The Role of Biomolecules and Cell
Structures in Gaucher Disease
Goals
•
•
Study a specific disease as a vehicle to
understand the interrelationships of biological
molecules and various cell organelles.
To learn cell function to figure out what causes
a debilitating disease.
Gaucher Disease On-Line
Resources
Good overview of Gaucher Disease for the
layperson:
» http://www.gaucherdisease.org
An in depth scientific resource concerning Gaucher
disease:
» http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?230800
Gaucher Disease: A Clinical Trial of Gene Therapy
» http://neuro-www2.mgh.harvard.edu/gaucher/genetherapy.html
Gaucher Disease
 An inherited condition
» Affects 1 in 60,000 people
» Affects 1 in 500 Jews of Eastern European origin;
1 in 10 are carriers of the gene!
 GD Results in....
»
»
»
»
»
Anemia  Always tired
Huge spleen and liver  Enlarged abdomen
Weak bones  Bent or curved legs
Neurological problems  Cross-eyed + head back
Premature death
GD is a genetic disease
 Recently a therapy has been developed
» Costs $100,000 per year.
» The therapy turned 11 year old Brian Berman's life around.
 Before therapy
» too weak to walk up stairs.
» anemia threatened his life
 After Therapy
» Hemoglobin count is normal
» Purple belt in karate
» Plays basketball and football
Reference: Biotechnology vol 9, p 600 1991 B.J.Spalding.
Gaucher Disease

Due to the inability of macrophages to digest the glycolipid,
glucocerebroside(GC).
The detailed structure of an animal cell’s plasma membrane
Breakdown of GC in Healthy People
The enzyme, GCase, breaks down glucocerbroside in most people.

GCase = Glucocerebrosidase.
GCase is defective in GD Patients
Why can’t GCase break down GC?
Why is it a problem if “the enzyme” doesn’t work?


Accumulation of GC in
macrophage cells causes
GD
More GC enters the cell
than can be broken down
Why do GD patients have the defective enzyme?
They inherited a mutant gene that
encodes the protein glucocerebrocidase
 a Gene is…

»
»

A segments of DNA in a chromosome
Carries information that can be decoded to
produce a specific kind of protein
DNA is a polymer of nucleotides
DNA Nucleotides

Four Kinds of nucleotides in DNA
»
»
»
»

A
T
G
C
=
=
=
=
Adenine
Thymine
Guanine
Cytosine
Why is the order of nucleotides in a gene Important?
Central dogma of Biology:
1. Order of nucleotides in a gene determines the order of amino acids in a protein
2. Order of amino acids in a protein determines…..
A.
B.
Nucleotide Structure

Nucleotides are....
the building blocks
(monomers) of DNA and
RNA
»
As monomers they
transfer energy to power
almost all processes in all
cells
»
–e.g.
ATP
~30,000 genes
code for
proteins that
perform all
life functions
GD Patients have defective Macrophage Cells
called……

Gaucher cells
»

Derived from Macrophages (big eater)
– A kind of WBC that eats dying cells , invading microbes, and
damaged tissue
Gaucher cells 10x’s larger than normal
» 100 mm vs. 10 mm
» What is accumulating within the lipid bilayer to make them so large?
» How does it get there?

Let’s review the functions of cell organelles.
Eukaryotic Cell Structure
1. Nucleus:
»
Site of DNA, the
genetic material
2. Smooth Endoplasmic
Reticulum:
»
Makes lipids and cell
membranes
3. Ribosomes:
»
Site of protein
synthesis
Eukaryotic Cell Structure
4. Rough
ER:
Makes proteins found in
membrane bound
organelles exported from
the cell
5. Golgi Apparatus:
Modifies newly made
proteins, lipids, and
carbohydrates
6. Vesicles:
Membrane-bound
“balloons” that
transport and store
substances in cells
Eukaryotic Cell Structure
7. Lysosomes:
»
Sacs containing enzymes
that digest worn out cell
parts
8. Cytoskeleton:
»
Protein fibers that help a cell
maintain its shape
9. Mitochondria:
»
Harvest energy from
organic molecules (e.g.
sugars and fats) to produce
ATP
Molecules involved with GD


What does GCase do?
Which organelle probably contains
glucocerebrosidase in most people?
»

Where are digestive enzymes?
How does it get there?
» Starting with the GCase gene in the nucleus, trace the
pathway of the enzyme to this organelle.
GCase Pathway: from GCase gene to lysosome
GCase Pathway:
from GCase gene to lysosome
GCase Pathway
1.
2.
3.
4.
5.
6.
7.
8.
GCase Gene read to make RNA 
RNA goes to Rough E.R 
Ribosomes read RNA  GCase produced 
GCase packaged into vesicles 
Vesicles transported to Golgi 
GCase modified in Golgi 
GCase packaged into vesicles 
Vesicles fuse with lysosomes
Ribosome on Rough ER Producing a Protein


Ribosome reads mRNA
to produce a protein
ID of structures...
»
»
»
»
Red = ?
Blue = ?
Brown = ?
Yellow = ?
Rough E.R. to Golgi Apparatus
Transport from Golgi Apparatus
 Proteins modified by
Golgi Apparatus are
either...
Used inside cell
e.g.?
Or
Exported from cell
e.g.?
Membrane Bound GC


Glycolipid
molecules
(GC) are
normally
found on the
surface of cells.
GC helps cells
recognize each
other
Synthesis of GC in Cells

Which organelle synthesizes GC?
»

Well, where are lipids made in cell?
Where are sugars added to newly made
biochemicals?
» I.e. where do chemical modifications occur?

Trace the biosynthetic pathway of the glycolipid,
GC, through the cell to its final home in the plasma
membrane
GC Synthesis and Transport
GC Synthesis and Transport
Digestion of GC
Now we've got GCase in a lysosome of macrophage
cells and GC in the plasma membrane of the cells
they eat.
 How does GC get into Macrophage lysosomes?
 Let’s take a look at this happens.......

Phagocytosis—a macrophage snacking on bacteria
Macrophages:
“Big Eaters”



Eat dead, injured,
and foreign cells
These cells have GC
in their membrane
Engulfed cells
transported to
lysosome for
digestion
ID of ...
1=
2=
3=
4=
5=

The formation and functions of lysosomes (Layer 1)
The formation and functions of lysosomes (Layer 2)
The formation and functions of lysosomes (Layer 3)
Lysosome Fusing with a Vesicle containing a cell
 Lysosomal digestive
enzymes digest cell
» Green = _______________
 Where is GC?
» GC is in the membrane
of_____________________
 What happens to the
GC in….
» normal people?
» GD patients?
Gaucher Cells
Macrophage with Defective GCase
 Undigested GC builds up in….

» Lysosome membrane
– Affected lysosomes become ____________ in size
» Plasma membrane of macrophage
– Affected macrophages become ________________in size
and are known as “Gaucher Cells.”
Why does the presence of Gaucher cells in the
bone marrow cause the following symptoms?
Anemia
 Huge spleen and liver
 Weak bones
 Neurological problems

»

Cross-eyed + head back
Premature death
Gaucher Cells before and after therapy
GD Therapies
How would the following therapies work at the
cellular level to overcome disease symptoms?
1. Enzyme Therapy
2. Cell Therapy
3. Gene Therapy
1. Enzyme Therapy




What would happen to the enzyme in a pill?
It would be subjected to degradation in the gut just
like any other protein.
So its given by intravenous injection.
» Obtain the enzyme from human placentas or from
genetic engineering in prokaryotes.
Need treatment every two weeks—why?
Problems Involved with Enzyme Therapy




Even if it got to the bone marrow, where would it have to go in
the cell?
Has got to get into the lysosome.
» How do we get it there?
A special signal needs to be added to proteins that are
"supposed" to end up in the lysosome. This signal is a special
sugar (called mannose phosphate).
This signal is recognized by macrophages which take it from
body fluids and put it into lysosomes.
2. Cell Therapy

Could transplant
bone marrow
from well person
to sick person.
» This works—
why?
Problems with Cell therapy
Finding a compatible donor
 20% risk of fatal complications
 Cell transplantation therapy does work!
Therefore.....

»

the presence of normal cells can reduce the
severity of the disease.
How can we make normal cells?
3. Gene therapy
1.
2.
3.
4.
5.
Isolate blood stem cells from GD patient
Insert functional gene for GCase into stem
cells using a viral vector
Put these genetically engineered cells back
into the patient
Patient’s blood stem cells reproduce to form
normal macrophage cells
Gene therapy should work because the
enzyme makes symptoms go away.
Genetically
Stem
cells modified
reproduce
to to patient
cells returned
produce normal
macrophage
cells
Cells removed
from bone marrow
Genetically modified cells
returned to patient
How gene
therapy
works
Virus inserts the normal
gene into one of the stem
cells chromosomes
Isolate blood
stem cells
Stem cells infected with virus carrying the therapeutic gene
Possible Problems
with Gene Therapy
Virus might start replicating and cause disease
 Immune system might overreact to the virus

» e.g. Jesse Gelsinger (died Fall 1999)
GC gene might go into patient's DNA in a place
that causes a mutation that results in cancer or
disrupt genes needed for life .
 Clinical trials are now being done.
