Transcript Glycosaminoglycans and Ocular Structures

Glycosaminoglycans and ocular
structure
Class 8 Dr. Pittler
PHOTORECEPTOR LIPIDS
Photoreceptors transduce
light energy into a neuroelectrical signal that is sent
to area 17 of the brain which
is perceived as light.
The early part of transduction,
involving rhodopsin and its
G-protein (transducin), requires
the presence of a highly fluid
membrane in the disks or disklike structures.
The way in which nature has seen fit to give sufficient flexibility
to photoreceptor membranes is to insert significant quantities
of cervonic acid into the disk and disk-like membranes. This
fatty acid has 6 double bonds and can nearly twist backwards
upon 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
CERVONIC ACID
three of the phospholipids.
(dicosahexaenoic acid)
22:6D4,7,10,13,16,19
Cervonic acid is synthesized from lenolenic acid (18:3) and
this precursor must be obtained from the diet. The general
pathway (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, the
very long chain fatty acid is transported to the inner segment
of the photoreceptors. There it is incorporated into phospholipids.
Since this fatty acid is vital to visual transduction, there exists
a “sparing” effect for it in the photoreceptors. As you will see,
it is preserved after removal from photoreceptor membranes
and taken back up again into photoreceptor inner segments.
Due to the high metabolic rate of
photoreceptors, the disks of the
outer segments are replaced about
every ten days. The turnover of
disk membranes in photoreceptors
was demonstrated over 20 yrs ago
by R. Young using radiolabelled
membrane precursors. In a series
of experiments, it is possible to
see the initial precursor assembly
at the inner segment (1 and 2),
incorporation into disk membranes
(3), transport to the distal end
of 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 long
chain, unsaturated fatty acids such as cervonic acid. It is already
known that sparing (i.e., re-use) of both opsin and vitamin A
occurs.
It was not known, at first, what occurred with the highly unsaturated
fatty acids that are vulnerable to oxidation of their double bonds
and 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 the
transport and sparing of [3 H] 22:6n3, a tritiated form
of cervonic acid, in its progression through the neural
retina using frog retinas as an animal model.
The figure on the right shows the
incorporation as radioactive black dots.
After 4 hours, uptake can be seen in
the 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 no
uptake into cone cells by this period
(as shown by the black asterisk) which
reflects the typical slower turnover of
cone cells.
The data compare the uptake of
radiolabelled cervonic acid in the
outer segments (top graph) with
that in the inner segments (lower
graph). By comparing time and
grain density, it can be seen that
incorporation in the inner segments
precedes incorporation in the outer
segments. By 6 hr, incorporation had
not been made to outer segment
rods, but was seen in the inner
segments. Although not shown here,
data also showed a bidirectional
labelling (at the inner segments that
suggested sparing of cervonic acid).
Cervonic acid turnover in the retina. Dietary 18:3 is incorporated
into the liver where 22:6PL is synthesized and transported to the
photoreceptor inner segments. 22:6PL is incorporated into outer
segment disks (or disk-like membranes) and removed with disk
shedding. The 22:6PL is returned to the inner segment via the
interphotoreceptor 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 &
vitreous
4. 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:
6
5
4
1
3
2
20-60 stands for the number
of two sugar units found in
this GAG
NOTE:
1) the N-acetylated group in the right hand sugar
2) the presence of negative charges on the carboxylate and sulfate groups
3) the alternating beta 1-> 3 and beta 1-> 4 linkages
4) a carboxylate in the position is uronate; in the position is an iduronate
5) sulfation make take place in several positions – each adds another negative charge
Here are four basic GAG units that occur
in 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 the
cornea, like CS -- has galactose in the
left hand unit, but the N-glucosamine
in the right hand unit.
Dermatan sulfate – also a component
of the cornea – contain iduronic acid,
but is otherwise like chondrointin
sulfate.
These small differences do not seem
to make much difference in the
corneal components except for the
accumulation of negative charges. One other
point is that the corneal GAGs have relatively short lengths (~120 glycan units) whereas
hyaluronate is composed of ~50,000 glycan units per molecule. It can, therefore, be
contained 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 you
would have an idea of what one hyaluronate molecule looks like in
the 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 the
general classification:
GLYCOPROTEIN
(a protein to which sugars are bound)
GLYCOPROTEIN
(has bound oligosaccharides)
PROTEOGLYCAN
(has GAGs bound to it)
Here’s the difficulty: a proteoglycan may refer to the holoprotein
or the apoprotein (protein less the GAGs) and the literature is
not always clear in making the distinction. HERE we will always
refer to the holoprotein. Also glycoprotein may be general or
refer to a protein bound to oligosaccharides.
GLYCOPROTEIN
EXAMPLE:
Rhodopsin
PROTEOGLYCAN
EXAMPLE:
Lumican
LINK OLIGOSACCHARIDE: GALGAL-XYLOSE (-Ser-PROTEIN)
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.
Collagen types V/VI seem to have
two roles: they limit type I
fiber diameters and they connect
type I collagen with proteoglycans.
Proteoglycan “fibers” are shown at right angles
to the collagen type I fibers in A (see red arrows).
The fibers were stained with copper blue and
MgCl2. The diagram in B represents how GAGproteoglycans act as spacers between collagen
fibrils. Collagens types V/VI are the “go between”
molecules that connect type I fibers with the
proteoglycans (not shown in B).
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 hybrid
collagen, and (hybrid type IX/XI “FACIT” collagen)
the hyaluronate is
depicated as
“spaghetti”
placed
between the
collagen. The
hyaluronate
contains large
volumes of
water and swells the
(type II)
two components. This gel is
both elastic and readily transmits the IOP.
Here is a better view of type II
collagen in the vitreous (A&B).
Coming out from the fibers are
thin lines of type IX/XI collagen
(red arrow). A diagram of that
collagen is seen below the
figure. This collagen has areas
that are non-collagenous (FACIT
Yellow arrows indicate possible NCs.
collagen = fibril associated
collagen with interrupted triple
helices) and are designated “NC”.
In the type IX/XI hybrid, NC4 is
considered to be the noncollagenous area that associates
with hyaluronate. As can be seen the organization of collagen
and GAG are much less organized than in the corneal stroma.
USELESS GLYCOSAMINOGLYCANS….WHERE DO OLD GAGs
GO WHEN THEY DIE?
Generally, when molecules turnover, due to partial breakdown
and 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 reason
some of the degradative enzymes are either missing or nonfunctional. This has also been seen, for example in metabolism
in the case of galactosemia where one of three enzymes may be
non functional.
Sometimes the enzymes are made, but due to signalling
defects the enzymes fail to be transported to the lysozymes
and may even be mistakenly moved out of the cell.
When glycosaminoglycans run into this problem, the disease
that results from it are called mucopolysaccharidoses. All of
these diseases are rare and all of them involve defects of
degradative enzymes.
It is often the case that the GAGs are only partially broken
down and the process halts when the step involving the defective
enzyme is encountered. These diseases often involve the eye,
particularly the cornea and/or the retina. The pathology occurs
due to the fact that the partly degraded GAGs accumulate in the
lysosomes, engorge the cells and then spill out into nearby tissues.
This situation is particularly devastating to limbs and joints as well
as brain tissues. If the disease begins at birth (and often does) then
mental retardation quickly establishes itself.
Diseases of this type are also known as metabolic storage diseases
and can involve lipids and well as polysaccharides.
MPS DISEASES AND THEIR FEATURES
FREQUENCY
Each lysosomal storage disease is genetically caused and
comparatively rare. However, the accumulated cases of
all types is not so rare, but each one may be very difficult
to diagnose. Some examples:
Hurler’s disease
Sheie disease
Hurler/Sheie disease
30 cases/year in the U. S.
10 cases/year in the U. S.
30 cases/year in the U. S.
There are about 28 variations of all of the lysosomal
storage diseases.
HURLER’S DISEASE
Let us take us one example, Hurler’s disease. This disease
has its onset in infancy. There is a linear arrest in growth at
~1 year of age. There is psychomotor retardation. Since the
GAGs pile up in the joints and internal organs, there is a
distorted facial appearance, deformed and stiff joints with
an enlarged liver and spleen. In the eyes, the cornea
becomes cloudy and the optic nerve degenerates. Also
glaucoma may occur. The disease is usually fatal within a few
years due to congestive heart failure and respiratory
pulmonary infections. Biochemically, this is the disease in
which 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 of
the enzymes associated
with various GAG
degradations. An
important point is the early
involvement of alpha-Liduronidase in the degradative
sequence (red arrow). This
essentially leaves the molecule in
a large, nearly intact form to
pile 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 joints
are common. The cornea is seen at the right. In particular, note
the 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?