The First Bioactive Wnt1 Protein · 2020. 2. 20. · The First Bioactive Wnt1 Protein: WHITE PAPER...
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The First Bioactive Wnt1 Protein: WHITE PAPER Abstract e.g. Wnt3a, Wnt5a/b and Wnt8a, can be overexpressed. Wnt1, however, seems to be primarily associated with the plasma membrane of Wnt producing cells and is not detected in the conditioned media of CHO cells overexpressing Wnt1. We discovered that sFRP expression, in Wnt1 producing cells, can liberate membrane tethered Wnt1 from the cell surface. Co-expression of sFRP1 or sFRP5 with Wnt1 in CHO cells results in detection of active Wnt1 in conditioned media. Co-culturing of sFRP1 or sFRP5 expressing CHO cells with Wnt1 expressing CHO cells also resulted in active Wnt1 in the conditioned media arguing against a mechanism of sFRP facilitating the ER/Golgi secretion pathway of Wnt1. Wnt1 and sFRP form a non-covalent complex which Liwen Xiong, Guo Guang Du, Camrin Tracy, Thomas Stuart, Charles Geng, John Humphrey, Ming Bi, Anthony Person R&D Systems Protein Development Department | 614 McKinley Place NE, Minneapolis, MN 55413, USA Introduction state 1 . These growth factors generally associate with the outer membrane of the Wnt producing cells resulting in limited secretion and signaling away from the Wnt production source. In 2003, Roel Nusse’s lab published a procedure to purify palmitoylated Wnt proteins using CHAPs detergent to maintain Wnts in a hydrophobic environment which proved essential for retaining Wnt activity 2,3 . The structure of Xenopus laevis Wnt8 (XWnt8) critical for articulation with the Fz8 receptor 4 . Secreted frizzled-related proteins (sFRPs) were originally described as Wnt inhibitory proteins 5-7 but were later shown to also enhance the diffusion of Wnt ligands and expand their signaling range 8-9 . There are 5 sFRPs in the human and mouse genomes. They all show structural similarity to the Wnt binding cysteine rich domain (CRD) of Frizzled receptors. Unlike 7-pass transmembrane Frizzled receptors, sFRPs are secreted proteins that contain a netrin-like domain downstream of the CRD. We have demonstrated that sFRPs can serve as Wnt1 chaperone proteins and form a biological active Wnt1/sFRP
Transcript of The First Bioactive Wnt1 Protein · 2020. 2. 20. · The First Bioactive Wnt1 Protein: WHITE PAPER...
The First Bioactive Wnt1 Protein:
WHITE PAPER
Abstract
e.g. Wnt3a, Wnt5a/b and Wnt8a, can be
overexpressed. Wnt1, however, seems to be primarily associated with the plasma membrane of Wnt producing cells and is not detected in the conditioned media of CHO cells overexpressing Wnt1. We discovered that sFRP expression, in Wnt1 producing cells, can liberate membrane tethered Wnt1 from the cell surface. Co-expression of sFRP1 or sFRP5 with Wnt1 in CHO cells results in detection of active Wnt1 in conditioned media. Co-culturing of sFRP1 or sFRP5 expressing CHO cells with Wnt1 expressing CHO cells also resulted in active Wnt1 in the conditioned media arguing against a mechanism of sFRP facilitating the ER/Golgi secretion pathway of Wnt1. Wnt1 and sFRP form a non-covalent complex which
Liwen Xiong, Guo Guang Du, Camrin Tracy, Thomas Stuart, Charles Geng, John Humphrey, Ming Bi, Anthony PersonR&D Systems Protein Development Department | 614 McKinley Place NE, Minneapolis, MN 55413, USA
Introduction
state1. These growth factors generally associate with the outer membrane of the Wnt producing cells resulting in limited secretion and signaling away from the Wnt production source. In 2003, Roel Nusse’s lab published a procedure to purify palmitoylated Wnt proteins using CHAPs detergent to maintain Wnts in a hydrophobic environment which proved essential for retaining Wnt activity2,3. The structure of Xenopus laevis Wnt8 (XWnt8)
critical for articulation with the Fz8 receptor4. Secreted frizzled-related proteins (sFRPs) were originally described as Wnt inhibitory proteins5-7 but were later shown to also enhance the diffusion of Wnt ligands and expand their signaling range8-9. There are 5 sFRPs in the human and mouse genomes. They all show structural similarity to the Wnt binding cysteine rich domain (CRD) of Frizzled receptors. Unlike 7-pass transmembrane Frizzled receptors, sFRPs are secreted proteins that contain a netrin-like domain downstream of the CRD. We have demonstrated that sFRPs can serve as Wnt1 chaperone proteins and form a biological active Wnt1/sFRP
Figure 1. Wnt Proteins can be Either Secreted into Media or Associated with the Outer Membrane of the Wnt Producing Cells. A
B
Table 1: sFRP1 or sFRP5 Liberates mWnt1 Protein from the Cell Surface When Wnt1 and sFRP1/5 are Expressed in the Same or Different Cells.
9000
Wnt
Rep
orte
r Act
ivity
(RFU
)
8000
7000
6000
5000
4000CHO CM mWnt1 CM mWnt2B CM Wnt ReportermWnt3a CM
25000
Wnt
Rep
orte
r Act
ivity
(RFU
)
20000
15000
10000
5000
0CHO Cell mWnt1 Cell mWnt2B Cell Wnt ReportermWnt3a Cell
Treatment Reporter Activity
+
+
+
+
A B
Figure 2. Co-Expression of sFRP1 in Cells Expressing Wnt1 Results in Bioactive Wnt1 in the Conditioned Media. A
B
A-C AB
C BD-E
D E
sFRP-1
Complex Free sFRP-1
Anti-Wnt1
Anti-sFRP1
CHO CM CHO mWnt1/msFRP1-His CM
CHO mWnt1/msFRP1 CM
CHO mWnt1 CM
CHO msFRP1 CM
1 2 3 4 5
Wnt-1
sFRP-1
Wnt-1
14000 CHO CMCHO mWnt1 CMCHO mWnt1/msFRP1 CMCHO mWnt1/msFRP1-His CMCHO msFRP1 CM
Wnt
Rec
epto
r Act
ivity
(RFU
)
Relative Conditioned Media Volume
12000
8000
10000
6000
2000
4000
00.001 0.01 0.1 1 10
A
A B
B
D E
C
Complex is Resolved on an SDS-PAGE Gel. AB C
B C
mWnt-1
msFRP-1
SDS-PAGE
R
29
36
43
55
66
NR R NR R NR
Western Blots
C. Probed with anti-sFRP-1B. Probed with anti-Wnt-1A. Coomassie Blue Staining
Fold
Cha
nge
Recombinant Mouse Wnt-1/sFRP-1 Complex (µg/mL)
2.5
2.0
1.0
1.5
10-3 10-2 10-1 100
Figure 6. Model of Wnt1/sFRP Signaling A B
CD E
Cold Spring Harbor perspectives in biology 4
et al. Nature 423
et al.Nature 423
Science 337
Proc Natl Acad Sci 1
Cell 21
Cell 21
Development 136
Development 138
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