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Introduction
  
Prions are fatal neurodegenerative diseases that are caused by
aggregation of the prion protein. Prion diseases are caused by
the conversion of a normal cellular prion protein, PrP , to a
pathogenic and infectious isoform, PrPSC . The function of PrP is
not clear, however it¶s found to be involved in copper metabolism
and transport, as well as neuronal cell adhesion. It also binds to
glycosaminoglycan (GAG) at the KKRPK motif site. Familial or
inherited prion diseases, which account for 10-15% of human
prion diseases, is the result of mutations in the prion protein
gene, PRNP. These mutations are insertional, and occur solely
in the octapeptide-repeat region; wild-type human PrP has five
octapeptide repeats. The number of pathogenic insertions
ranges from two to nine. While the mechanism of how the prion
undergoes conformation is unknown, it¶s thought that mutant
prions are unstable and more prone to misfold and aggregate,
recruiting other proteins to misfold to become pathogenic PrPSC .
Recombinant bacterial produced rPrP with pathogenic mutations
is used as a model system to understand the conversion
process. Previous study shows that rPrP with a pathogenic
mutation of three additional insertions, rPrP8OR , binds better to
glycosaminoglycans (GAGs) and is more prone to oxidative
attack. This paper further studies the consequences of binding
GAG and and Cu2+ to mutant rPrPs.
Methods
Ä
 Previous reports have found that insertion mutant rPrPs
such as rPrP8OR and rPrP10OR bind much better to GAG than rPrP.
Furthermore, the level of GAG binding is proportional to the number of
inserts. This experiment therefore investigates whether heparin, a
GAG, promotes the aggregation of rPrPs, and whether the degree of
enhancement is proportional to the number of inserts. rPrP-KKRPK is a
mutant rPrP that lacks the KKRPK GAG-binding motif. The
experiments were carried out in NaCl solution at pH 7.4, at more
physiological conditions. Heparin-enhanced aggregations of rPrP,
rPrP8OR , rPrP10OR , and rPrP-KKRPK were mixed with various
concentrations of heparin under the given conditions. Enhanced
aggregation levels were recorded by measuring turbidity levels with
spectrophotometer.
Ä
 rPrP binds divalent cations, such as Cu2+ and Zn2+ . This
experiment determines whether Cu2+, Zn2+ , Mg2+ or Mn2+ promote
rPrP aggregation in a concentration dependent manner. Assays are
carried out at pH 7.4, with 1 µM of rPrP, rPrP8OR , rPrP10OR , and rPrP-
KKRPK mixed with various concentrations of the stated metal ions.
Enhanced aggregation is measured by increased turbidity levels
measured by spectrophotometer.
Ä
  This experiment determines the role of heparin in enhancing
rPrP aggregation. GAG was tested to see if it promotes aggregation of
 Sucrose-gradient centrifugation to compare the relative sizes of and PrP10OR±Cu2+ of
rPrP-51-90 , which lacks the octapeptide region; rPrP-KKRPK , which lacks
the KKRPK motif GAG-binding site; and of rPrP as a control. Different
concentrations of each were mixed with a set concentration of heparin
to test for increased percentage of starting turbidities. This test also
confirms the significance of the octapeptide-repeat regions.
Ä

 This experiment measures the relative sizes of aggregates mixed with Cu2+, all the immunoreactivity is detected in the bottom fraction.
formed with rPrP, rPrP10OR ,and rPrP-51-90 when bound to Cu2+ , and
detect aggregate formation in these without the presence of Cu2+ .
rPrP-51-90 is used as a control. Aggregates are measured here by
centrifugation, noting where they fraction off. The greater the
aggregates formed, the lower in the fraction they appear.
Results 
 recombinant wildtype PrP
 : recombinant PrP with deletion of octapeptide-
Ä
 Aggregation of rPrP is enhanced repeat region
by heparin. A) At pH 7.4, heparin enhances
the aggregation of all three rPrPs, and the
 : recombinant PrP with deletion of GAG binding
enhancement is greatest for rPrP10OR
motif,KKRPK
followed by rPrP8OR and then rPrP. Heparin
does not promote the aggregation of rPrP-
 : recombinant PrP with 10 octapeptide-repeats
KKRPK, which lacks the GAG-binding motif,
KKRPK, the first five amino acids at the N-
 : recombinant PrP with 8 octapeptide repeats
terminus. B) Kinetics of the heparin-
enhanced aggregations of rPrP, rPrP8OR,
rPrP10OR and rPrP-KKRPK.
Conclusion
Ä
. Aggregation of rPrPs is ‡ Aggregation of PrP is an essential step in the
conversion of PrP to PrPSC
enhanced by metal ions. One micromole
rPrP, rPrP8OR or rPrP10OR was mixed with
‡In the presence of PrP ligands, specifically GAGs or
various concentrations of CuCl2 (A), ZnCl2
Cu2+, rPrPs aggregate in proportion to the number of
(B), MnCl2 (C) and MgCl2 (D). Assays
octapeptide inserts;
carried out at pH 7.4. Cu2+ and Zn2+ , but
not Mg2+ or Mn2+ , promote rPrP
‡rPrPs with insertional mutations, such as rPrP8OR and
aggregation in a concentration-dependent
rPrP10OR form more and larger aggregates with faster
manner (Fig. A-D). The levels of
enhancement are proportional to the kinetics than wild-type rPrP;
number of inserts.
‡GAG-induced aggregation requires the GAG-binding
motif; Cu2+-induced aggregation requires the
octapeptide repeat
‡The octapeptide-repeat region is essential for both
GAG and Cu2+ promoted rPrP aggregation
‡Aggregation induced by GAG and Cu2+ share common
features, yet each has its own distinctions too. This
suggests there are multiple pathways leading to rPrP
aggregation.
‡Because these aberrant features are proportional to the
number of insertions, these findings provide a
biochemical explanation for the observation that patients
Ä
 . Heparin enhances
with more octapeptide-repeat insertions have an earlier
aggregation of rPrP, but not rPrP-
KKRPK and rPrP-51-90. A) Enhanced disease onset and shorter disease duration.
aggregation upon heparin binding
shown as an increased percentage
of starting turbidities. B) Detection
of rPrP-51-90 binding to heparin by
ELISA. At higher protein
concentrations, rPrP-51-90 and rPrP
References
have comparable GAG-binding
activity. ‡ ³Ligand binding promotes prion protein aggregation ±
role of the octapeptide repeats´
Authors: Shuiliang Yu, Shaoman Yin, Nancy Pham, Poki
Wong, Shin-Chung Kang, Robert B. Petersen, Chaoyang
Li, Man-Sun Sy
Ä

rPrP±Cu2+ aggregates. Ten fractions were drawn from top to bottom. An equal volume of each
Published in FEBS Journal: Volume 275, Issue 22, pg.
fraction was loaded onto 12%SDS/PAGE and the PrPs were detected by immunoblotting with
5564-5575, November 2008.
mAb 8H4. rPrPǻ51-90 was used as a control. Without Cu, rPrP, rPrPǻ51-90 and rPrP10OR were
detected in the top fractions. By contrast, when rPrP is mixed with Cu2+, most of the PrP
immunoreactivity is detected in the bottom fractions. However, upon longer exposure, PrP
immunoreactivity is also present in the intermediate fractions (not shown). When rPrP10OR is