uses a different protein named amelogenin.

c) Enamel has a higher mineral content than bone.

Types of Collagen

Type Composition Tissue distribution

I [1(I)]2

2(I) Skin, tendon, bone, dentine

II [1(II)]3 Cartilage

III [1(III)]3 Extensible connective tissues (skin, arteries)

IV selection of 6 chains Basement membraneV

1(V)2(V)3(V) Associated with Type I

VI 1(VI)2(VI)3(VI) Associated with Type I

VII [1(VII)]3 Epithelial anchors

VIII 1(VIII)2(VIII)2 Hexagonal lattice in endothelium

IX 1(IX),2(IX),3(IX) Associated with Type II, bound to

glycosaminoglycansX [1(X)]3 Hexagonal lattice in mineralizing cartilage

XI 1(XI)2(XI)1(II) Associated with Type II

XII [1(XII)]3 Associated with Type I

XIII [1(XIII)]3, many variants Membrane bound

XIV [1(XIV)]3 Associated with Type I

XV [1(XV)]3 Many tissues

XVI [1(XVI)]3 Many tissues

XVII [1(XVII)]3 Skin, membrane bound

XVIII [1(XVIII)]3 Many tissues

XIX [1(XIX)]3 RhabdomyosarcomaProckop and Kivirikko, Ann. Rev. Biochem. 1995, 64:403-34.

fibril

50 nm5 nm

Type VI

Anchoring fiber

Type IV collagen forms planar arrays

and makes basement membranes

Type VII collagen anchors basement membrane to underlying connective tissue (stromal) cell layer.

Type III collagen

Sometimes called elastic collagen or extensible collagen

Collagen type III micrograph visualized in polarized light, showing crimped organization of the fibrils.

Osteogenesis Imperfecta

Blue sclera

Dentinogenesis Imperfecta

Opalescent and cracked teeth

Defects in Type I collagen cause:

Type I: associated with OI.

Will probably need full crown coverage.

Abraded teeth in Dentinogenesis Imperfecta

Chronic bone fractures in Osteogenesis Imperfecta

An example of a mutation underlying Osteogenesis Imperfecta

988

Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala

CCG GGT CCT CGC GGT CGC ACT GGT GAT GCT

Pro Cys Pro Arg Gly Arg Thr Gly Asp Ala

CCT TGT CCT CGC GGT CGC ACT GGT GAT GCT

True or False?

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a) DNA is a polymer of bases named A,T,G, and C.

b) The sequence of bases in a gene determines the sequence of the polypeptide that will be produced.

c) A “mutation” is a heritable change to the sequence of a gene that causes the encoded polypeptide to have altered function.

d) Some individual residue changes may have large effects on the protein function, whereas other changes may have little or no effect.

e) Sickle cell anemia is an example of an inherited disease caused by a single residue change in a protein.

Ehlers-Danlos Syndrome

Hyperplasticity of the skin

Disorders due to defects in collagen.

Disorder Collagen Defect Clinical Manifestations

Ehlers-Danlos IV Defect in type III Arterial, intestinal, or uterine rupture; thin,easily bruised skin

Osteogenesisimperfecta

Decrease in type I Blue sclerae, multiple fractures, low bonemass

Dentinogenesisimperfecta

Found for some type I defects Discoloration; enamel chips off the dentin;defect is in the dentin.

DystrophicEpidermolysis Bullosa

Defect in type VII Blistering of skin and mucous membranes

Chondrodysplasia(various types)

Defect in type II Short-limbed dwarfism, skeletal deformity

Alport syndrome Defects in type IV (specializedminor forms)

Kidney disease, hearing loss, ocular lesions

Osteoporosis (1-3%) Defect in type IOsteoarthritis (subset) Defect in type II or type IXSchmidt MetaphysealChondrodysplasia

Defect in type X Short limbs, bowing of legs

Ehlers-Danlos VII Amino terminal propeptide presentdue to COL1A mutation

Hyperextensible, easily bruised skin, hipdislocations

Scurvy Decreased hydroxyproline Poor wound healing, deficient growth;increased capillary fragility

Ehlers-Danlos VI Decreased hydroxylysine Hyperextensible skin and joints, poor woundhealing, musculo- skeletal deformities

Ehlers-Danlos V Decreased cross-linking Skin and joint hyperextensibility

Steps in collagen biosynthesis:

•Translation on rough ER and entry into ER.

•Hydroxylation in the ER.

•Triple helix assembly.

•Glycosylation, transport to Golgi, further glycosylation.

•Secretion.

•Removal of propeptides.

•Assembly into fibrils.

•Crosslinking.

Collagen hydroxylation

Perifollicular abnormalities. Gingival abnormalities

Scurvy

True or False?

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What are preliminary steps to get procollagen polypeptides intothe ER?

a) Depending on the synthetic cell type, transcription factors willselect specific preprocollagen genes from a family of such genes be transcribed and translated.

b) The preprocollagen polypeptide is released into the cytoplasm.c) A special sequence on the N-terminus called the ‘signal peptide’

directs the preprocollagen to a pore in the ER through which itenters the ER.

Examples of collagen nomenclature:

An individual polypeptide: 1(I) procollagen

Assembled triple helix with propeptides still on: Type I procollagen

Triple helix after propeptide removal:

Type I tropocollagen

Assembled into 50 nm fiber:

Collagen microfibril

A third lysine (from a third tropocollagen) can add to make pyridinoline.

Crosslinks involving hydroxylysine are more stable than those involving lysine.

There is also a crosslink involving histidine.

Steps in collagen biosynthesis:

•Translation on rough ER and entry into ER.

•Hydroxylation in the ER.

•Triple helix assembly.

•Glycosylation, transport to Golgi, further glycosylation.

•Secretion.

•Removal of propeptides.

•Assembly into fibrils.

•Crosslinking.

+ (or else degrade)

vitamin C

+

HOlys HOpro

++

Elastin:

Where: elastic connective tissues.

Cellular location: extracellular matrix

Function: add elasticity to connective tissue

Structure: beta spiral

Crosslink: desmosine

Facial features associated with Williams Syndrome

Dental features include small widely spaced teeth and malocclusion.

Genetic defects in elastin underlie Williams Syndrome

Dynamic structure of elastin:

The structure of elastin is called a spiral, and is loosely held together by the hydrophobic force.

It is easily deformed to an extended configuration, but will relax back to a compact conformation.

Repeating unit: PGVGV

Fibrillin

Where: connective tissue; extracellular matrix

Structure: forms outer envelope of elastin microfibrils

Genetic defects in fibrillin result in Marfan’s Syndrome, characterized by a tall gaunt appearance, joint problems, an often leading to death by aortic aneurysm.

Abraham Lincoln is thought to have had Marfan’s syndrome.

Aortic Aneurysm

Enzymes that turn over connective tissue

Common names: collagenase, gelatinase, elastase

Formal names: Matrix Metalloproteases (MMPs)

Function: degrade connective tissue in support of tissue remodeling, wound healing, and cell migration (including during metastasis).

Family of Zn++ -requiring zymogens embedded in connective tissue. They can be activated in a cascade starting from a cell surface MMP.

True or False?

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Zymogens are inactive forms of enzymes that will become activated by cleavage to remove a propeptide. Zymogenes are also called “proenzymes”. Examples of zymogens are:

a) trypsinogen

b) collagen

c) coagulation factors

d) DNA polymerase

e) cathepsin

Proteoglycan aggregate

Where: major component of ground substance, the material within which collagen and other fibers are assembled to form connective tissue.

Function: make space, absorb water and allow compressibility through water flow, act as reservoir of Ca++ prior to mineralization.

Proteoglycan aggregate

True or False?

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Which of these are glycosaminoglycans?

a) b)

c) d)

Proteoglycan breakdown associated with mineralization

Adhesion proteins

Where: cell surface

Functions: adhere cells to extracellular proteins (or to other cells), sense presence of extracellular proteins, sense mechanical stress in tissues.

Integrins

cell membrane

inactiveOutside in signaling

Inside out activation

True or False?

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a) Integrins would be a good choice of adhesion protein to use for a cell like a platelet that has to very rapidly switch from a nonbinding to a tight binding mode.

Integrins bind the sequence RGD in ligand proteins. We’veseen that in:

b) bone proteinsc) matrix metalloproteasesd) keratin

Tissues of Teeth