Cation-Dependent Mannose 6-Phosphate Receptor: Tag, You’re it!
Brown Deer SMART Team: Maxwell Bord, Carolyn Hermsen, Wongsai Heur, Christopher Jones, Erica Kennedy, William Keslin, May Khang, Hannah Leedom,
Andrew LeMense, David McMurray, Evan Naber, Danielle Parish, Mary Elizabeth Rice, Alana Rodgers, Charles Rosio, Jordan Schubert, Alexandra Smith
Teacher: Gina Vogt
Mentors: Nancy Dahms, Ph.D., Linda Olson, Ph.D. and Jung-Ja Kim, Ph.D., Medical College of Wisconsin
Abstract
4
Approximately 50 different lysosomal storage disorders affect
one in every 5000 births, limiting life expectancy and quality of
life. Therapies are available for a handful of these disorders.
Sixty different lysosomal enzymes are responsible for
recycling macromolecules in cells. In normal functioning cells,
lysosomal enzymes are synthesized in the endoplasmic
reticulum and are transported to the lysosome. Transportation
of lysosomal enzymes occurs when a mannose 6-phosphate
(Man-6-P) tag is placed on these enzymes, which is
recognized by the cation-dependent mannose 6-phophate
receptor (CD-MPR). The CD-MPR binds to the Man-6-P tags
through a recognition site (Y45, Q66, H105, R111, E133,
R135, Y143) and transports the enzymes from the Golgi
apparatus to an endosome where enzymes are released from
CD-MPR due to a more acidic environment. Enzymes are
finally transported via vesicles to the lysosome to accomplish
their function. Without the Man-6-P tags, CD-MPR cannot
bind to the enzymes, prohibiting transportation to an
endosome and ultimately the lysosome. If lysosomal enzymes
are not properly transported, they are secreted out of the cell,
causing lysosomal storage disorders due to buildup of
macromolecules in the lysosome. The Brown Deer SMART
Team (Students Modeling A Research Topic) created a
physical model of CD-MPR using 3D printing technology.
Figure 2
2
Pompe’s disease is caused when there is less than
30% lysosomal enzymatic activity of acid αglucosidase. Lower percentages of activity
correlates to earlier development of the disease, due
to faster rate of glycogen build up.
What is the Structure of CD-MPR?
A model of the quaternary structure of CD-MPR (Figure 3) depicts mannose 6-phosphate (magenta)
bound to its binding site (lime). Loop D (yellow) forms the side of the binding pocket and participates
in an inter-subunit salt bridge (E19 - K137) (cyan). Disulfide bonds are highlighted in orange, with the
C106-C141 disulfide bond tying loops C and D together and partially stabilizing the binding pocket.
When mannose 6-phosphate leaves the binding pocket, the unbound structure shows that loop D
folds into and stabilizes the pocket by keeping the position of the
Fig. 3: CD-MPR
(bound)
sugar binding residues unchanged. This movement of loop D also
breaks the inter-subunit salt bridge, causing the two subunits to twist.
Figure 1
Plasma Membrane
*
Sometimes an enzyme is not properly
transported to the endosome and is secreted
out of the cell through the secretory pathway.
This occurs in the lysosomal storage disorder
mucolipidosis II in which the enzyme that adds
the Man-6-P tag is defective, resulting in
lysosomal enzymes lacking in
the Man-6-P tag.
Nucleus
Lysosomes with
macromolecule build up
3
CD-MPR transports the
enzymes via vesicles to the
endosome.
Early/Late
Endosome
Golgi
Apparatus
Who is Affected by Lysosomal Storage Disorders?
7
Taylor
Lysosomal storage disorders lead to
cardiorespiratory complications, cognitive
disabilities, skeletal abnormalities, and a decrease
in life expectancy. These disorders are
inherited recessively. Tests for these disorders
are available but are not commonly utilized. Embryos affected by
such disorders may result in miscarriages or still
births. The onset of lysosomal storage disorders
normally occurs at birth or shortly after, however
adult onset is possible. The movie Extraordinary
Measures, starring Harrison Ford and
6 Jenny
Brendan Fraser, highlights Pompe’s
disease in hope of generating greater
research and understanding to
enhance the present enzyme
replacement treatments available to
5 Stacy
those who are affected.
9
The Crowley Family
and Harrison Ford
2
4b
In the Golgi
apparatus, a Man-6-P
tag is placed on the
newly synthesized
enzymes. CD-MPR
then binds to the
Man-6-P tag.
After CD-MPR
releases the
enzyme in the
endosome, it
travels back to the
Golgi in order to
transfer more
enzymes.
Lysosome
Lysosomal enzymes
are synthesized at a
ribosomal site on the
endoplasmic
reticulum.
1
Figure 5
Endoplasmic
Reticulum
4a
5
In the endosome, the
enzyme is released from
CD-MPR due to a drop in
pH of this compartment
compared to the pH in the
Golgi.
Enzymes are then
transported via
vesicles from the
endosome to the
lysosome.
mannose 6-phosphate
tag
Ribosome
What are the Structural Differences Between
Bound and Unbound CD-MPR?
Figure 6
A
B
The CD-MPR in panel A represents
a side view of the receptor sitting
on a cell membrane, whereas
panel B is rotated 90o toward the
reader showing the top of the
receptor. CD-MPR’s structure
differs significantly between its
unbound and bound states. Two
simultaneous changes occur when
Man-6-P binds to CD-MPR. The
first is a scissoring motion, pivoting
on the dimer's center of mass on
the XZ plane (Fig. 6A). The second
is a twisting motion in which the
end of the molecule on the top of
axis Z, near the CD-MPR's binding
site, rotates clockwise around axis
Z, and the bottom of the molecule
near the membrane rotates
counter-clockwise around axis Z
(Fig. 6B).
How Does the Binding Activity of CD-MPR Change
When the Salt Bridge is Mutated? 1
Figure 7
A
Research has been done to find
the function of CD-MPR without
a functioning intermonomer salt
bridge (E19/K137). The figures
show the comparison between
the binding activity of normal
CD-MPR (Fig. 7A) and mutated
CD-MPR (Fig. 7B). The
mutation of CD-MPR (E19Q/
K137M) results in loss of binding
activity.
How Does CD-MPR Function in a Cell?
Secretory Pathway
8
Fig. 4: CD-MPR
(unbound)
A model of the quaternary structure of CD-MPR (Figure 4) depicts
the unbound state when mannose 6-phosphate is not present. This
1KEO.PDB
model of CD-MPR’s unbound state contains all features of the bound
state, excluding mannose 6-phosphate. The various aspects of the model highlighted are hydrogen
bonds in lemon chiffon, backbone in light steel blue, alpha helices in midnight blue, and beta sheets in
dark orchid. Note the difference in shape between the bound and unbound forms of CD-MPR. The view
shown in Figure 3 and Figure 4 is looking down onto the top of the receptor as it would be sitting on the
2RL8.PDB cell membrane (similar view as depicted in Fig. 6B).
What is a Lysosome?
A lysosome is a cellular organelle responsible for recycling cellular
macromolecules. Macromolecules are degraded into reusable parts
by enzymes found in the lysosome. When a person is diagnosed
with a lysosomal storage disorder, the degradative enzyme is not
properly transported to the lysosome or is not active. The lysosome
is unable to perform its function without this enzyme, which results
in a buildup of macromolecules in the lysosome. The cell’s
response to this buildup is to produce more lysosomes, however
these lysosomes also accumulate macromolecules (Fig. 1).
When is the Onset of Pompe’s Disease?
enzyme
cation-dependent
mannose 6-phosphate
A SMART Team project supported by the National Institutes of Health Science Education Partnership Award (NIH-SEPA 1R25RR022749) and an NIH CTSA Award (UL1RR031973).
sCD-MPR
B
E19Q/K137M
Conclusion
Cation-dependent mannose 6-phosphate receptor (CD-MPR) plays
a major role in the transportation of lysosomal enzymes to the
lysosome. Intravenous enzyme replacement therapies are
available and replace enzymes deficient in the body due to missing
genetic material. As research continues on CD-MPR, more
efficient treatments may be found for lysosomal storage disorders.
Figure 8 depicts a normal cell (Fig. 8A) compared to a cell suffering
from Mucolipidosis II
(Fig. 8B) and the
reduction of lysosomes
(red) in the cell due to
enzyme replacement
A
B
C
therapy (Fig. 8C).
8 Figure 8
References
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Feb. 2012. <http://medicaljournal.blogspot.com/2007/05/i-cell-disease-mucolipidosis-type-ii.html>.
8. Otomo, et al. (2011) The Journal of Biological Chemistry, Vol. 286, NO. 40, 35283-35290
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(AP Photo/ Evan Agostini)