Glycosaminoglycans and ocular structure

Class 8 Dr. Pittler

PHOTORECEPTOR LIPIDS

Photoreceptors transducelight energy into a neuro-electrical signal that is sentto area 17 of the brain whichis perceived as light.

The early part of transduction,involving rhodopsin and itsG-protein (transducin), requiresthe presence of a highly fluidmembrane in the disks or disk-like structures.

The way in which nature has seen fit to give sufficient flexibility to photoreceptor membranes is to insert significant quantitiesof cervonic acid into the disk and disk-like membranes. This fatty acid has 6 double bonds and can nearly twist backwardsupon itself. This places considerable disorder into the membranes into which it is inserted as a phospholipid. From the table it can be seen that marked percentages of 22:6 exist in three of the phospholipids. CERVONIC ACID(dicosahexaenoic acid) 22:64,7,10,13,16,19

Cervonic acid is synthesized from lenolenic acid (18:3) andthis precursor must be obtained from the diet. The generalpathway (through many steps) is:

18:3 ---------------> 22:5 -------------->24:6 -----------------> 22:6 linolenate docosapentate tetracosahexate cervonate

Synthesis takes place in the liver, and after transport, thevery long chain fatty acid is transported to the inner segment of the photoreceptors. There it is incorporated into phospho-lipids.

Since this fatty acid is vital to visual transduction, there existsa “sparing” effect for it in the photoreceptors. As you will see,it is preserved after removal from photoreceptor membranesand taken back up again into photoreceptor inner segments.

Due to the high metabolic rate ofphotoreceptors, the disks of theouter segments are replaced aboutevery ten days. The turnover of disk membranes in photoreceptors was demonstrated over 20 yrs agoby R. Young using radiolabelled membrane precursors. In a seriesof experiments, it is possible tosee the initial precursor assemblyat the inner segment (1 and 2),incorporation into disk membranes(3), transport to the distal endof the rod outer segment (3->4),and shedding to a PE organelle (4->5).

time

At this point (incorporation into PE organelles), there is a choice to be made regarding the disk components including the longchain, unsaturated fatty acids such as cervonic acid. It is alreadyknown that sparing (i.e., re-use) of both opsin and vitamin Aoccurs.

It was not known, at first, what occurred with the highly unsaturatedfatty acids that are vulnerable to oxidation of their double bondsand destruction of the acid into shorter chain aldehydes. Logically,there is good reason to naturally preserve and re-use as many of the fatty acids as possible since:

1)their synthesis is limited and metabolically complicated;2)the retina has a significant supply of anti-oxidants (vitamin E)

With that in mind, investigators decided to follow thetransport and sparing of [3 H] 22:6n3, a tritiated formof cervonic acid, in its progression through the neuralretina using frog retinas as an animal model.

The figure on the right shows theincorporation as radioactive black dots.After 4 hours, uptake can be seen inthe inner segment (green arrow) of one type of rod cell (502). However, the 435-rods had already taken up cervonic acid into their outer segments (red arrow). There was nouptake into cone cells by this period(as shown by the black asterisk) whichreflects the typical slower turnover of cone cells.

The data compare the uptake ofradiolabelled cervonic acid in the outer segments (top graph) with that in the inner segments (lowergraph). By comparing time and grain density, it can be seen thatincorporation in the inner segmentsprecedes incorporation in the outersegments. By 6 hr, incorporation hadnot been made to outer segmentrods, but was seen in the inner segments. Although not shown here,data also showed a bidirectional labelling (at the inner segments thatsuggested sparing of cervonic acid).

Cervonic acid turnover in the retina. Dietary 18:3 is incorporatedinto the liver where 22:6PL is synthesized and transported to thephotoreceptor inner segments. 22:6PL is incorporated into outersegment disks (or disk-like membranes) and removed with diskshedding. The 22:6PL is returned to the inner segment via theinterphotoreceptor matrix or to the liver for re-transport to the retina.

PHOTORECEPTOR LIPIDS SUMMARY & STUDY GUIDE

1. Why is it important to have highly unsaturated fatty acids in disks and disk-like membranes of photoreceptors? [One word is not an answer]2. How would you explain what cervonic acid is?3. What is meant by the “sparing” effect for cervonic acid? Why is it important?4. Describe an experiment that shows the incorporation of cervonic acid into photoreceptors. Further explain how the same experiment could be used to show the sparing effect.5. Can you diagram cervonic acid turnover with enough detail to make it meaningful?

OCULAR GLYCOSAMINOGLYCANS

USEFUL & USELESS

What we are going to consider in this lecture:

1.Basic structural properties of GAGs and their functional properties in tissues.2.How GAGs associate with glycoproteins 3.How holo-glycoproteins (proteins + GAGs) are formed and assembled in the eye (cornea & vitreous4. GAG pathology (general and ocular)

The connective, extracellular tissue that we find in the eye has two sides: the collagen side and the glycosaminoglycan side – both must work together to form tissues.

Glycosaminoglycans (GAGs) were formerly called mucopolysaccharides since they were originally discovered in mucous or mucoid material such as nasal discharges and around sensitive areas of body openings. From that term we have the diseases that are known as mucopolysaccharidoses, a term that is still used today. GAGs are polymers that consist of repeating two sugar units of a sugar and an amino sugar.

An example unit is shown here:

123

6

4

5

NOTE:1)the N-acetylated group in the right hand sugar2)the presence of negative charges on the carboxylate and sulfate groups3)the alternating beta 1-> 3 and beta 1-> 4 linkages4)a carboxylate in the position is uronate; in the position is an iduronate5)sulfation make take place in several positions – each adds another negative charge

20-60 stands for the numberof two sugar units found inthis GAG

Here are four basic GAG units that occurin the eye. Hyaluronic acid (hyaluronate)-- a component of the vitreous -- has one less charge per unit and contains N-acetyl glucose rather than N-acetyl galactose.

Keratan sulfate -- a component of thecornea, like CS -- has galactose in theleft hand unit, but the N-glucosaminein the right hand unit.

Dermatan sulfate – also a componentof the cornea – contain iduronic acid, but is otherwise like chondrointinsulfate.

These small differences do not seemto make much difference in the corneal components except for theaccumulation of negative charges. One otherpoint is that the corneal GAGs have relatively short lengths (~120 glycan units) whereashyaluronate is composed of ~50,000 glycan units per molecule. It can, therefore, becontained in a much larger volume.

There are six glycan units in this partial structue of hyaluronate.If you can imagine a molecule with 50,000 of these units, then youwould have an idea of what one hyaluronate molecule looks like inthe vitreous. The point is that these molecules (in very twisted and curved forms) absorb tremendous volumes of water and help the gel to have viscoelastic properties. A viscoelastic property allows deformation with the ability to return to an original shape with the same volume.

GAGs ARE BOUND TO GLYCOPROTEIN APOPROTEINS

This may sound like double-talk, but it is inherited with the difficulty that arose from the literature over the years. Here is thegeneral classification:

GLYCOPROTEIN (a protein to which sugars are bound)

GLYCOPROTEIN PROTEOGLYCAN(has bound oligosaccharides) (has GAGs bound to it) Here’s the difficulty: a proteoglycan may refer to the holoproteinor the apoprotein (protein less the GAGs) and the literature is not always clear in making the distinction. HERE we will alwaysrefer to the holoprotein. Also glycoprotein may be general or refer to a protein bound to oligosaccharides.

LINK OLIGOSACCHARIDE: GAL-GAL-XYLOSE (-Ser-PROTEIN)

GLYCOPROTEIN EXAMPLE:Rhodopsin

PROTEOGLYCAN EXAMPLE:Lumican

CORNEAL PROTEOGLYCANS

There are two proteoglycans in the human corneal stroma:decorin and lumican. Each protein has a molecular weight of~40,000 D and can bind 1-3 GAGs. Strangely enough, these proteoglycans are also glycoproteins – in the sense that there is an oligosaccharide at one end of the molecule and a “link” oligosaccharide that connects the protein with each GAG. Lumican binds only keratan sulfate while decorin may bind to either chondroitin sulfate, dermatan sulfate or keratan sulfate.

These molecules act as molecular spacers between type I collagen fibers and also exhibit some viscoelasticity.

Shown here are two collagen tissue sections from the corneal stroma (on the left) and the sclera (on the right). The illustrations indicate that the fibers are separated, even though the separation of scleral fibers is less organized. So spacing is a characteristic of fiber separation in both tissues.

This is an immunoelectron micrograph using a labelled (dark splotches) antibody to decorin in the human sclera.

Proteoglycan “fibers” are shown at right anglesto the collagen type I fibers in A (see red arrows).The fibers were stained with copper blue andMgCl2. The diagram in B represents how GAG-proteoglycans act as spacers between collagenfibrils. Collagens types V/VI are the “go between”molecules that connect type I fibers with theproteoglycans (not shown in B).

Collagen types V/VI seem to have two roles: they limit type Ifiber diameters and they connecttype I collagen with proteoglycans.

CURRENTLY, cross-sectional areas of the corneal stroma (within a lamella) have been proposed to have the geometrical pattern shown on the right. Each collagen fiber is shown in green. The proteoglycans are indicated as six lines radiating from each fiber by blue lines. The water between each fiber is indicated by light purple coloring. This is suggested as being the best possible model to maintain equidistances from one fiber to the next.

GAGs IN THE VITREOUS --The GAG in the vitreous is hyaluronate. It is not associated with a proteoglycan apoprotein, but it does seems to be bound to the type IX/XI connecting collagen that surrounds the main type II collagen found in the vitreous.

The drawing on the right is the general pattern of type II collagen fibers in the vitreous and indicates that GAGs (namely hyaluronate) is sandwiched in between the fibers.

This is an enlarged segment of vitreous and shows better details of the relationship between collagen and GAGs. Note that the collagen fibers are type II, the fibrils are the type IX/XI hybridcollagen, andthe hyalu-ronate is depicated as“spaghetti”placed between thecollagen. Thehyaluronatecontains largevolumes ofwater and swells thetwo components. This gel isboth elastic and readily transmits the IOP.

(hybrid type IX/XI “FACIT” collagen)

(type II)

Here is a better view of type IIcollagen in the vitreous (A&B). Coming out from the fibers arethin lines of type IX/XI collagen(red arrow). A diagram of thatcollagen is seen below the figure. This collagen has areasthat are non-collagenous (FACITcollagen = fibril associated collagen with interrupted triplehelices) and are designated “NC”.In the type IX/XI hybrid, NC4 isconsidered to be the non-collagenous area that associateswith hyaluronate. As can be seen the organization of collagenand GAG are much less organized than in the corneal stroma.

Yellow arrows indicate possible NCs.

USELESS GLYCOSAMINOGLYCANS….WHERE DO OLD GAGsGO WHEN THEY DIE?

Generally, when molecules turnover, due to partial breakdownand loss of funtion, they are transported to lysosomes where low pH and a host of about 30 degradative enzymes will convert them to simpler molecules that can be reused.

Problems arise, however, when for a genetic or other reasonsome of the degradative enzymes are either missing or non-functional. This has also been seen, for example in metabolismin the case of galactosemia where one of three enzymes may benon functional.

Sometimes the enzymes are made, but due to signalling defects the enzymes fail to be transported to the lysozymesand may even be mistakenly moved out of the cell.

When glycosaminoglycans run into this problem, the diseasethat results from it are called mucopolysaccharidoses. All of these diseases are rare and all of them involve defects ofdegradative enzymes.

It is often the case that the GAGs are only partially broken down and the process halts when the step involving the defectiveenzyme is encountered. These diseases often involve the eye,particularly the cornea and/or the retina. The pathology occursdue to the fact that the partly degraded GAGs accumulate in thelysosomes, engorge the cells and then spill out into nearby tissues.This situation is particularly devastating to limbs and joints as wellas brain tissues. If the disease begins at birth (and often does) thenmental retardation quickly establishes itself.

Diseases of this type are also known as metabolic storage diseasesand can involve lipids and well as polysaccharides.

MPS DISEASES AND THEIR FEATURES

FREQUENCY

Each lysosomal storage disease is genetically caused andcomparatively rare. However, the accumulated cases ofall types is not so rare, but each one may be very difficultto diagnose. Some examples:

Hurler’s disease 30 cases/year in the U. S.Sheie disease 10 cases/year in the U. S.Hurler/Sheie disease 30 cases/year in the U. S.

There are about 28 variations of all of the lysosomalstorage diseases.

HURLER’S DISEASE

Let us take us one example, Hurler’s disease. This diseasehas its onset in infancy. There is a linear arrest in growth at~1 year of age. There is psychomotor retardation. Since theGAGs pile up in the joints and internal organs, there is a distorted facial appearance, deformed and stiff joints withan enlarged liver and spleen. In the eyes, the cornea becomes cloudy and the optic nerve degenerates. Alsoglaucoma may occur. The disease is usually fatal within a fewyears due to congestive heart failure and respiratory pulmonary infections. Biochemically, this is the disease inwhich alpha-iduronidase is deficient. There is an accumulation of dermatan sulfate and heparan sulfate (2:1)in the tissues (including the eyes), blood and urine.

This diagram shows a typical sequence of degradation for the GAG: dermatan sulfate.Here you can see some ofthe enzymes associatedwith various GAG degradations. An important point is the early involvement of alpha-L-iduronidase in the degradative sequence (red arrow). This essentially leaves the molecule in a large, nearly intact form topile up in tissues and fluids. The odd sequence of dermatan sulfate (in case you didn’t catch it [blue arrow]) is typical of GAGs.

Here are some typical appearances of Hurler disease children.The blank, unknowing stare – the swollen organs and jointsare common. The cornea is seen at the right. In particular, notethe ring of GAG deposits in the tissue.

This picture shows the accumulation of GAGs in a histiocyte of the brain of a Hurler’s patient. A histiocyte is a type of phagocyte derived from bone marrow that invades other tissues. Note the engorgement of GAGs in the vacuoles of the cell (one colored orange).

ASSAY FOR HURLER’S DISEASE

In the assay, an artificial substrate replaces dermatan sulfate (or other GAG) with a fluorgen bound to iduronic acid. When the enzyme lyses the substrate, the product: 4-methylumbelliferone fluoresces at 446 nm in proportion to the activity of the enzyme.

TREATMENT

There are two forms of treatment:

1)bone marrow transplant. The earlier this is done the better to avoid brain irreversible damage. The bone marrow makes the missing enzyme and can be successful, but it is risky.

2)enzyme replacement therapy. This has limited success and often will not correct neural damage. It’s long term outcome is not known.

FOR REVIEW:1) Basic GAG structure and function . Do not memorize specific structures.2) Can you explain the difference between a glycoprotein and a proteoglycan?3) What is decorin and lumican?4) What is known about the nature of GAG/proteoglycans and collagens in the spacing of collagen fibers in the corneal stroma? How are collagens V/VI involved in this association? What kind of a spacing structure (geometric) is proposed for collagen and proteoglycan spacing?5) What is known about hyaluronate and type II associations? What about type IX/XI collagen there? 6)How does GAG degradation occur and where does it go wrong in the mucopolysaccharidoses?7) Can you thoroughly explain Hurler’s disease? How is it assayed?