Báo cáo khoa học: Epitope analysis of the rat dipeptidyl peptidase IV monoclonal antibody 6A3 that blocks pericellular fibronectin-mediated cancer cell adhesion pot

Epitope analysis of the rat dipeptidyl peptidase IV
monoclonal antibody 6A3 that blocks pericellular
fibronectin-mediated cancer cell adhesion
Ting-Ting Hung
1,
*, Jun-Yi Wu
1,
*, Ju-Fang Liu
1
and Hung-Chi Cheng
1,2
1 Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
2 National Cheng Kung University Hospital Cancer Center, National Cheng Kung University, Tainan, Taiwan
Introduction
Specific endothelial ⁄ cancer cell–cell adhesions dictate
organ-preference cancer metastases [1]. We previously
demonstrated that the blood-borne cancer cells become
arrested in the lung vasculature via adhesion between
the lung endothelial adhesion receptor dipeptidyl
peptidase IV (DPP IV) and pericellular polymeric
Keywords
dipeptidyl peptidase IV; epitope mapping;
monoclonal antibody; polymeric fibronectin;
steric hindrance
Correspondence
H C. Cheng, Department of Biochemistry
and Molecular Biology, College of Medicine,
National Cheng Kung University, Tainan
70101, Taiwan
Fax: +886 6 274 1694
Tel: +886 6 235353

E-mail:
*These authors contributed equally to this
work.
(Received 9 June 2009, revised 27 July
2009, accepted 3 September 2009)
doi:10.1111/j.1742-4658.2009.07352.x
We previously showed that the rat dipeptidyl peptidase IV (rDPP IV)
monoclonal antibody (mAb) 6A3 greatly inhibits the pericellular polymeric
fibronectin-mediated metastatic cancer cell adhesion to rDPP IV.
L
311
QWLRRI in rDPP IV has been proposed as the putative fibronectin-
binding site. However, the inhibitory mechanism of 6A3 has been elusive.
Epitope mapping of 6A3 may help to understand the interaction between
fibronectin and rDPP IV. In the present study, we showed that 6A3 spe-
cies-specifically recognized rDPP IV but inhibited fibronectin ⁄ rDPP IV-
mediated cell adhesions of various cancer types and species, which was
independent of rDPP IV enzymatic activity. The 6A3 epitope was stably
exposed in both native and denatured rDPP IV. On the basis of the
resolved structures and the species variations in DPP IV sequences, we
finely mapped the 6A3 epitope to a surface-exposed Thr331-dependent
motif D
329
KTTLVWN, only 11 amino acids away from L
311
QWLRRI on
the same plane as the fifth b-propeller blade. The functionality of 6A3
epitope in rDPP IV was ultimately demonstrated by the ability of 6A3-rec-
ognizable fragments to interfere with the inhibitory effect of 6A3 on full-
length rDPP IV binding to pericellular polymeric fibronectin. On the basis

of structural analysis, and the fact that the preformed fibronectin frag-
ment ⁄ rDPP IV complex was co-immunoprecipitated by 6A3 and fixing the
rDPP IV structure with paraformaldehyde did not avert the inhibitory
effect, the mechanism of 6A3 inhibition may not be the result of complete
competition or conformational change.
Structured digital abstract
l
MINT-7261577: DppIV (uniprotkb:P14740) binds (MI:0407)toFNIII14 (uniprotkb:P04937)
by anti bait coimmunoprecipitation (
MI:0006)
Abbreviations
ADA, adenosine deaminase; DPP IV, dipeptidyl peptidase IV; FACS, fluorescence-activated cell sorting; FN, fibronectin; hDPP, human
dipeptidyl peptidase; Hm, human mutant; mAb, monoclonal antibody; MBP, maltose-binding protein; mDPP, mouse dipeptidyl peptidase;
Mm, mouse mutant; pAb, polyclonal antibody; PFD, paraformaldehyde; polyFN, polymeric fibronectin; rDPP, rat dipeptidyl peptidase.
6548 FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS
fibronectin (polyFN) [1–5]. This cancer cell adhesion to
DPP IV can be greatly blocked by monoclonal antibody
(mAb) 6A3 directed against rat DPP IV (rDPP IV) [2,3].
DPP IV is a homodimeric type II transmembrane
serine protease with multiple functions [6]. For exam-
ple, it plays roles in proteolytic cleavage and inactiva-
tion of glucagon-like peptide 1 to maintain glucose
homeostasis [7] and in inactivating other bioactive pep-
tides and various cytokines for sustaining normal phys-
iological conditions [8]. Much of the drug development
carried out with respect to type II diabetes has aimed
to effectively block the enzymatic activity of DPP IV
[9]. However, the binding between DPP IV and pericel-
lular polyFN appears to be independent of this enzy-
matic activity [3,4]. Indeed, the putative FN-binding

site in DPP IV has been proposed as a seven amino
acid sequence, L
311
QWLRRI, located in the fifth blade
of the b-propeller domain, which is structurally distinct
from the a ⁄ b hydrolase domain that harbors the cata-
lytic triad [6,10].
FN, a large, multifunctional glycoprotein, is
secreted as the soluble, dimeric plasma FN or as the
insoluble polyFN. PolyFN is either deposited in the
extracellular matrix or assembled on the surfaces of
metastatic cancer cells in the lung to which the endo-
thelial adhesion receptor DPP IV binds [11,12].
A consensus DPPIV-binding motif in FN has been
located in the 13th, 14th and 15th FN type III
repeats [1]. Recently, we demonstrated that the assem-
bly of hematogenous cancer pericellular polyFN is
regulated by protein kinase Ce [5].
Several mAbs against DPP IV have been found to
interrupt extracellular matrix attachment and to arrest
the cell cycle of DPP IV-expressing cancer cells [13,14].
One of these mAbs was engineered into humanized
mAb and exerted an inhibitory effect on tumor growth
in a xenograft model [15]. Before considering 6A3 as a
base for designing anti-adhesion drugs [16,17], we need
to better understand how 6A3 exerts its inhibitory
effect on the interaction between DPP IV and polyFN.
For example, although metastatic cancer cells of vari-
ous species adhere to DPP IV [1], it is unclear whether
the inhibitory effect of 6A3 is also a cross-species phe-

nomenon in preventing cancer cell adhesion to DPP
IV. Although 6A3 strongly inhibits DPP IV ⁄ FN bind-
ing, the inhibitory mechanism of 6A3 still remains elu-
sive. Furthermore, although DPP IV enzymatic
activity plays such important roles in many physiologi-
cal functions [18], it is not known whether 6A3-binding
affects this catalytic activity. Moreover, because the
structural stabilities of eptiopes recognized by mAbs
are indispensable to the drug efficacy [19,20], it is
important to examine the stability of the 6A3 epitope.
Epitope mapping of 6A3 may be the most direct
approach for answering the above questions [21].
In the present study, we first show that the inhibi-
tory effect of 6A3 on rDPP IV ⁄ FN is a general phe-
nomenon. 6A3 species specifically recognized a
structurally stable epitope in both native and dena-
tured rDPP IV, independent of DPP IV enzymatic
activity, which is suggestive of a noncompetitive inhibi-
tion mechanism. The 6A3 epitope in full-length rDPP
IV was finely mapped to the surface-exposed Thr331-
dependent eight amino acid sequence D
329
KTTLVWN
near the putative FN-binding site, L
311
QWLRRI, and
only 11 amino acids apart. According to structural
analysis and co-immunoprecipitation of the preformed
FN fragment ⁄ rDPP IV complex with 6A3, we suggest
that the competitive mechanism is not responsible for

6A3 inhibition. Preventing conformational change of
the rDPP IV structure did not avert the inhibitory
effect of 6A3. These observations suggest that the
inhibitory effect of 6A3 is a result of steric hindrance
rather than conformational change.
Results
The inhibitory effect of 6A3 on rDPP IV
⁄
cancer
pericellular polyFN-mediated cell adhesion is a
general phenomenon
To determine whether the 6A3 inhibition is a general
phenomenon, we selected another two metastatic can-
cer cell lines, human breast cancer cells (MDA-MB-
231) and mouse melanoma cells (B16F10), for rDPP
IV adhesion assays. Similar to MTF7, MDA-MB-231
and B16F10 exhibited high adhesion activities to rDPP
IV, which was specifically abolished by 6A3 in a dose-
dependent manner (Fig. 1A). The adhesion activities
exerted by these cells of various species and cancer
types were also greatly blocked by a polyclonal anti-
body (pAb) that recognizes FN of multiple species [3]
(and data not shown). We next reconfirmed the inhibi-
tory effect of 6A3 on binding between rDPP IV and
pericellular polyFN. After incubation with 6A3, solu-
ble rDPP IV lost the ability to bind to immobilized
MTF7 cell surfaces to the same degree that polyclonal
anti-FN serum inhibited rDPP IV-binding (Fig. 1B).
These data suggest that the decreased adhesion activity
by 6A3 was indeed a result of the blockage of binding

between rDPP IV and pericellular polyFN. Biochemi-
cally, we also demonstrated that the same FN
co-immunoprecipitated with polyclonal anti-FN serum
was affinity-precipitated by DPP IV-conjugated beads,
which was abrogated by preincubating the beads with
6A3 (Fig. 1C). This rDPP IV-precipitated FN was
T T. Hung et al. Epitope of monoclonal DPP IV antibody
FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS 6549
demonstrated to be pericellular polyFN because it was
removed from the cell surfaces by pretreating the
[
35
S]methionine metabolically labeled cells with a-chy-
motrypsin (Fig. 1C). These results suggest that 6A3
directly inhibits binding of rDPP IV to pericellular
polyFN of cancer cells.
6A3 species-specifically recognizes both native
and denatured rDPP IV without interfering with
its enzymatic activity
We next examined the species-specificity of 6A3-recog-
nition of DPP IV in immunoprecipitation and in
immunoblotting assays. We found that 6A3 strongly
recognized rDPP IV but only negligibly bound to
native human (h)DPP IV and mouse (m)DPP IV in
immunoprecipitation assays (Fig. 2A–C). In immuno-
blotting assays where DPP IVs were subjected to the
denatured condition in SDS–PAGE, 6A3 exclusively
recognized rDPP IV (Fig. 2D). The ability of 6A3 to
recognize both native and denatured rDPP IV suggests
that the 6A3 epitope is stably exposed in full-length

rDPP IV. By contrast to the species-conserved FN-
binding sequence, the rat-specific recognition of DPP
IV by 6A3 implies that the 6A3 epitope does not
totally overlap with the putative FN-binding site. This
possibility was further supported by the fact that,
although the FNIII14 (a DPP IV-binding competent
FN fragment) [1] did not to bind to the 6A3-preincu-
bated rDPP IV (data not shown), the pre-bound
FNIII14 ⁄ rDPP IV complex was co-immunoprecipitat-
ed by 6A3, even in the presence of high salt (200 mm
NaCl) solution (Fig. 2E). To determine whether the
inhibitory effect of 6A3 interferes with the peptidase
function, we set out to measure DPP IV enzymatic
activity in the presence of 6A3. In line with the previ-
ous results showing that FN ⁄ DPP IV binding is inde-
pendent of DPP IV catalytic activity [3], 6A3-binding
of rDPP IV also had no effect on its enzymatic activ-
ity, indicating that this process may occur outside the
enzymatic active site (Fig. 2F).
The eight-bladed b-propeller domain harbors the
6A3-binding site
To identify the 6A3-binding site, we first generated
recombinant maltose-binding protein (MBP)-fusion
fragments of extracellular rDPP IV based on the
reported structural and functional domains of the pre-
dicted version 1 [22] and the resolved version 2 [23]
(Fig. 3A). These two versions differ mainly in the
numbers of blades in the b-propeller domain.
Although version 1 was predicted to be seven-bladed
No DPP IV

Bio-DPP IV alone
Bio-DPP IV + Control
50 50300 300
Bio-DPP IV + 6A3 (µg·mL
–1
)
Bio-DPP IV +
α
FN pAb (µg·mL
–1
)
DPP IV binding
(O.D. 492 nm)
–+
IP:
α
FN
+Control
+6A3
DPP IV-conjugated Affi-Gel
–+–+
α
-chymotrypsin
FN monomer
180 kDa
Cells alone
Control
6A3 50 µg·mL
–1
6A3 100 µg·mL

–1
6A3 300 µg·mL
–1
MDA-MB-231
MTF7
B16F10
% adhesion activity
A
B
C
Fig. 1. 6A3 inhibits pericellular FN-mediated lung-metastatic
cancer cell adhesion. (A) rDPP IV adhesion activities of MDA-MB-
231, MTF7 and B16F10 (5 · 10
4
cells per well) in the absence or
presence of various concentrations of 6A3 or 0.3 mgÆmL
)1
non-
immune isotype IgG1 (control) at 37 °C for 30 min were mea-
sured as described in the Experimental procedures. (B) Binding of
soluble biotinylated rDPP IV (2 lg) to MTF7 (5 · 10
4
cells per
well), grown at 37 °C overnight on 96-well plates (after PFD fixa-
tion) in the absence or presence of 6A3 (50 or 300 lgÆmL
)1
), the
same isotype mouse IgG1 (control; 300 lgÆmL
)1
) or polyclonal

FN antibody (50 or 300 lgÆmL
)1
), was detected with horseradish
peroxidase-conjugated streptavidin. (C) Whole cell lysates from
1 · 10
6
2-h recovered suspended S
35
-labeled MTF7 cells, pretreat-
ed without ()) or with (+) 10 lgÆmL
)1
a-chymotrypsin (1 h at
37 °C), were immunoprecipitated with 2 lg polyclonal FN antibody
or pulled-down with DPP IV-conjugated Affi-Gel 10 beads in the
presence of 0.3 mgÆmL
)1
6A3 or control before being subjected
to SDS–PAGE and radiography. Note that Affi-Gel conjugated with
control protein phosphorylase b did not pull down any pericellular
polyFN [1] (and data not shown) and the FN monomeric bands
shown in the radiography were reduced from high molecular
weight pericellular polyFN by b-mecaptoethanol [3,37].
Epitope of monoclonal DPP IV antibody T T. Hung et al.
6550 FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS
(amino acids 131–502) [22], version 2 has been shown
to be eight-bladed (amino acids 49–502) [24] (Fig. 3A).
The reason that we took the predicted version into
consideration was that other members of the propyl
oligopeptidase family to which DPP IV belongs con-
tain a seven-bladed b-propeller domain and that delet-

ing portions of the predicted N-terminal a ⁄ b-hydrolase
domain (amino acids 29–130; the first propeller blade
of version 2) resulted in a loss of enzymatic activity
and protein integrity, implying that this region,
although structurally belonging to the eight-bladed
b-propeller domain, may be functionally involved in
a ⁄ b-hydrolase catalytic activity [22]. Because the
a ⁄ b-hydrolase domains of the two versions both
include certain portions of the N- and C-terminal
regions, we constructed two chimeric MBP-fusion frag-
ments, A+D and G+D, to cover these domains
(Fig. 3A). After purification, we found that all but
fragment A were endogenously degraded (Fig. 3B),
which is not uncommon in bacterial systems expressing
mammalian proteins [25]. The high structural stability
of fragment A explains why it is required for stabiliz-
ing the a ⁄ b-hydrolase structure and enzymatic activity
even within the b-propeller domain [22,24]. From the
results of both immunoblotting and immunoprecipita-
tion, we found that fragments B and C, and extracellu-
lar full-length, were recognized by 6A3 (Fig. 3B),
suggesting that the 6A3 epitope resides within the last
seven propeller blades in the eight-bladed b-propeller
domain. The fragments containing the a⁄ b-hydrolase
IP:
mαhDPP IV
6A3
mαhDPP IV
6A3
Control

IB: IF7
hDPP IV-Jurkat
Mock-Jurkat
IB: 6A3
Control
6A3
CU31
IP:
Rat Kidney Extract
6A3
rα
α
mDPP IV
IP:
IB: r
α
mDPP IV
Control
Rat IgG
Mouse Kidney Extract
AB
CD
IB: 6A3
Rat
Mouse
Human
DPP IV
F
DPP IV + 6A3
DPP IV alone

O.D. 405 nm
IP: 6A3
IB: MBP
MT
MBP-
FNIII14
WT
MT
WT
NaCl 150 mM 200 mM
E
110 kDa
110 kDa
110 kDa
110 kDa
55 kDa
110 kDa
IP: 6A3
IB: 6A3
1.00
0.25
0.50
0.75
0.00
Fig. 2. 6A3 specifically recognizes both native and denatured rat DPP IV independently of its enzymatic activity. (A) Three hundred micro-
liters of Sprague-Dawley (SD) rat kidney extracts (one kidney per milliliter being homogenized, as previously described [3]) was subjected
to immunoprecipitation at 4 °C overnight with 2 lg of purified control mouse IgG1, 6A3 mAb or CU31 pAb and then to immunoblotting
with 6A3. (B) Three hundred microliters of C57BL6 mouse kidney extracts (two kidneys per milliliter [3]) was subjected to immunoprecipi-
tation at 4 °C for overnight with 2 lg of purified 6A3, control, mouse DPPIV (mDPP IV) mAb from rat or rat IgG, and then to immunoblot-
ting with mDPP IV mAb from rat. (C) Three hundred microliters of human Mock-Jurkat or DPP IV-Jurkat cell lysates (1mgÆmL

)1
from
1 · 10
7
cells) was subjected to immunoprecipitation at 4 °C for overnight with 2 lg of 6A3, control, or human DPP IV (hDPP IV) mAb
from mouse and then to immunoblotting with hDPP IV mAb IF7 from mouse. (D) Mouse, rat kidney extracts or DPP IV-Jurkat cell lysates
(made as described in A, B, and C) were subjected to immunoprecipitation at 4 °C for overnight with 2 lg of 6A3, mDPP IV mAb from
rat or hDPP IV mAb from mouse, respectively, and then to immunoblotting with 6A3. (E) rDPP IV preincubated with DPP IV-binding com-
petent FN fragment MBP-FNIII14 (WT) or with DPP IV-binding sequence-mutated MBP-FNIII14 (MT) [1] were co-immunoprecipitated by
6A3 in the presence of 150 or 200 m
M NaCl and then subjected to immunoblotting with a-MBP pAb (upper panel) or with 6A3 (lower
panel). (F) DPP IV enzymatic activities were measured in the absence or presence of 6A3 as described in the Experimental procedures.
T T. Hung et al. Epitope of monoclonal DPP IV antibody
FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS 6551
domain were not recognized by 6A3, which is consis-
tent with the results showing that 6A3-binding did not
interfere with DPP IV enzymatic activity (Fig. 2E).
6A3-binding epitope is located within a
Thr331-dependent linear eight-amino acid
region near the proposed FN-binding site
Because 6A3 specifically recognized rDPP IV
(Fig. 2A–D), we next compared the aligned secondary
structures and sequences of the three species and
selected three nonconserved rDPP IV regions to test
6A3-binding (Fig. S1A). Unfortunately, amino acid
swapping in these three regions in rDPP IV with those
of hDPP IV did not affect 6A3 recognition (Fig. S1B).
Therefore, we began with a serial deletion scheme and
generated two truncated B fragments: B-144 and
B-272. 6A3 only bound to B-144 but not to B-272

(Fig. S2A), suggesting that the 128 amino acid region
between Val231 and Ala358 contains the 6A3 epitope.
To further narrow down the 6A3 epitope, we then
identified two nonconserved clusters within this 128
amino acid region (Fig. S2B). Because the first cluster
harboring B ⁄ c was invalid for 6A3-binding (Fig. S1),
we focused on the second cluster, according to which
we generated B-159 and B-166, and a 28 amino acid
IB:6A3
IP:6A3
IB:MBP
IB:MBP
29 131 503 7671
3749 503 7671
Ver. 1
Ver. 2
7
7
AB D
DCG
A
exF
L
G + D
A + D
D
C
B
50
100

75
kDa
50
75
100
50
75
100
*
*
*
*
*
*
*
*
*
*
*
*
A
B
50
75
kDa
IB:6A3
B-159
B (329~358)
B-272
B-166

T331I
V334T
B-166
75
kDa
IB:6A3
IB:6A3
75
WT
Hm
Mm
B-166
kDa
C
D
E
Fig. 3. 6A3 recognizes the linear Thr331-dependent eight-amino acid epitope in the eight-bladed b-propeller domain. (A) Scheme of rDPP IV
molecular dissecting for constructing MBP-fusion proteins based on the predicted version 1 [22] and resolved version 2 [24] of the reported
structural and functional domains. In both versions, black rectangles represent intracellular domains and white rectangles transmembrane
domains. Although fragments A and G belong to N-terminal portions of a ⁄ b-hydrolase domains in both versions of DPP IV, fragment D is the
invariant C-terminal a ⁄ b-hydrolase domain. Fragment B represents the seven-bladed b-propeller domain of version 1 and fragment C is the
eight-bladed b-propeller domain of version 2. Chimera fragments A+D and G+D represent the complete a ⁄ b-hydrolase domains. exFL repre-
sents the extracellular full-length DPP IV fragment. (B) 6A3 immunoblotting (upper panel), 6A3 immunoprecipitation followed by anti-MBP
immunoblotting (middle panel) and anti-MBP immunoblotting (lower panel) for 50 ng of amylose agarose bead-purified MBP-fusion fragments
as described in the Experimental procedures. As a result of general protein degradations, all fragments were loaded so that the amounts of
full-length proteins were approximately equal. Note that full-length fragments are indicated by an asterisk to the right of the individual protein
bands. (C) 6A3 immunoblotting of B-159, B-166, B-272 and B(329–358) (for detailed positioning, see Fig. S2B). Note that binding of B-166
and B(329–358) to 6A3 indicates that the eight-amino acid sequence (Asp329 to Asn336) within the 106-amino acid region between Val231
and Asn336 of the eight-bladed b-propeller domain harbors the 6A3 epitope. (D) 6A3 immunoblotting of T331I and V334T mutants (for
detailed positioning, see Fig. S2D). (E) 6A3 immunoblotting of wild-type (WT), Hm and Mm (for detailed positioning, see Fig. S2E).

Epitope of monoclonal DPP IV antibody T T. Hung et al.
6552 FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS
fragment B (amino acids 329–358) (Fig. S2B). We
found that 6A3 recognized all of them (Figs 4C and
S2C), indicating that the 6A3 epitope is located within
the linear eight-amino acid region between Asp329 and
Asn 336. This conclusion was well supported by the
fact that swapping the eight-amino acid sequence in
B-166 (wild-type) with that of human (Hm) or mouse
(Mm) (Fig. S2D) lead to a loss of their 6A3-binding
abilities (Fig. 3D). We then constructed B-166
mutants, T331I and V334T, where the only amino
acids distinct from those in Mm were individually
swapped with Ile and Thr (Fig. S2E). The results
demonstrate that 6A3 recognizes V334T but T331I
(Fig. 3E), strongly suggesting that the 6A3-binding
epitope lies within a Thr331-dependent eight-amino
acid region.
In the compiled ribbon and surface representations
of the resolved DPP IV structure, this region is located
at the fifth propeller blade of the eight-bladed propel-
ler domain (Fig. 4A, B). Consistently, it resides at the
A
C
D
R
315
R
316
I

317
T
331
2
1
3
4
5
6
7
8
T
331
B
T
331
T
331
T
331
Cells alone
Control
6A3 50 µg·mL
–1
6A3 100 µg·mL
–1
6A3 300 µg·mL
–1
MTF7 cell adhesion to PFD-fixed DPP IV
% adhesion activity

E
Fig. 4. 6A3 epitope is in close proximity
with the proposed FN-binding site
L
311
QWLRRI but is far from the enzymatic
active site. (A) Ribbon representation for the
eight-bladed b-propeller domain (dark yellow)
of the resolved rDPP IV (Protein Data Bank
code: 2GBC). The view is from the top of
the propeller domain with individual propel-
ler blades being alphabetically numbered.
The Thr331-dependent eight-amino acid 6A3
epitope located at upper portion of the fifth
propeller blade is labeled in green with
Thr331 highlighted in red. The proposed
FN-binding sequence L
311
QWLRRI in cyan
is located at lower portion of the fifth pro-
peller blade. The rest of the fifth propeller
blade is labeled in blue. (B) Side view of the
eight-bladed b-propeller domain as
presented in ribbon mode (upper panel) or
in surface contour mode (lower panel). (C)
Top view and (D) side view of the surface
contour image for the entire rDPP IV struc-
ture. The catalytic triad (S631, D709 and
H741) labeled in marine blue can be visual-
ized through the narrower channel that is

formed by the eight b-propeller blades (the
white arrow) where the catalytic dipeptide
products leave (C), or through the open
active site cleft into which DPP IV sub-
strates may enter (the yellow arrow) (D). All
the views were rendered with
PYMOL 0.99
and color representations are the same as
those shown in (A). (E) rDPP IV-coated wells
were first pre-treated with 2% PFD at room
temperature for 30 min. Percent specific
adhesion activities of MTF7 were then
measured similar to Fig. 1A.
T T. Hung et al. Epitope of monoclonal DPP IV antibody
FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS 6553
outer surface of the eight-bladed propeller domain,
which is totally opposite to the inner surface of the
a ⁄ b-hydrolase domain (Fig. 4C, D). Interestingly, this
6A3 epitope and the proposed FN-binding site,
L
311
QWLRRI, both belong to exactly the same propel-
ler blade, and are only 11 residues apart (Fig. 4A, B).
This structural proximity suggests that the inhibitory
effect of 6A3 is a result of steric hindrance. To rule
out the remote possibility that the inhibitory effect is a
result of conformational change, we used paraformal-
dehyde (PFD), which forms methylene bridges between
any two residues with an amino group in their side
chains [26], to prevent conformational change of the

DPP IV structure. 6A3 was still able to inhibit the can-
cer cell adhering to the PFD-fixed DPP IV (Fig. 4E),
further supporting the idea that 6A3 most likely
exerts steric hindrance in the inhibition of FN ⁄ DPP IV
binding.
The Thr331-dependent linear eight-amino acid
region mediates the 6A3-binding in full-length
DPP IV
Although we identified the 6A3 epitope in a loop area
of the rDPP IV molecule according to the in silico
analysis (Fig. 4A–D), we did not know whether this
epitope is available for 6A3-binding in the full-length
rDPP IV structure. Therefore, we first ectopically
expressed full-length rDPP IV on HEK293 cell sur-
faces. After preincubation with 6A3, B-166 wild-type
but not Hm, Mm or T331I, greatly inhibited 6A3
immunofluorescent staining of rDPP IV-expressing
HEK293 cells (Fig. 5A). Next, purified full-length
rDPP IV together with reference mouse IgG were
immunoblotted with 6A3 that was preincubated with
B-166 wild-type, Hm or Mm. Normalized with the
55 kDa IgG heavy chain, the wild-type demonstrated a
greater blocking effect compared to the other two
mutants (Fig. 5B). Consistently, 6A3 ⁄ rDPP IV immu-
noprecipitation was inhibited in the presence of the
wild-type fragment (Fig. 5C). The essential role of
Thr331 in 6A3-recognition was reconfirmed by the
results showing that T331I failed to inhibit 6A3 ⁄ rDPP
IV binding in immunofluorescent staining, immunocy-
tochemistry, immunoblotting and immunoprecipitation

assays (Fig. 5A–C). To further corroborate the specific
binding between 6A3 and its epitope in rDPP IV, we
performed competition assays between DPP IV protein
and the T331I orV334T B-166 mutant peptides for
6A3 binding. T331I, but not V334T, blocked the inhi-
bition of cancer cell adhesion to DPP IV and soluble
DPP IV binding to polyFN-expressing cancer cells
(Table 1). Taken together, these data indicate that the
Thr331-dependent linear eight-amino acid region
indeed mediates 6A3-binding in full-length DPP IV.
Discussion
Specific adhesion between cancer cells and endothelia
contributes to organ-preference cancer metastasis [1,3].
We previously generated rDPP IV mAb 6A3 that
blocks the DPP IV ⁄ FN-mediated adhesion of lung-
metastatic cancer cells in the lungs [3]. In the present
study, we finely mapped the 6A3 epitope and analyzed
the inhibitory mechanism of 6A3.
One of the three mechanisms, namely competition,
conformational change and steric hindrance, may
explain the inhibitory effect of 6A3. The distinct spe-
cies-specificities of the 6A3 epitope and the FN-binding
and the co-immunoprecipitation of preformed
FN ⁄ DPP IV complex with 6A3 (Fig. 2E) make it less
likely that 6A3 inhibits the FN ⁄ rDPP IV binding via
competition. Nevertheless, before the putative
FN-binding sequence is firmly validated, we cannot
totally rule out that both binding sites partially overlap.
On the other hand, most residues in the putative
FN-binding sequence L

311
QWLRRI [10] are apparently
buried in the propeller core except the R
315
RI (Fig. 4B,
lower panel). FN remains to bind to the PFD-fixed
DPP IV, where no conformation can be changed to
expose the buried residues (Fig. 4E), indicating that
additional residues, other than R
315
RI, may contrib-
ute to the FN-binding. However, the DPP IV frag-
ment B, which contains L
311
QWLRRI, did not
exhibit significant FN-binding activity (data not
shown), suggesting either that the true FN-binding
site is located outside the fragment B and affected by
6A3 via conformational change or that L
311
QWLRRI
is part of the true-binding site, the presentation of
which might only be supported by the full-length
DPP IV structure and affected by 6A3 via steric
hindrance.
Although the exact inhibitory mechanism of 6A3
cannot be proclaimed for certain, the preservation of
the inhibitory effect on FN binding to PFD-fixed DPP
IV by 6A3 (Fig. 4E) appears to disfavor the effect of
conformational change. By contrast, the R

315
RI
together with several nearby sequences appear to
belong to a discrete surface-exposed motif, which is
located near the 6A3 epitope at a distance of approxi-
mately 30 A
˚
(Fig. S3A, C), likely representing a dis-
continuous FN-binding domain and favoring the
mechanism of steric hindrance. Interestingly, based on
structural comparison and superimposition of rDPP
IV (2GBC) and hDPP IV (1NU6), this putative
FN-binding motif appears to be relatively conserved
Epitope of monoclonal DPP IV antibody T T. Hung et al.
6554 FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS
(Fig. S3A–D), implying that this motif may be a more
appropriate FN-binding domain.
General application of antigen-specific mAbs in can-
cer therapy is highly anticipated [27]. For example,
bevacizumab is generally used to inhibit angiogenesis
in treating patients with various cancer types [28]. In
line with this concept, 6A3 inhibits the rDPP IV-bind-
ing of rat and human breast cancer cells, mouse mela-
noma cells (Fig. 1A) and several other types of cancer
cells (data not shown). However, the failure of 6A3 in
recognizing hDPP IV makes it impossible to use for
direct application in cancer therapies. Nevertheless, the
superimposition of rDPP IV and hDPP IV reveals that
the overall conformations of the two molecules are
rather similar, with subtle differences as a result of

amino acid side chain variations (Fig. S3D). We specu-
late that mAbs generated against peptides containing
the 6A3 epitope-corresponding sequence D
331
ESS-
GRWN in hDPP IV may be evaluated in the future
for therapeutic purposes. One important consideration
is that, before clinical application, pretests of these
mAbs in animal models are required. Accordingly, the
antigenic peptide sequences should be carefully deter-
mined so that the generated mAbs will also recognize
rDPP IV and ⁄ or mDPP IV and block lung metastases
of cancer cells in these animals.
To serve as a safe drug, a potential mAb is expected
to exert its effect without causing adverse consequences
of physiological functions [29]. DPP IV is involved in
adenosine deaminase (ADA)-mediated T cell prolifera-
tion [6]. Antibodies recognizing epitopes including resi-
dues Leu294 and Val341 were found to have inhibitory
effects on ADA-binding of hDPP IV [30]. These epi-
topes do not overlap with the 6A3 epitope-correspond-
ing sequence in hDPP IV, implying that the mAb
generated against this hDPP IV sequence should not
interrupt ADA binding to hDPP IV. Although mono-
clonal DPP IV antibody 4D10 arrests the cell cycle
A
C
Binding of 6A3 preincubated with B-166 mutants
MBP
WT

Hm
Mm
T331I
IB: 6A3 preincubated with B-166 mutants
WT
Hm
T331I
V334T
IP: 6A3 preincubated with B-166 mutants
Ig heavy chain
rDPP IV
rDPP IV
B-166 mutants
WT (A.F.I. = 8.75) T331I (A.F.I. = 22.53)Mm (A.F.I. = 20.46)
Control (A.F.I. = 1)
MBP (A.F.I. = 20.26)
Hm (A.F.I. = 20.26)
B
Fig. 5. 6A3-recognizable DPP IV fragments
block the bindings of 6A3 to both native and
denatured full-length DPP IV. (A) FACS
analyses of ectopically full-length rDPP
IV-expressing HEK293 cells by staining the
cells with 6A3 preincubated with MBP,
B-166 Hm, Mm, wild-type (WT), T331I
mutants or the same isotype mouse IgG
(control) as described in the Experimental
procedures. The dot plots were generated
with PE-Cy5 fluorescent intensities (repre-
senting 6A3-binding abilities) against FSC-H.

The arbitrary fluorescent intensities (A.F.I.)
of the DPP IV-positive HEK293 cells are
calculated as average fluorescent inten-
sity · total DPP IV-positive HEK293 cell
numbers for each staining. Representative
images of each immunofluorescent staining
results are inserted inside each correspond-
ing dot plot. (B) Immunoblotting of the
purified full-length rDPP IV and the mouse
IgG heavy chain as a quantitative reference
protein with 6A3 preincubated with the
same MBP-fusion DPP IV fragments as in
(A). (C) 6A3 immunoblotting of the immuno-
precipitates where 0.2 lg of purified full-
length DPP IV was immunoprecipitated with
0.5 lg of 6A3 preincubated with 2 lgof
WT, Hm, T331I or V334T mutants. Note
that, although the 6A3 binding-competent
WT and V334T blocked rDPP IV immuno-
precipitation by 6A3, Hm and T311I, which
were incapable of binding to 6A3, did not
exert this inhibitory effect.
T T. Hung et al. Epitope of monoclonal DPP IV antibody
FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS 6555
progression of cancer cells and reduces in vivo tumor
growth, it does not cause any apparent adverse effect
in nude mice [15]. It is likely that it causes distinct
effects of cytotoxicity against DPP IV-expressing can-
cer cells and normal cells. Altogether, we propose a
hypothesis that a mAb raised against the 6A3 epitope-

corresponding sequence in hDPP IV may functionally
be unique and safe in cancer patients.
The surface-exposed mAb epitope must be stable
enough in the circulation to resist those cancer-associ-
ated protein modification factors, such as the reactive
oxygen species resulting from pro-inflammatory oxida-
tive stress [31] and mechanical stress toward endothelia
[32,33]. Together with shear stress from the capillary
circulation and the respiratory pressure of the lungs,
all the above inflammatory factors are potent with
respect to causing various degrees of conformation
alterations of endothelial proteins [34]. Because 6A3 is
able to recognize either the native form of DPP IV in
immunoprecipitation assays or the denatured form in
immunoblotting assays (Fig. 2A–D), the 6A3 epitope
in DPP IV appears to be relatively stable. This stabil-
ity is further confirmed by the ability of 6A3 to recog-
nize cell-surface expressed DPP IV either in live cells
(Fig. 5A) or formaldehyde-fixed tissues [2,4,35].
In conclusion, we have successfully identified the
epitope of the potent anti-metastatic mAb 6A3 that
blocks metastatic cancer cell adhesions to rat lung
endothelial DPP IV, most likely via steric hindrance.
The 6A3-epitope corresponding sequence in hDPP IV
may potentially be used to generate functionally
unique and safe monoclonal anti-metastatic sera.
Experimental procedures
Cell lines, antibodies and reagents
Rat lung-metastatic MTF7 cells, mouse B16F10 cells
and human MDA-MB-231 cells were obtained from

Dr B. U. Pauli (Cornell University, Ithaca, NY, USA) [1].
They were grown in DMEM (Invitrogen, Carlsbad, CA,
USA) containing 5% fetal bovine serum (FBS). Mock
(Mock-Jurkat) or hDPP IV-expressing (hDPP IV-Jurkat)
Jurkat cells were generous gifts from Dr C. Morimoto
(University of Tokyo, Tokyo, Japan). They were grown in
RPMI 1640 medium (Invitrogen) containing 5% FBS. mAb
6A3 and pAb CU31 were generated against rDPPIV [1].
mDPP IV mAb from rat was from R&D Systems (Minne-
apolis, MN, USA); hDPPIV mAb from mouse was from
Santa Cruz Biotechnology (Santa Cruz, CA, USA); human
FN pAb from rabbit was from Sigma (St Louis, MO,
USA); rabbit polyclonal anti-MBP serum and amylase aga-
rose beads were from New England Biolabs Inc. (Ipswich,
MA, USA); and IF7 mAb was a generous gift from Dr
C. Morimoto (University of Tokyo, Tokyo, Japan). All
other reagents were purchased from Sigma. [
35
S]methionine
was from ICN Biochemicals (Irvine, CA, USA) and
Affi-Gel 10 beads were obtained from Bio-Rad (Hercules,
CA, USA).
Plasmid construction
pMAL-c2 vector and full-length rDPP IV cloned in pRC-
CMV vector were obtained from Dr B. U. Pauli [1]. All
MBP-fusion fragments of DPP IV were PCR-amplified and
inserted into the EcoRI and HindIII sites of pMAL-c2 vec-
tor as described previously [1]. Using wild-type B-166 as
template, overlap extension PCR amplification was per-
formed to generate B-166 Hm mutant, B-166 Mn mutant,

B-166 T331I (T331) and B-166 V334T (V334T) as MBP-
fusion proteins, as described above [1]. The amino acids
included in the PCR-amplified fragments are described
in Doc. S1.
Purification of MBP-fusion fragments and rDPP IV
The fusion protein purification procedures have been
described previously [1]. Briefly, the cell lysates of Escheri-
chia coli cells expressing various MBP-fusion proteins were
passed through amylose columns and eluted with 10 mm
maltose in column buffer [1]. Immunoblotting with poly-
clonal anti-MBP serum was used to verify the purified pro-
teins. Rat lung DPP IV was purified from rat lung
extracts by 6A3 immunoaffinity chromatography [3].
[
35
S]methionine metabolic labeling of MTF7
for rDPP IV-conjugated Affi-Gel 10 affinity
precipitation
MTF7 Cells were first starved for methionine before addi-
tion of 50–100 lCi of [
35
S]methionine at 37 °C overnight.
Cells were recovered in DMEM containing 20% FBS as
previously described [3]. Before making cell lysates [36],
Table 1. 6A3-recognizable DPP IV fragments competitively neutral-
ize the 6A3 inhibitory effects on cancer cell adhesion to DPP IV.
Status of 6A3 DPP IV binding (A
492
)
a

DPP IV adhesion (%)
b
No 6A3 1.76 ± 0.18 79.1 ± 6.85
6A3 + B-166 V334T 0.67 ± 0.06
c
11.2 ± 6.49
c
6A3 + B-166 T331I 1.60 ± 0.20 69.23 ± 14.75
a
Binding of biotinylated rDPP IV to MTF7 in the absence or pres-
ence of 6A3 preincubated with B-166 mutants.
b
rDPP IV% specific
adhesion activities of MTF7 in the absence or presence of 6A3
preincubated with B-166 mutants.
c
Compared to the values of
assays without 6A3, these values are significantly decreased
(P < 0.05).
Epitope of monoclonal DPP IV antibody T T. Hung et al.
6556 FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS
some cells were first treated with 10 lgÆmL
)1
a-chymotryp-
sin for 30 min at 37 °C. Cell lysates were then subjected to
rDPP IV-conjugated Affi-Gel 10 affinity precipitation in the
absence or presence of 0.3 mgÆmL
)1
6A3 and then to SDS–
PAGE and autoradiography. The aliquots of cell lysates

were also subjected to immunoprecipitation with polyclonal
anti-FN serum [3].
DPP IV enzymatic activity assays
Purified rDPP IV (0.5 lg per assay) in the presence or
absence of a two-fold molar ratio excess of 6A3 was incu-
bated in 250 lL of assay buffer for 30 min at 37 °C, fol-
lowed by stopping the reaction with 750 lLof1m acetate
buffer and then measuring the absorption at 405 nm [4,35].
Cancer cell adhesion assays
rDPP IV adhesion activities of MDA-MB-231, MTF7 and
B16F10 (5 · 10
4
cells per well) in the absence or presence
of 0.3, 0.1 or 0.05 mgÆmL
)1
6A3 or the same isotype IgG1
(control) were measured at 37 °C, for 30 min in 96-well
plates coated with 100 lgÆmL
)1
purified rDPP IV as
described previously [1,3,35].
Immunoprecipitation and immunoblotting
[
35
S]methionine metabolically labeled MTF7 cell lysates
(1–3 mgÆmL
)1
) or purified MBP-fusion fragments
(0.1 lgÆmL
)1

) were incubated with various antibodies (1–
2 lg). The immunoprecipitates were subjected to immuno-
blotting, as described previously [36]. Full-length rDPP IV
(0.2 lg) and 0.05 lg of mouse IgG were subjected to 6A3
immunoblotting (dilution 1 : 1000) in the presence of vari-
ous MBP-fusion fragments (0.1 lg).
ELISA
A modified ELISA [1,3] was used to measure the binding
of soluble biotinylated rDPP IV (2 lg) to MTF7
(5 · 10
4
cells per well) grown at 37 ° C overnight on 96-well
plates (after PFD fixation) in the absence or presence of 50
and 300 lgÆmL
)1
polyclonal anti-FN serum, 6A3 or the
same isotype mouse IgG1 (control) with horseradish peroxi-
dase-conjugated streptavidin.
Immunofluorescent staining and fluorescence-
activated cell sorting (FACS) analysis
rDPP IV-expressing HEK293 cells were stained with
2 lgÆmL
)1
control mouse IgG2a or 6A3 preincubated with
10 lgÆmL
)1
MBP, B-166 wild-type, Mm, Hm, T331I or
V334T mutants, followed by PE-Cy5-conjugated goat
anti-mouse IgG and FACS analysis (detected using a FL3
photonmultiplier tube) with FACSCallibur (BD Bio-

sciences, San Jose, CA, USA) [1,36].
Software for DPP IV structural analysis
The ribbon- and surface-representations of rDPP IV(Pro-
tein Data Bank code: 2GBC) X-ray structure was visualized
with pymol, version 0.99 ( The
superimposition of rDPP IV and hDPP IV was performed
using friend 2.0 ( />php).
Acknowledgements
These studies were supported by National Science
Council Grant NSC94-2314-B-006-122, National Sci-
ence Council Grant NSC 95-2320-B-006-075-MY3,
and The Program for Promoting Academic Excellence
& Developing World Class Research Centers and Cen-
ter of Excellence for Clinical Trial and Research in
Oncology Specialty DOH-TD-B-111-004. The authors
wish to thank Dr Iain C. Bruce for his professional
editing on our manuscript and Dr Ming-Derg Lai for
his thoughtful discussion regarding the manuscript.
The authors also thank Ms. Megan Cheng for her
language assistance in manuscript writing and Mr
Chien-Fang Kuo and Mr Chu-kuei Lin for their help
in preparation and assembly of figures.
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Supporting information
The following supplementary material is available:
Fig. S1. The putative 6A3-binding epitope is not
located in the third- and the fourth-bladed b-propeller
domains.
Fig. S2. 6A3 recognizes the linear Thr331-dependent
eight-amino acid epitope.
Fig. S3. Structural comparison and superimposition of
rDPP IV and hDPP IV.
Doc. S1. Amino acids included in the PCR-amplified
fragments.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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may be re-organized for online delivery, but are not

copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
T T. Hung et al. Epitope of monoclonal DPP IV antibody
FEBS Journal 276 (2009) 6548–6559 ª 2009 The Authors Journal compilation ª 2009 FEBS 6559