An efficient and selective in vitro solid phase assay

An efficient and selective in vitro solid phase assay for PCSK4 activity:
Potential diagnostic tool in fetal and placental growth restriction
Caroline Muia, Chunyu Lu and Dr.Ajoy Basak
Health Science Department, University of Ottawa
Introduction
Methods
Nearly half of all infant mortality occur in the first 28 days after birth
with Intrauterine Growth Restriction (IUGR) syndrome being one of
the leading causes. Even today despite advancements in research
and our better understanding of the mechanism of this condition, it
still remains in the forefront of reproduction research. IUGR, also
called Fetal Restricted Syndrome (FRS), leads to abnormal
development and growth of placenta and fetus. It is linked to the
proteolytic
activity
of
PCSK4
(Proprotein
Convertase
Subtilisin/Kexin type 4) enzyme, also known as PC4. This enzyme
is exclusively present in reproductive tissues/organs including
ovary and placenta. It is present on the surface of plasma
membrane overlying the acrosome of the sperm. It cleaves inactive
larger pro-Insulin growth factor2 (proIGF2) to generate its mature
shorter form IGF2, which plays a crucial role in fetal and placental
growth/ development during pregnancy. Decreased PCSK4 activity
leads to less production of mature IGF2 causing IUGR condition.
Thus monitoring PCSK4 activity using a selective and sensitive
method will be useful for early detection of IUGR.
Design & Preparation of a fluorescent peptide: A 5-amino acid long peptide
sequence mimicking the recognition motif of PCSK4 enzyme was selected from the
cleavage site of proIGF2 (human) which is described as the most potent
physiological substrate of PCSK4.
-The above peptide also contains a linker (Adoa) and Cys-attached Fluorescence
moiety as shown below.
-The peptide thus designed was synthesized using Solid Phase Peptide Chemistry
using Intavis MultiPep instrument and PEGA resin on which the it remain
immobilized.
Fluorescent & non-fluorescent PEGA resins under microscope
Fluorescent resin
(fluorescent filter)
Fluorescent resin
Non-fluorescent resin
MS of supernatants showing fragments after enzyme digestion & control
+ Water (Control)
Enzyme assay: The above immobilized fluorescent peptide on resin (~100
mg) was incubated with various enzymes including recombinant PCSK4 available in
the lab
Intensity
Fluorescent
group
0
PCSK4 cleavage site
1000
2000
50
Caroline-
25
0
Linker
-Cys-
0
0
Linker
1000
1000
2000
2000
+PCSK4
15
N
S
D
H
D
637
S
mPC2/PCSK2
D
H
N
RESULTS
814
ykexin
S
PCSK4 cleavage site
Fluorescent
group
654
753
rPC4A/PCSK4
D
H
N
S
Linker
783
N
S
Lys-Ser-Glu-Arg
-Cys-
mPC1/PCSK1
D H
25
Linker
Asp
20
15
N
Resin
1877
S
- Aqueous sample containing active PC4
- Incubate at 40C for 3 - 4 hours
rPC5A/PCSK5
969
rPC5B
1052
D H
N
S
692
C-terminal
piece
hPACE4A/PCSK6
D
H
D
H N
N
N-terminal piece
S
hSKI-1/PCSK8
S
Signal peptide
P-domain
Cytoplasmic
GF/cytokine receptor
Pro-segment
Ser/Thr rich
Amphipathic
RGD(S) sequence
Trans membrane
Cys-rich domain
N-glycosylation
Catalytic domain
Asp
Lys-Ser-Glu-Arg
-Cys-
hNARC1/PCSK9
12.5
1460.5
1302.8
731 (Dye)
10
7.5
1000
1000
2000
2000
1500
1500
1000
1500
1500
2500
2500
200
200
m/z
m/z
+ Pepsin
915
+ Chymotrypsin
1500
2000
1464.8
3000
2500
1306.7
Intensity
N
1314.6?
731 (Dye) 928.3?
1000
1000
hfurin/PCSK3
D H
200
1712.9
965.7?
5
794
S
1464.8 (4)
10
rPC7/PCSK7
D H
2155 0
0
00
1088.7
1100.2 (1)
Intensity
382
D H
2000 1000
Intensity
Subtilisin
1500
30
Amino acids
S
4000
4000
+ Trypsin
Intensity
N
3000
3000
m/z
Resin
Adoa - Lys-Ser-Glu-Arg-Asp - Adoa
Adoa=8-Amino 3,6-Dioxa-Octanoic acid), serves as an 8-atom linker/spacer chain
D H
4000
365.2 (Matrix)
75
1000
Enzyme
3000
40
30
1183.0
1522.4
20
Resin
1100.2
Car
10
1000
1000
C-terminal
N-terminal highly fluorescent
fragment (Supernatant)
1500
1500
2000
2000
+Endo-Lysine-C
1000
2000
3000
750
Decreased level or impaired PCSK4 protease activity is
responsible for production of low mature IGF2 level leading to
IUGR.
Cleavage by Proteases
Intensity
1093.8 (1)
Hypothesis
30
Objective
1250
5000
1500
30
1117.7 (M+Na)
Caroline-Mar07-201
Car
20
20
10
750
750
2000
2000
1000
1000
3000
3000
1250
1250
4000
4000
1500
1500
5000
5000
m/z
S
CH2
H2N-Cys-Adoa-Lys-Ser-OH
Summary
MW 1198
- A novel solid phase fluorescence based method has been
developed for in vitro assay of PCSK4 activity
Develop an efficient, highly selective and rapid solid phase
fluorescence based method for monitoring PCSK4 protease
activity in vitro.
S
CH2
H2N-Cys-Adoa-Lys-Ser-Glu-OH
1
Aim 1: Design and Synthesis of a Highly Flourescent Peptide
4000
1093.8 (M)
40
1000
1000
MW 1111
1000
40
10
S
CH2
H2N-Cys-Adoa-Lys-OH
3000
3000
m/z
m/z
C-terminal non fluorescent fragment
+ undigested peptide (Resin)
All possible cleavages and the expected Mol Wts of all fragments thus produced
and released into the medium (supernatant).
2500
2500
2
3
4
5
MW 1327
S
CH2
H2N-Cys-Adoa-Lys-Ser-Glu-Arg-Asp-Adoa- PEGA-Resin
- The method is very simple and can be made selective and
sensitive by using dextro amino acid incorporation and the use of
protease inhibitors
- Application of this method for examination of PCSK4 activity in
Placenta tissue is under progress.
MW 1733
Acknowledgement
Aim 2: Application of the Fluorescent Peptide for selective In
Vitro Assay of PCSK4 Activity
I thank Chunyu Lu for his skilled technical assistance as well as
other lab members. Thanks are also due to the University of Ottawa
for the award of UROP fellowship and Dr. Ajoy Basak for his
guidance, supervision and the opportunity to work in his laboratory.
Aim 3: Potential Application of the above method for
monitoring PCSK4 activity in biological samples
under IUGR vs Control conditions
S
CH2
H2N-Cys-Adoa-Lys-Ser-Glu-Arg-Asp-OH
MW 1598
S
CH2
H2N-Cys-Adoa-Lys-Ser-Glu-Arg-OH
MW 1483
References
Qiu et al PNAS, 102, 11047-52, 2005; DeChiara et al Cell 64, 849-59, 1991; Baker, J. et al Cell 75, 73-82, 1993; Constancia, M. et al Nature 417,
945-48, 2002; Fowden, A. L. Placenta 24, 803-12, 2003; Saving Lives at Birth: A Grand Challenge for Development, Grand Challenge Development, 2014;
Basak et al Methods in Molecular Biology 768

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