unit (fig. S8A) or linker region (fig. S8B) could
bind to TNFR2, suggesting that the binding domain of PGRN may span granulin unit and linker.
We first expressed each granulin with its immediately adjacent downstream or upstream linker
and observed only weak binding of granulin F-P3,
P4-granulin A, and P5-granulin C to TNFR2 (fig.
S9, A and B). We then linked all three fragments
identified above to generate an engineered mutant (referred to as FAC) (fig. S10A), which exhibited an even stronger binding affinity to
TNFR2 than did PGRN. F, A, and C are the
granulin domains most capable of independent
folding, and each domain has N- and C-terminal
subdomains that are structurally independent (32).
By deleting ever greater portions of each of the
granulin domains of FAC, we determined that a
mutant composed of half units of granulins A, C,
and F plus linkers P3, P4, and P5 appears to be
the “minimal” engineered molecule that retains
affinity to TNFR2 (fig. S10). This molecule was
referred to as Atsttrin (antagonist of TNF/TNFR
signaling via targeting to TNF receptors). Y2H
assay revealed that PGRN associated weakly with
other members of TNFR subfamily, whereas
Atsttrin selectively interacted with TNFR1 and
TNFR2 (fig. S11). Atsttrin was expressed in
bacteria as a glutathione S-transferase (GST) fusion protein, purified on glutathione agarose resin,
and eluted with the use of Xa factor (there is a Xa
Fig. 3. Deletion of PGRN exacerbates, whereas recombinant PGRN prevents, the spontaneous development of
inflammatory arthritis in TNF transgenic (TNF-Tg) mice. (A)
Incidence of arthritis in TNF-Tg, TNF-Tg/Grn+/−, and TNF-Tg/Grn−/− mice (n = 8 mice per group). (B) Clinical arthritis
scores. Data are presented as the mean clinical score T
SEM. P < 0.01 and P < 0.001 versus the control
TNF-Tg group. (C) Photographs of paws of TNF-Tg mice
with mild arthritis treated with either PBS or rhPGRN
for 4 weeks. (D) Effect of PGRN in TNF-Tg mice. TNF-Tg
mice with established mild arthritis (clinical score of ~5)
were treated with PBS or rhPGRN (n = 8 mice per group).
The treatment type was then switched between the two
groups, and the switch time point is indicated with
arrows. Development of arthritis was then scored. The
data are presented as the mean clinical score T SEM. The
statistics were compared between the untreated (PBS)
and the rhPGRN-treated group before the switch time
point (black stars). After that, statistics were compared to
the switch time point in each group (green stars). P <
0.05, P < 0.01, P < 0.001.
Fig. 4. Atsttrin exhibits selective TNFR binding and
inhibits TNFa-TNFR interactions. (A) FastStep Kinetic
Assay for binding of Atsttrin and TNFa to TNFR1 and
TNFR2. Samples were by FastStep injection, and dissociation of analyte-ligand complexes was monitored.
KD for each interaction was indicated. (B) Atsttrin inhibits the binding of TNFa to TNFR1 and TNFR2
(solid-phase binding). Microtiter plate coated with
TNFa was incubated with TNFR1 or TNFR2 in the presence of various amounts of Atsttrin, and the bound
TNFR to TNFa was detected by corresponding antibodies. Values are means T SD. (C) Flow cytometric
analysis of Raw264.7 cells after staining with 50 ng
of biotinylated human TNFa (Bt-TNFa) in the presence of different doses of Atsttrin.
factor cleavage site between GST and Atsttrin)
(fig. S12A). Reversed-phase HPLC showed high
purity (~90%), indicating one major isoform of
Atsttrin (fig. S12B). Of 17 cysteine residues within Atsttrin molecule, five exist as free thiols. When
compared to TNFa, recombinant Atsttrin exhibited higher binding affinity for TNFR2, but lower
affinity for TNFR1 (Fig. 4A). Atsttrin demonstrated dose-dependent inhibition of the interaction between TNFa and TNFR1/TNFR2 (Fig. 4,
B and C). Furthermore, Atsttrin also inhibited the
binding of lymphotoxin a (LTa) to TNFR1 and
TNFR2 (fig. S13).
Atsttrin could inhibit several downstream events
of TNF-TNFR signaling. Atsttrin inhibited TNFa-