Crystallins, Cataract, Collagens, Sclera

What are crystallins?

- water-soluble proteins that compose over 90% of the proteins within the lens

- they form soluble, high molecular weight aggregates that pack tightly in lens fibers, thus increasing index of refraction of the lens while maintaining its transparency

List the different types of crystallins from largest to smallest

a, b, g

Which crystallins' primary structure have post-translational modifications?

a and b (N-terminus acetylated)

What are the different crystallins' secondary structures composed of?

a: a-helical and b-sheets

b: b-sheets

g: b-sheets

What are the molecular weights of the different types of crystallin?

a: 750,000 Da

b: 50,000 or 160,000 Da (depending on subtype)

g: 20,000 Da

What is the cysteine content in the different crystallins?

a: 16/1000 residues

b: 25/1000 residues

g: 41/1000 residues

What are the pI's (pH at which all + and - charges are equal) in the different crystallins?

a: 4.9

b: 6.4

g: 7.6

What is the relative lens occurrence for the different crystallin types?

a: 35%

b: 55%

g: 10%

What does acetylation on the N-terminus on a & b crystallins do?

protects from protein degradation by adding a carbonyl group

What does phosphorylation on the serines of a crystallins do?

adds negative charges to each change; could change structure

Which crystallins does glycosylation occur on?

a and b crystallins (on serines)

Which crystallins does deamidation occur on?

a and b crystallins (conversion of Asn to Asp and Gln to Glu)

Which crystallins does oxidation occur on?

a and b crystallins

Which are the simplest crystallins structurally?

g crystallins (consist of 2 beta sheets)

Describe the structure of b-crystallin

four sets of b-sheets in which there are two N-terminal and two C-terminal ends (complex form of g-crystallin)

Describe the structure of a-crystallin

symmetrical, triple hybrid structure

What are cataracts?

lens opacification resulting from aggregation of crystallins, causes scattering of light

Which type of crystallin has chaperone activity?

a-crystallin

How can the chaperone activity of a-crystallins help b-crystallins?

adding a-crystallin to a b-crystallin solution prevents the denaturation of the b-crystallin, preventing light scattering in the lens

After a lens fiber cell is formed from a lens epithelial cell, why does it no longer have the capacity for translation?

lens fiber cell loses all its protein generating "machinery" and cell slowly loses water content with age

What are three protective mechanisms in the eye that may help prevent cataract formation?

1) chaperone activity of a-crystallin (in retina too)

2) anti-oxidative activity of glutathione (reduced GSH) (protects from H2O2 in aqueous that causes oxidation)

3) high concentration of Vitamin C in aqueous

What damaging effects can post-translational phosphorylation have on crystallins?

1) phosphorylation may weaken chaperone properties of a-crystallins by opening its structure

2) phosphorylation may make all crystallins subject to oxidation (disulfide bond formation) and the beginning of protein complex formation (and aggregation)

Why is it important to prevent crystallin oxidation?

- when the crystallin conformation is opened up, it is possible for the -SH groups in Cys to react with one another to form disulfide bonds

- if the process continues, then aggregate formation occurs and may lead to cataract formation

What are the three types of age-related cataracts?

1) cortical

2) nuclear

3) posterior subcapsular (PSC) (subtype of nuclear)

Breifly describe the mechanism of cortical cataract formation

protein aggregation, membrane rupture, debris formation

What is the protein 26k?

intrinsic membrane protein that binds to crystallin aggregates

What is the protein 43k?

extrinsic membrane protein that binds to crystallin aggregates

What is the structure of collagen? What repeating triplet is found in the molecule?

triple helix (right-handed); Pro-Hyl-Gly

Where is Type I collagen found?

cornea, sclera, skin, tendon, bone, ligaments, dentin, interstitial tissues

Where is Type II collagen found?

cartilage, vitreous humor

Where is Type III collagen found?

lens of the eye, skin, cartilaginous tissues, muscle, blood vessels (often found with Type I)

What are the three classes of collagen?

Class I: banded, fibrillar collagens that exhibit long, uninterrupted domains

Class II: contain interrupted triple helices and include "FACIT" collagens (Fiber Associated Collagens with Interrupted Triple Helices)

Class III: not attached to fibers, such as anchoring rods

Which type of collagen is the most abundant?

Type I

How is the structure of the corneal stroma arranged?

- vast majority of corneal stroma consists of 200-500 layers of flattened collagenous lamellae extending from limbus to limbus, some crossing the apex of the corneal dome

- in the anterior 1/3rd of the stroma, collagen lamellae are thin, run obliquely to the corneal surface, and sometimes split into 2-3 sub-layers that become interwoven

- in the posterior stroma, collagen lamellae tend to be arranged parallel to the surface and are thicker

What is stromal lamellae? What purpose does it serve?

- biggest corneal layer

- holds collagen fibers together

How are the lamellae in the corneal stroma connected to each other?

Four methods:

1) disulfide crosslinks (fiber to fiber)

2) fibers that cross over and weave into the fibers of adjacent lamellae

3) lamellae that often divide into smaller components that later merge with adjoining lamellae

4) sometimes lamellae split into two to three sub-layers that become interwoven

What are glycosaminoglycans (GAGs)?

long, unbranched, linear polysaccharides with hexose repeats

Where are GAGs found?

cell surfaces in the extracellular matrix

What are GAGs used for?

several are used as biomaterials: heparin, chondroitin sulfate, hyaluronic acid

What are the four most common GAGs in the eye?

- hyaluronic acid

- dermatan sulfate

- chondroitin-4-sulfate

- chondroitin-6-sulfate

What is unusual about the corneal stroma's hyaluronic acid content?

corneal stroma contains no hyaluronic acid except at the limbus where there is a gradual increase in hyaluronic acid concentration towards the sclera

How do GAGs play a role in collagen fiber arrangement?

GAGs bind to collagen fibers at specific binding sites that are essential to the spacing of the fibrils

How does the cornea regulate the size of its Type I collagen fibers?

- the more type V fibers that bind to the type I fiber, the more type I is blocked from expanding

- type V fibers are acidic at the N-termini. this or possibly steric hindrance may inhibit new type I fibers

How does the organization of collagen fibers maintain corneal clarity?

maintained by equidistant fiber spacing regulated by proteoglycans

What are the roles of Type V/VI collagen?

1) limit Type I fiber diameters

2) connect Type I collagen with proteoglycans

What are the Bowman's membrane and the Descemet's membrane?

the bowman's membrane and the descemet's membrane are extracellular matrices that are meant to act as barriers for the epithelium and endothelium

Which collagen types is the Bowman's membrane made of?

a condensation of types I and VII together with type III

Which collagen types is the Descemet's membrane made of?

types V, VIII, IX, XII

How are Type II collagen fibers organized in the vitreous humor?

type II fibers run anterior to posterior inside the globe - attaching most strongly at the vitreous base (by the ciliary body near the ora serrata) and at the posterior retina near the macula

Which GAG is located in the vitreous? What is it bound to?

hyaluronate; no associated with a proteoglycan apoprotein, but is bound to type IX/XI connecting collagen that surrounds the main type II collagen found in the vitreous

What is the role of hyaluronate in the vitreous?

drives IOP by swelling and contracting elastic gel

In the Type IX/XI hybrid, what is the non-collagenous area (FACIT collagen) that associates with hyaluronate?

NC4