© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685

TOOLS AND TECHNIQUES

Tailored placement of a turn-forming PA tag into the structured
domain of a protein to probe its conformational state

ABSTRACT
Placement of a tag sequence is usually limited to either terminalend of the target protein, reducing the potential of epitope tags forvarious labeling applications. The PA tag is a dodecapeptide(GVAMPGAEDDVV) that is recognized by a high-affinity antibody
NZ-1. We determined the crystal structure of the PA-tag–NZ-1
complex and found that NZ-1 recognizes a central segment of the PA
tag peptide in a tight β-turn configuration, suggesting that it iscompatible with the insertion into a loop. This possibility was tested
and confirmed using multiple integrin subunits and semaphorin. Morespecifically, the PA tag can be inserted at multiple locations within the
integrin αIIb subunit (encoded by ITGA2B) of the fibrinogen receptor
αIIbβ3 integrin (of which the β3 subunit is encoded by ITGB3) without
affecting the structural and functional integrity, while maintaining its
high affinity for NZ-1. The large choice of the sites for ‘epitope grafting’enabled the placement of the PA tag at a location whose accessibility
is modulated during the biological action of the receptor. Thus, we
succeeded in converting a general anti-tag antibody into a special
anti-integrin antibody that can be classified as a ligand-induced
binding site antibody.
KEY WORDS: Epitope tag, Integrin, Site-specific labeling, β-turn,
Reporter antibody

INTRODUCTION

Monoclonal antibodies show exquisite specificity toward theirrespective antigen by displaying a binding pocket with highchemical and geometrical complementarity to the antigen surface.
The pocket is lined by a set of spatially arranged residues in thecomplementarity-determining region (CDR) loops of the antibody
variable region. On the antigen, the epitope residues also need to becorrectly arranged in three dimensions, rather than just being closely
positioned in a primary sequence. As the conformation of a peptide
segment in the structured protein is usually very different from thatof free oligopeptide with the same sequence in solution (Dyson
et al., 1988), it can be difficult to obtain monoclonal antibodies thatare capable of recognizing native protein with high affinity by using
a synthetic linear peptide as the immunizing or screening antigen(Spangler, 1991). For antibodies raised against small peptide
fragments of proteins in order to generate reactivity with the cognatesequence of the intact protein, it is generally believed that they haveto be targeted to highly mobile segments (Tainer et al., 1984).
1

Laboratory of Protein Synthesis and Expression, Institute for Protein Research,
2
Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Departmentof Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan.
*Author for correspondence (takagi@protein.osaka-u.ac.jp)
Received 29 June 2015; Accepted 10 February 2016

1512

However, anti-peptide monoclonal antibodies are indifferent fromother anti-protein monoclonal antibodies in their requirement forcorrect three-dimensional (3D) arrangement of epitope residues,
other than the fact that all crucial residues are provided from a single
stretch of primary sequence. Meanwhile, numerous monoclonal
antibodies recognizing short linear peptide with high affinity and/or
specificity have been successfully obtained. Some such peptide andanti-peptide monoclonal antibody pairs have been incorporated into
research tools collectively called ‘epitope tag system’, with which
one can detect, label, capture and even purify tagged target proteinsvery easily (Wood, 2014). Use of such a system is vital to muchbiomedical research, especially when good antibodies against thetarget molecule are unavailable.
In addition to the utility as an immunological detection agent,monoclonal antibodies can sometimes be used as probe or reporter
for certain functional states of a protein, particularly when theprotein undergoes a large conformational change during its
biological action (Dennison et al., 2014; Humphries et al., 2003;
Irannejad et al., 2013; Walker et al., 2004). If the 3D structure of theprotein is known, it is theoretically possible to place a short peptide
tag at a desired position and to use a monoclonal antibody against
the tag to determine the function of the protein, as well as tomodulate the function of the protein. However, this is not practicallypossible because the tag insertion into the middle of a target protein
can often destroy its local conformation or alter its function. For thisreason, affinity tags are fused to either the N- or C-terminus of atarget protein in most applications. Even when the tag sequenceinsertion is not expected to affect the function of target protein, suchattempts are rare, because the inserted peptide itself does not usuallywork as a tag any more owing to the limited accessibility of theantibody and/or the altered conformation of the tag. However, wesometimes come across cases where protein termini are important
for the function, or are buried and not accessible to a binding
molecule or agent, in which case we have to seek ways to attach tags
somewhere else. There are several studies reporting successful
insertion of tag sequence into the middle of a protein domain bytargeting a flexible loop region that does not have secondary
structure. In these reports, however, either very long loops werechosen as the insertion point (Dinculescu et al., 2002; Morlacchi
et al., 2012; Smith et al., 2004) or the inserted tag was flanked bylong linker sequences (Facey and Kuhn, 2003; Kendall and
Senogles, 2006), limiting general applicability of this approach.
Therefore, there are unmet needs for the development of a generaltagging system that is compatible with the insertion or ‘grafting’ intoa structured protein domain for various cell biology andbiochemical applications.
Recently, we have reported the successful construction of a novel
epitope tag system, by combining a 12-residue peptide (PA tag)
derived from human podoplanin and a high affinity monoclonal
antibody (NZ-1) against it (Fujii et al., 2014). The binding affinity

Journal of Cell Science

Yuki Fujii1,2, Yukiko Matsunaga1, Takao Arimori1, Yu Kitago1, Satoshi Ogasawara2, Mika K. Kaneko2,
Yukinari Kato2 and Junichi Takagi1,*

of NZ-1 toward its epitope peptide is one to two orders of magnitude
higher than that of other popular anti-tag monoclonal antibodies,including the anti-FLAG M2 antibody. We found that the NZ-1–
PA-tag system, which has been made available from commercial
source, can be applied to a wide variety of applications, including
western blotting, immunoprecipitation and immunoaffinity
purification. Particularly important is its compatibility withpurification, where low-abundance proteins present in cell culture
supernatants or in cell lysates can be purified to near homogeneity injust one step. Detailed binding analysis has revealed that NZ-1
recognizes the core central portion of the PA tag, independently of
the sequence or structure of the peptides flanking both sides. This
property encouraged us to investigate the possibility of using the PA
tag system in the ‘epitope grafting’ application.
In this study, we determined the crystal structure of the NZ-1–PAtag complex and found that NZ-1 recognizes a β-turn structure thatis formed at the center of the PA peptide. The structure indicates that
the PA tag is compatible with the insertion into exposed loops
without accompanying linker sequences, which was experimentallyconfirmed in multiple target proteins. Furthermore, insertion of the
PA tag in a pinpointed manner enabled tailored placement of theNZ-1 epitope into the αIIb integrin subunit (encoded by ITG2AB) ofa platelet fibrinogen receptor, αIIbβ3 integrin (where the β3 subunit isencoded by ITGB3), as well as into the mouse β1 integrin,transforming a general anti-tag monoclonal antibody into a
specialized reporter and/or activator antibody that can beclassified as a ligand-induced binding site (LIBS) antibody.
RESULTS
Structure of the NZ-1 Fab in complex with the PA tag peptide

The NZ-1 fragment antigen-binding (Fab) fragment was crystallized
either in the presence of an excess concentration of a 14-residue
PA (PA14) peptide (EGGVAMPGAEDDVV, residues in boldcorrespond to the PA tag sequence) or on its own, leading to thedetermination of high-resolution structures of the complex (1.70 Å)and of the apo form (1.65 Å), respectively (Table 1). Both crystals
contained two copies of the molecules in the asymmetric unit,
deriving two independent Fab structures for each form.
Surprisingly, the structure of the variable (Fv) region was nearly
identical, not only between two molecules in the same crystal [Cαroot-mean-square deviation (RMSD) of 0.237 Å and 0.123 Å forthe complex and apo form, respectively], but also between thecomplex and apo form (Cα RMSD < 0.466 Å) (Fig. 1A). As the
structural difference between the two forms was minimal, even at
the level of the conformation of the side chains involved in the directcontacts with the antigen (Fig. 1A, right), the complementaritydetermining region (CDR) of NZ-1 seems to be poised at the‘binding competent’ conformation, partly explaining its very highaffinity. We also noted that some surface-bound H2O molecules
involved in a hydrogen bond network at the antibody–peptide
interface were already immobilized in the apo form (Fig. S1), further
highlighting the surface compatibility of the NZ-1 antigen-binding
groove with the peptide. In the two complex structures, clear
electron densities were visible for the C-terminal 11 or 12 residuesof the bound PA14 peptide (Fig. 1B). Because the unseen Nterminal two residues are not contained in the regular PA tag
constructs (Fujii et al., 2014), here, we have used peptide numbering
based on the 12-residue PA tag peptide. The PA peptide was dockedin the cleft that formed between the variable regions of the heavy
and light chains (VH and VL, respectively) of NZ-1 (Fig. 1C), andburied ∼1200 Å2 of the total solvent-accessible surface area (ASA)
at the interface. As predicted, NZ-1 recognized the central region of

Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685

Table 1. Data collection and refinement statistics

Data collection
Space group
Unit cell parameters

Resolution [Å]Number of uniquereflections
Completeness [%]Rsym or Rmerge [%]bRedundancy
I/σ(I )
Refinement
Resolution range [Å]Number of atoms
Proteins
Ligands
H2O molecules
Rwork [%]c
Rfree [%]dRMSD bond length [Å]RMSD bond angle [°]RMSZ bond length
RMSZ bond angle

Complexapoa

P21
a=42.2 Å,
b=80.0 Å,
c=132.5 Å,
β=96.5°40.0–1.56(1.65–1.56)
121,358 (18,070)

P1
a=42.9 Å,
b=57.2 Å,
c=101.5 Å,
α=97.9°,β=101.5°,
γ=105.4°
31.5–1.58(1.61–1.58)
118,170 (5308)

97.2 (90.2)
5.7 (126.3)
7.5 (7.2)
17.5 (1.5)

97.7 (89.0)
13.0 (76.7)
6.0 (3.8)
24.5 (2.3)

40.0–1.56(1.58–1.56)

31.5–1.58(1.60–1.58)

6404
175
499
17.9 (38.1)
21.4 (43.2)
0.018
1.50
0.87
0.86

6478
0
610
16.8 (27.5)
20.4 (30.9)
0.017
1.55
0.80
0.89

Values in parentheses correspond to the highest-resolution shell. RMSZ, rootmean-square value of the Z-scores.
a
Two dataset collected from two independent crystals were merged.
b
Rsym=100×Σ|Ihkl−<Ihkl>|Σ Ihkl, <Ihkl> is the mean value of Ihkl.
c
Rwork=100×Σ||Fo|−|Fc||/Σ|Fo|. dRfree was calculated from the test set (5% of thetotal data).

the PA peptide, and the conformation of the region spanning
residues Val2 and Val11 of the bound peptide was indistinguishable
between the two molecules (Fig. 1B). Within the binding pocket of
NZ-1, the PA peptide assumes a type II β-turn conformation at the‘MPGA’ sequence (Fig. 1C). There are numerous contacts formed
between the PA tag and NZ-1, primarily between residues 4–10 of
the peptide and the NZ-1 heavy chain (Fig. 1D). Among them, theresidue Met4 and Asp10 side chains provide the most prominentcontribution to the interaction, consistent with the result of thesurface plasmon resonance experiments using an alaninesubstituted peptide reported previously (Fujii et al., 2014).
Outside of the core interface, peptide residues 2, 3 and 11 also
make direct or indirect hydrogen bonds with NZ-1 using theirbackbone, indicating that the presence of residues at these sites, but
not a specific amino acid side chain, is important for the recognition.
Insertion of the PA tag into loop regions of integrin andsemaphorin

The unique binding mode visualized in the NZ-1–PA-peptide
complex, where the tight turn of the peptide was tucked into thebinding groove with both the N- and C-termini projecting away fromthe antibody, indicated an interesting possibility that the epitope
structure could be maintained when presented as a loop within atarget molecule. In order to test this hypothesis, we first tried toinsert the PA tag into exposed flexible loops of two test proteins,
integrin α6β1 (the α6 and β1 subunits are encoded by ITGA6 andITGB1, respectively) and semaphorin 3A (Sema3A). Although the
3D structure of integrin α6β1 is not available yet, a reasonable
1513

Journal of Cell Science

TOOLS AND TECHNIQUESTOOLS AND TECHNIQUES

Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685

homology model can be built by using integrin α5β1 (the α5 subunit
is encoded by ITGA5) as a template (Nagae et al., 2012). A longprotruding loop predicted to be present in the thigh domain of the α6
subunit for α6β1 integrin (Fig. 2A) was chosen as an insertion point.
For Sema3A, for which the crystal structure is known, we chose aloop in the N-terminal Sema domain (Fig. 2B). Both insertions are
predicted to have no effect on the function of the respective proteins,because they are located far from the interfaces involved in theinteraction with physiological binding partners (Fig. 2A,B, arrows).
In fact, these mutants were expressed well at the same or even betterlevels than the wild-type counterparts (Fig. 2C,D, lanes 5 and 6)
and exhibited normal biological activities (data not shown).
Furthermore, both insertions were functionally active as epitope
tags, because the secreted PA-tagged version of both proteins could
be efficiently immunoprecipitated in their native state with the NZ-1
antibody from the culture medium (Fig. 2C,D, lane 2). These results
clearly demonstrate that the 12-residue PA tag peptide inserted into
the middle of a long flexible loop is able to assume the β-turnconformation that is required for recognition by NZ-1 and can fully
function as epitope tag.
1514

Next, we aimed to see if the PA tag could be inserted into loops
with a higher degree of structural restriction. The αIIb subunit ofintegrin αIIbβ3 comprises four large all-β domains and bears many
surface-exposed loops that are ideal for evaluating the effect ofloop length on the applicability of the PA tag insertion (Fig. 3A)(Zhu et al., 2008). First, we chose the 3-4 loop of blade 2 [referred
to as W2, where a four-stranded antiparallel sheet is represented by‘W’ (Springer, 1998)] of the β-propeller domain as the insertion
point. As this four-residue (PESG) loop is at one end of a β-hairpinthat connects neighboring strands with an end-to-end distance of
only 5.2 Å (Fig. 3B), a simple insertion of the PA peptide, which
has an end-to-end distance of 10.2 Å in the NZ-1-bound state,might not be structurally compatible. We therefore tested several
constructs, inserting the PA tag dodecapeptide flanked by linker
segments of varying lengths at the W2 site (Fig. S2A). The
insertion itself did not seem to destabilize the integrin because allinsertion mutants were expressed normally regardless of the linker
length (Fig. S2B, lanes 8–12). Surprisingly, all these mutants
could be successfully immunoprecipitated by the NZ-1 antibody(Fig. S2B, lanes 2–6), indicating that even the minimum (i.e. no

Journal of Cell ScienceFig. 1. Structure of the antigen-binding site of anti-PA
tag antibody NZ-1. (A) Four structures of the NZ-1 Fabderived from apo and PA-tag complex forms (two molecules
for each) are superposed at the Fv region and shown in Cα
tracings with different colors. A close-up view of the antigenbinding pocket (dotted oval) is shown in the right, withresidues that are in direct contact with the PA-tag peptide
shown in the stick models. Bound peptide has been
removed for clarity. CDR-H and CDR-L, CDRs of the heavy
and light chains, respectively; Mol, molecule.
(B) Conformation of the PA-tag peptide bound by NZ-1.
Structural models for the peptide in two molecules areshown as stick model with the weighted 2Fo-Fc electron
density map (magenta wireframe) contoured at 1.0 σ. The
NZ-1 Fab heavy (green) and light (cyan) chains are shown incartoon models. (C) The PA-tag peptide (stick model) boundin the pocket on top of the NZ-1 Fab (surface model) is
shown, with the main chain hydrogen bond maintaining thetype II β-turn (yellow dotted line). Colors are as inB. (D) Residue-wise contacts between the NZ-1 Fab (thincolors) and the PA peptide (thick colors), analyzed by using
the LIGPLOT program. The bond-length cut off was set to3.35 Å for hydrogen bonds (denoted by green dotted line)
and an inter-atomic distance range of 2.9–3.9 Å was usedfor van der Waals contacts (orange eyelash). Oxygen,nitrogen, carbon and water molecules are shown as red,
blue, black and cyan spheres, respectively.

TOOLS AND TECHNIQUES

Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685

linker) PA tag inserted at a very tight hairpin loop remained
reactive with NZ-1.
Next, we expanded the target location of the insertion to see if theabove property of the PA tag is generally applicable. We chose ninemore sites, including five β-hairpins similar to W2 (W1, cap, W3,
W6, Calf1_XZ), three inter-sheet loops with intermediate lengths(Thigh_FG, Calf1_EF, Calf2_FG) and one very long loop thatharbors internal furin cleavage site (Calf2_XY) (Fig. 3C; Fig. S3).
First, the effect of these mutations on the structural integrity of theprotein was evaluated by comparing the level of expression in thecontext of the soluble ectodomain fragment of the αIIbβ3-integrin
heterodimer. As shown in Fig. 4A, all mutants except for W3 andW6 could be expressed and secreted into the medium, indicating thecompatibility of these eight insertions to the native structure. The
location of the W3 and W6 insertions were very close to the Ca2+binding sites known to be important for the correct folding of the
β-propeller domain of α integrin subunits (Mitchell et al., 2003),
suggesting that the correct metal ion coordination geometry was
severely perturbed by these two mutations. Among the eightmutants that could be expressed and secreted successfully, seven

were immunoprecipitated with NZ-1 at the same efficiency as thatwith the pan-β3-integrin monoclonal antibody 7E3 (Fig. 4B),
confirming the successful insertion and presentation of the epitope
structure at a variety of locations in αIIbβ3 integrin. The cap mutant
was reactive with NZ-1 but at a reduced level compared to that of theother constructs (Fig. 4B, lane 4). To gain a more quantitative
assessment on the effect of loop insertion, we purified six αIIbβ3integrin mutants and measured their binding affinity for NZ-1.
Kinetic binding analyses using biolayer interferometry (BLI)
derived KD values ranging from 0.6 to 3.6 nM (Fig. 4C), whichwere similar to the values obtained previously with various
terminally PA-tagged proteins (Fujii et al., 2014). As it is natural
to expect slight variations in the affinity value of a monoclonal
antibody for a peptide when the epitope is placed in different
structural contexts, we conclude that insertion of the PA tag is
generally applicable to a wide range of loop and turn positions in avariety of target proteins. It is also noteworthy that a functionallyactive PA-tag epitope can be readily inserted into the middle of aloop of target proteins without extensive optimization of linker
length (Fig. S2), which has great technical advantages.
1515

Journal of Cell ScienceFig. 2. The PA tag can be inserted into long flexible loops. Loops selected for the PA-tag insertion (asterisk) are shown in the homology model of the α6β1integrin headpiece fragment (A) and the crystal structure of mouse Sema3A (Protein Data Bank ID: 4GZ8) (B). The actual amino acid sequences for wild-type(WT) and insertion mutant (PAins) near the insertion positions are shown below the structure, with the inserted PA-tag sequence and the loop segments shown inthe structure colored red and magenta, respectively. Locations of known protein–protein interfaces that are important for the function of these proteins areindicated by arrows. (C,D) Immunoprecipitation of the PA-tagged α6β1 headpiece fragment (C, α6+β1) and Sema3A (D) by NZ-1. The PA-tagged, but not the WT,
version of each protein could be specifically captured by NZ-1 immobilized beads and eluted by the PA peptide (lanes 2 and 3), whereas both bound to beads
immobilized with anti-α6-integrin antibody GoH3 (C, lanes 5 and 6) or to Ni-NTA agarose (D, Ni-NTA, lanes 5 and 6). Bands labeled with an asterisk were present
in all samples, including that obtained from mock-transfected cells (lanes 1 and 4) and, hence, represent nonspecific contamination.

TOOLS AND TECHNIQUES

Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685

Fig. 3. Surface-exposed loops in the αIIbβ3integrin ectodomain selected for the tag
insertion. (A) Crystal structure of the αIIbβ3-integrin
ectodomain (Protein Data Bank ID: 3FCS) is shownin red (αIIb) and gray (β3) cartoon models, with the ten
PA-tagged loops labeled. (B) Conformation of the βhairpin at the W2 site is shown side by side with thatof the reverse U-shaped PA-tag peptide taken fromthe PA–NZ-1 complex structure molecule
2. (C) Amino acid sequences of the segments
containing the insertion position (indicated by //).

property of the PA tag. In order to test the uniqueness of the PA tag,two popular peptide tags, Myc and FLAG, were inserted into threelocations (W2, Thigh_FG and Calf1_XZ) and the functionality wascompared with that of the PA tag. As evident from the successful
Fig. 4. The PA tag can be inserted into multiple
αIIb-integrin loops. (A) Soluble ectodomain
fragments of integrins comprised of various αIIbmutant and wild-type β3 subunits were expressed in
HEK293T cells and immunoprecipitated with the antivelcro-tag antibody 2H11, followed by 7.5% SDSPAGE analysis under reducing conditions andvisualization with the Oriole fluorescent protein stain.
Note that the mobilities of the Calf1_XZ and Calf2_XY
mutant αIIb chains were different from that of the wild
type, owing to the elimination of an N-linked
glycosylation site at residue Asn680 (for Calf1_XZ)
and a furin-cleavage site within 855-KRDRR-859 (forCalf2_XY), respectively. (B) The PA-tagged mutant
αIIbβ3 ectodomain fragments were expressed as in A
and immunoprecipitated (IP) with the anti-β3-integrin
antibody 7E3 (upper panel) or NZ-1 (lower panel),followed by immunoblotting with the anti-velcro
polyclonal antibody. (C) Determination of the affinity of
the NZ-1 antibody toward each insertion mutant. The
αIIbβ3-integrin ectodomain fragments with inserted PA
tag were purified and subjected to kinetic binding
analyses using biolayer interferometry. Eachsubpanel shows the overlaid binding curves obtainedwith four different concentrations (10, 5, 2.5 and1.25 nM) of the PA-bearing integrin. Dissociation
constant (KD) values were derived by global curve
fitting (black thin lines) to the actual binding curves(cyan) and are shown as mean±s.d. (n=3). (D) PA(lanes 1 and 4), FLAG (lanes 2 and 5) and Myc (lanes
3 and 6) tags were inserted at three select sites,
immunoprecipitated with the 7E3 antibody (lanes 4–8)
or corresponding anti-tag antibodies (lanes 1–3), andvisualized by anti-velcro immunoblotting. In B and D,
SDS-PAGE analysis was conducted under nonreducing conditions so that the αIIbβ3 integrin migrated
as a single ∼250 kDa band (arrow).

1516

Journal of Cell Science

Although we believe that the insertion compatibility of the PA tag
is attributable to its turn-forming propensity revealed by the crystal
structure, one could think that the compatibility merely reflects theflexible nature of the selected loops rather than reflecting a special

expression and secretion of the mutant integrins (Fig. 4D, lanes 5
and 6), insertion of these tags was structurally tolerated, as in thecase of the PA tag, which is not surprising considering the shorter
length of these tags (eight and ten residues for FLAG and Myc,
respectively). In contrast to the PA -tagged versions, however, bothinserted sequences completely lost their reactivity with theirrespective monoclonal antibodies (Fig. 4D, lanes 2 and 3). This
result strongly suggests that Myc and FLAG sequences can nolonger function as epitope tags upon presentation within theseloops, underscoring the uniqueness of the PA tag.
Utility of the PA-tag–NZ-1 system to explore theconformational state of cell surface integrin

To explore the possibility of using the PA tag as a reporter system forreceptor conformation, seven of the insertion mutations wereintroduced in the context of the cell surface αIIbβ3-integrin
heterodimer. As judged by the flow cytometry analyses of cells
stained with an antibody against αIIb integrin (Fig. 5A, upper panels),
all mutants were expressed well on the cell surface. The two mutants(W1 and Calf2_FG) that had exhibited decreased levels of expression
and secretion in the context of the soluble ectodomain fragment(Fig. 4A) were also expressed well, suggesting that the negative effectof these insertions on the structural integrity of the receptor was notsevere. More importantly, these PA-tagged integrins exhibited normal
biological activity, as monitored by the activation-dependent bindingof the ligand-mimetic antibody PAC-1 (Fig. 5B).

Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685

As in the case of soluble ectodomain fragments, all these PAtagged mutant integrins were reactive with NZ-1, as judged by flow
cytometry analysis (Fig. 5A, lower panels). However, we found that
one mutant (Calf1_EF) showed consistently weak staining with
NZ-1, in spite of its high overall expression. We reasoned that thisreflects limited accessibility to the inserted PA tag in the context ofcell surface integrin, because this site is predicted to be partiallyhidden in the ‘bent’ conformation of resting integrin (Fig. 6A) andprevious experiments showed that the affinity of NZ-1 for this site is
not particularly low compared to other mutants (Fig. 4C). In fact,
NZ-1 reactivity toward this mutant was increased upon the treatmentof cells with Mn2+ and RGD peptide, a known ‘activating’ condition
that induces a global conformational change in integrins (Takagi
et al., 2002), and such effect was not observed with two othermutants (Fig. 6B). Although the net increase in the NZ-1 reactivity
was relatively small compared to the total binding signal, this assay
can report a range of integrin conformational changes caused bydifferent concentrations of soluble ligand (Fig. S4). A group of
antibodies recognizing integrin whose binding is upregulated upon
integrin activation or ligand-binding are called LIBS antibodies,even though the binding can be upregulated by certain cations suchas Mn2+ in the absence of ligands. The above result clearly
demonstrates that NZ-1 can behave as one of these LIBS antibodies,when combined with an appropriate PA-tag insertion design.
Cell surface integrins are known to exist in a conformational
equilibrium between bent (low affinity) and extended (high affinity)

Fig. 5. Grafting the PA-tag epitope in the context of αIIbβ3 integrin expressed on the cell surface. (A) Various PA-tagged full-length αIIbβ3 integrins weretransiently expressed on podoplanin-null HEK293T cells (293T-PA−) and co-stained with the anti-αIIb-antibody 10E5 (upper panels, green histograms) and NZ-1
(lower panels, orange histograms), followed by flow cytometry analysis using Alexa-Fluor-488- and phycoerythrin (PE)-labeled secondary antibodies againstmouse and rat IgGs, respectively. Signals from untransfected parental cells (gray area in the upper panels) were used to define 10E5-positive cell populations(brackets, ∼2000 cells for all mutants), and NZ-1 staining was evaluated only for those cells. The mean fluorescence intensity (MFI) values are shown. (B) Ligandbinding activity of the PA-tagged αIIbβ3 integrin. 293T-PA− cells that had been transiently transfected with the PA-tagged αIIbβ3 integrin were left untreated(blue solid line) or incubated with DTT (red solid line) to activate the integrin and were then co-stained with Dylight680–AP3 ( pan-β3-integrin monoclonal antibody)and FITC–PAC-1. The histograms show the FITC fluorescence (i.e. PAC-1 binding) from the cell populations that were positive for AP3 staining. WT, wild-type
αIIbβ3-integrin construct.

1517

Journal of Cell Science

TOOLS AND TECHNIQUESTOOLS AND TECHNIQUES

Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685

Fig. 6. The ‘LIBS’ nature of the PA tag inserted at
the Calf1_EF site. (A) Location of the loop insertion.
The entire αIIbβ3-integrin ectodomain in the bent form
(Protein Data Bank ID: 3FCS) is shown as a surface
model, with αIIb-integrin and β3-integrin chains in red
and gray, respectively. Locations of PA-tag insertions
into loops used in the experiments are highlighted incyan and labeled. Note that the Calf1_EF site is
located inside the bend, partially occluded by thebowed head, comprising the β-propeller domain.
(B) Conformation-dependent epitope exposure. 293TPA− cells that transiently expressed αIIbβ3-integrin withthe three different PA-tag insertions indicated wereincubated with Dylight680–AP3 alone (gray),
Dylight680–AP3 and FITC–NZ-1 (blue), or
Dylight680–AP3, FITC–NZ-1, 0.5 mM MnCl2 and1 mM RGD peptide (red). FITC fluorescencehistograms obtained with AP3-positive cell
populations are shown. (C) The ligand-binding activityof αIIbβ3-integrin with the Calf1_EF mutation can be
activated by NZ-1. 293T-PA− cells that had beentransiently transfected with the PA-tagged αIIbβ3integrin were stained with Dylight680–AP3 and FITC–
PAC-1 in the absence (gray) or presence (green) of
10 μg/ml unlabeled NZ-1. FITC fluorescencehistograms from the cell populations that were positive
for AP3 staining are shown. WT, wild-type αIIbβ3integrin construct.

1518(Fig. 7Bi, red). A rat monoclonal antibody recognizing β1 integrin(9EG7), a known LIBS antibody that can recognize both human andmouse β1-integrin chains (Bazzoni et al., 1995), also showed
increased binding upon Mn2+ treatment (Fig. 7Bii). However, themagnitude of the epitope exposure over a range of Mn2+
concentrations was greater for NZ-1 than for 9EG7 (Fig. 7C),
although the total maximum binding sites per cells should be higher
for 9EG7 because of the simultaneous recognition of transfectedmouse β1-integrin and endogenous human β1-integrin chains.
Therefore, the combination of the PA-tagged β1 integrin and NZ-1
offers a unique opportunity for the conformational probing of β1
integrins in various cell biology applications.
DISCUSSION

As a result of the structural analysis of the NZ-1–PA-peptide
complex, basis for the high affinity and specificity of this interaction
was justified. First, comparison between the structure of the apo state(i.e. without peptide) and the complex revealed that only a minimalchange is required for the unbound NZ-1 to accommodate the PAtag peptide, providing a structural basis for the very high affinity.
Another potential source for the high affinity likely resides in the
β-turn-forming propensity of the peptide. Because the Pro-Gly
sequence is known to occur very frequently in the type-II turn of
known protein structures (Guruprasad and Rajkumar, 2000), thispart of the epitope structure tends to be ‘preformed’, before theencounter with NZ-1, making the binding entropically favorable.
We discovered, however, that this turn-forming propensity of the PA
tag has a far more important implication in its unique utility in the‘loop insertion’ application. Inserting a functional tag sequence into
a protein has been difficult because the inserted tag would havedifficulty in assuming the correct conformation for the antibody

Journal of Cell Sciencestates, and shifting this equilibrium from inside the cell constitutes theregulatory mechanism of cell adhesion (Carman and Springer, 2003).
Because exogenous addition of antibodies that preferentially bind to
the extended conformation (such as LIBS monoclonal antibodies)can generally shift the equilibrium toward the high-affinity state, they
can often ‘activate’ integrins from outside of the cell (Humphries,2004). On the resting cell surface, wild-type αIIbβ3 integrin exhibited
negligible binding of the PAC-1 ligand, regardless of the addition of
NZ-1 (Fig. 6C, panel WT). However, the cells expressing theCalf1_EF mutant of αIIbβ3 integrin specifically bound to ligand upon
addition of a saturating concentration of NZ-1. This effect was notobserved in other PA-tagged mutants, indicating that the increased
ligand binding was due to the conformational change of integrin
induced by NZ-1 and not by the bound NZ-1 itself.
Encouraged by the successful conversion of NZ-1 into a LIBS
antibody for human αIIbβ3 integrin as described above, we next
designed an ‘activation reporter’ mutant for a different integrin
subunit. As most integrin LIBS antibodies available are specific forhuman integrins, development of antibodies that can reportconformational changes of mouse integrin is of great value. The
HUTS-4 mouse monoclonal antibody against human β1 integrin is anactivation-reporter antibody whose epitope residues have been
precisely defined (Mould et al., 2003). We inserted the PA tag at
the E-F loop of the mouse β1 hybrid domain that harbors Asn417,
which corresponds to the HUTS-4 epitope in human β1 integrin(Fig. 7A). When transiently transfected into HEK293T cells together
with the human α4-integrin subunit, the PA-tagged mouse β1 integrin
was expressed well on the cell surface and it could be stained with NZ1 (Fig. 7Bi, blue). When the cells were treated with 1 mM MnCl2, a
condition known to expose the activation epitope on β1 integrin(Takagi et al., 1997), the NZ-1 reactivity was upregulated, as expected

Fig. 7. Targeted insertion of the PA tag at a ‘LIBS’ site in the mouse β1
integrin. (A) A homology model of the headpiece portion of mouse β1 integrin(blue) in complex with the human α4-integrin chain (gray) is shown in a ribbon
model, with the three-residue portion replaced by the PA tag sequence in thedesigned mutant (PAins) shown in magenta with a stick model. Residue
Asn417 at the center of this segment (inset) corresponds to one of the crucial
epitope residues (Lys417) for the LIBS monoclonal antibody HUTS-4 in human
β1 integrin. Sequence alignment of human and mouse β1 integrin near theinsertion site is shown at the bottom of the panel. (B) Flow cytometry profiles of
NZ-1 (i) or 9EG7 (ii) binding to 293T-PA− cells expressing mouse β1-integrin(PAins) in the absence (blue) or presence (red) of 1 mM MnCl2. (C) The bindingof NZ-1 (i) or 9EG7 (ii) to either wild-type (WT) or PAins mutant versions ofmouse β1 integrin were evaluated as described in B in the presence of varying
concentrations of MnCl2. The data are presented as the mean fluorescenceintensity (MFI) of the total cell population. Data are mean±s.d. from three
independent experiments.

binding owing to the structural constraints. Certain high-affinity
antibodies against linear tag peptides recognize relatively long
stretches of peptide in an extended conformation (Krauss et al.,2008), which is difficult to achieve when inserted into a folded

Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685

protein domain. In contrast, NZ-1 recognizes the central region of
the PA tag when the tag is in a compact folded configuration,
enabling the presentation of a functional epitope structure in a loopof a folded globular domain. Furthermore, the PA tag can be inserted
into a very tight hairpin without a need for extensive optimization or
addition of linker residues. So far, we have successfully grafted the
PA tag onto several different targets, including α6-integrin, αIIbintegrin and β1-integrin chains, as well as into Sema3A (Figs 2, 3
and 7). Our ongoing research also suggests that the tag can beinserted into surface-exposed loops of multi-pass membraneproteins without compromising their biological functions (data notshown). Precise structural information regarding the target protein is
helpful but not essential for design of the insertion site, as long as
one can build a reasonable homology model with the correct
secondary structure assignments for the target.
The ‘insertion’ compatibility of the PA-tag system provides
several benefits that have been lacking with other epitope tag
systems. First, it can offer a way to attach purification tags to targets
whose N- and C-termini are not available for functional or structural
reasons. These include cases where termini are inaccessible toantibody in the native structure or cannot be modified because they
are an integral part of the active site of the target protein. Second, theutility of the inserted tag is not limited to the purification purpose.
Because one can insert the PA tag into a desired location of thetarget molecule in a pinpointed manner, it offers an easy way toachieve site-specific labeling in many applications. Third, the PA
tag (12 residues) is substantially smaller than GFP or otherinsertion-compatible protein domains and, hence, less likely tocause structural and functional alterations. Finally, owing to the very
slow dissociation rate of the PA-tag–NZ-1 interaction (Fujii et al.,2014), labeling with the Fab or the single chain variable fragment(scFv) of NZ-1 could be used to visualize individual domains or
subunits in electron microscopy analyses or to probe conformational
dynamics in Förster resonance energy transfer (FRET) experiments.
The advantage of the site-specific PA-tag insertion can also beappreciated in a unique application where a general anti-tag
monoclonal antibody is converted into a conformation reporter or
modulator, as exemplified by the results with the αIIb and β1
integrins presented here. Integrin experimental biology has greatly
benefitted from the use of various conformation-dependent
monoclonal antibodies. Numerous studies have identified socalled LIBS antibodies, which preferentially recognize activatedor high-affinity state integrin conformations (Humphries, 2004),
leading to the concept that the conformationally polymorphic
integrin undergoes global extension to expose LIBS epitopes during
activation and ligand binding (Takagi and Springer, 2002).
However, such monoclonal antibodies are obtained by chance,and LIBS antibodies are available for only a limited set of integrin
subunits. Particularly, LIBS antibodies that can react with mouse
integrins are in short supply, because most monoclonal antibodies
are still produced in mice. Another problem with the currently
available LIBS monoclonal antibodies is that it is impossible toperform quantitative comparisons between the patterns of epitope
exposure (i.e. conformational change) by using multiple antibodies
against different epitopes because of the inevitable difference intheir binding affinity and kinetics. In contrast, NZ-1 would bind to
the grafted PA epitope with similar binding affinity regardless of thelocation of the insertion (Fig. 4C). Therefore, the PA-tag–NZ-1
system will provide an opportunity not only to detect
conformational changes of integrins that have been previously
difficult to detect owing to the lack of appropriate antibody, but also
to gain insights into the real conformational dynamics of cell surface
1519

Journal of Cell Science

TOOLS AND TECHNIQUESintegrins by allowing comparative analysis of the exposure kinetics
for different epitopes. For applications involving cell-based assays,
one must avoid using human cells that express endogenous
podoplanin, the original antigen for the NZ-1 monoclonal
antibody. In order to get around this problem, we have establisheda HEK293T-derived cell line devoid of podoplanin expression(293T-PA−), which can be provided upon request. Moreover, NZ-1
does not react with podoplanin of mouse, rat, hamster or dog origin(Fujii et al., 2014), and is fully compatible with cells from theseanimals. Therefore, the uses described above are not limited tointegrin research and should be applicable to studies of any
signaling system that involves receptor conformational change.
Another yet unexplored possibility of the PA-tag–NZ-1 system is
its use as a tool for facilitating crystallization. Crystallization
chaperones are attracting much attention as a promising way toincrease the likelihood of obtaining well-ordered crystals of
biologically important and difficult targets for structural studies(Koide, 2009). The advantages of chaperone-mediatedcrystallization are thought to be twofold: firstly, it can reduce
conformational heterogeneity of target molecules by binding to a
specific conformation, and secondly can provide increased surface
area to promote crystal lattice formation (Koide, 2009). Monoclonal
antibodies are ideal candidates as crystallization chaperones, but they
have to be individually developed and screened for each target,
making the strategy very costly and unpredictable. The idea ofdeveloping a portable peptide tag and anti-tag ‘chaperoning’
antibody has been pursued by Lieberman and colleagues (Johnson
et al., 2015), but the technology has not been realized so far. As theinserted PA tag is a ‘foreign’ structure placed on the client protein,
NZ-1 might not have a strong stabilizing effect on the conformation,
as in the case of the target-specific chaperone antibody. If the inserted
loop is sufficiently short, however, the bound NZ-1 will haveminimum mobility while presenting sufficient surface for the latticecontacts. Therefore, use of NZ-1 Fab in assisting crystallization,
particularly for multi-pass membrane proteins where the PA tag isinserted into one of extramembranous loops, might prove highly
promising, and further studies are eagerly awaited.
MATERIALS AND METHODS
Antibodies and peptide

The monoclonal antibodies used in this study were: 2H11 (anti-ACID/BASE‘velcro’, kindly provided by Ellis Reinherz) (Chang et al., 1994), AP3 (anti-β3
integrin, HB-242, obtained from American Type Culture Collection)(Newman et al., 1985), 7E3 (anti-β3-integrin, kindly provided by BarryS. Coller) (Coller, 1985), 10E5 (anti-αIIb-integrin, kindly provided by
B. S. Coller) (Peerschke and Coller, 1984), GoH3 (anti-α6-integrin,
MAB1378, Merck Millipore), 9EG7 (anti-β1-integrin, 553715, BD
Biosciences) (Bazzoni et al., 1995), M2 (anti-FLAG, F-3165, SigmaAldrich) and 9E10 (anti-Myc, 017-21871, Wako Pure Chemical Industries,Ltd.). Anti-PA tag rat monoclonal antibody NZ-1 IgG was either purified fromhybridoma culture supernatants (Fujii et al., 2014) or purchased from Wako
Pure Chemical Industries, Ltd. (016-25861). The rabbit anti-velcro polyclonal
antibody (Takagi et al., 2001) was biotinylated using EZ-link Sulfo-NHS-LCBiotin (Thermo Scientific). Fluorescent labeling of AP3 was conducted using
DyLight 680 NHS Ester (Thermo Scientific) according to the manufacturer’s
recommendation. The FITC-conjugated mouse anti-αIIbβ3 ligand-mimetic
antibody PAC-1 (FITC–PAC-1) was obtained from Becton, Dickinson andCo. (340507). The Alexa-Fluor-488-conjugated goat anti-mouse IgG
polyclonal antibody (rat absorbed) and the phycoerythrin-conjugated goatanti-rat IgG polyclonal antibody (mouse absorbed) were obtained from
Invitrogen (A11029) and Southern Biotechnology (3010-09), respectively.
The PA14 peptide (EGGVAMPGAEDDVV) was synthesized and purified as
described previously (Fujii et al., 2014), and RGD peptide (GRGDSPK) was
purchased from Sigma-Aldrich.

1520

Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685

Preparation of NZ-1 FabPurified NZ-1 IgG was dialyzed against PE buffer (20 mM sodium andpotassium phosphate, 10 mM EDTA, pH 7.0) and incubated withimmobilized papain (Thermo Scientific) (0.1 ml per mg of IgG) in thepresence of 25 mM cysteine-HCl ( pH 7.0) for ∼4 h at 37°C under gentle
agitation. After the removal of the immobilized papain, the reaction mixture
was dialyzed against Tris-buffered saline (TBS; 50 mM Tris-HCl, 150 mM
NaCl, pH 7.5) and slowly added to PA14-peptide-conjugated Sepharose.
Bound Fab was eluted with 10 mM MES containing 3 M MgCl2, pH 6.0,
dialyzed against TBS, and further purified by performing gel filtration
chromatography on a Superdex 200 16/60 column equilibrated with TBS.
Structural analysis of NZ-1 with and without bound PA peptide

The purified NZ-1 Fab was concentrated to ∼10 mg/ml by ultrafiltration
using Spin-X UF (MWCO: 10,000) and subjected to the crystallization
trials. Initial screening of crystallization conditions was carried out using
Crystal Screen 1 and 2 (Hampton Research) by using the sitting drop vapordiffusion method, resulting in many hit conditions. Upon the optimization,
the diffraction quality crystals were grown under the condition of 0.1 M
sodium citrate buffer, pH 5.6, and 12–15% (w/v) PEG4000. Crystals were
cryoprotected with the same buffer containing 10% (v/v) glycerol and used
for data collection at BL17A of Photon Factory (Tsukuba, Japan) and
BL15A1 of The National Synchrotron Radiation Research Center (NSRRC,
Hsinchu, Taiwan). As the data sets collected at two beamlines have the same
crystal system (Table 1), these data were combined and processed using the
HKL2000 program package (Otwinowski and Minor, 1997). Initial phases
were determined by using the molecular replacement method using a rat
anti-NGF AD11 antibody Fab (IgG2a) structure (Protein Data Bank ID:
1ZAN) as a search model, by the Phaser-MR (Mccoy et al., 2007) program.
For the determination of the NZ-1–PA14 complex structure, NZ-1 Fab was
mixed with the PA14 peptide at a molar ratio of 1:3 and subjected to the
crystallization process. A diffraction quality co-crystal was grown under thecondition of 0.1 M sodium citrate buffer, pH 5.6, and 24% (w/v) PEG4000,
cryoprotected with 10% (v/v) ethylene glycol in the same buffer, and an
X-ray diffraction data set was collected at the beam line BL1A of Photon
Factory. The data were processed using XDS program package (Kabsch,2010). Initial phases were determined by using molecular replacement with
the NZ-1 Fab structure described above as a search model. The structuralmodels were built using COOT software (Emsley et al., 2010) with modelrefinement cycle with REFMAC5 (Murshudov et al., 2011) and phenix.
refine (Adams et al., 2010) programs. The models were validated using the
program MOLPROBITY (Chen et al., 2010). Data collection andrefinement statistics are listed in Table 1. The interaction between the
PA14 peptide and the NZ-1 Fab was analyzed using the program LIGPLOT(Wallace et al., 1995). All structural figures were prepared with the program
PyMOL (Schrödinger, LLC).
Generation of DNA constructs

cDNAs encoding human α6- and β1-integrin chains were truncated after
residues 612 and 481, respectively, to express a α6β1-integrin headpiecefragment. The 12-residue PA-tag sequence was inserted into a site shown in
Fig. 2A by performing overlap extension PCR. Similarly, PA-tagged versionof mouse Sema3A was constructed from mouse full-length Sema3A cDNA
N-terminally tagged with hexahistidine residues. The design of secreted theectodomain fragment of integrin αIIbβ3 was conducted by adopting thestrategy used for αVβ3 integrin, described previously (Takagi et al., 2002).
Briefly, ACID and BASE peptides were appended at the C-termini of thecomplete ectodomain of human αIIb- (residues 1–963) and β3-integrin(residues 1–692) chains, resulting in the formation of a disulfide-linked αhelical coiled-coil knot called ‘velcro’ (O’shea et al., 1993). The PA-tag
sequence was inserted at various sites of the αIIb subunit, as shown in
Fig. 3C. Each insertion position was designated based on the location in thedomain and secondary structure elements defined by Xiao et al. (2004). For
the cell surface expression of these integrins, mutated regions wereintroduced into the vectors coding for full-length αIIb and β3 subunits, as
described previously (Luo et al., 2004). For the design of the ‘activationreporter’ construct for mouse integrin β1, the PA tag was inserted into theE-F loop of the hybrid domain of the mouse full-length expression construct.

Journal of Cell Science

TOOLS AND TECHNIQUESTOOLS AND TECHNIQUESThe homology models of human α6β1-integrin (Fig. 2A) and mouse α4β1integrin (Fig. 7A) headpiece fragments were built by using the program
MODELLER (Eswar et al., 2008), using the structure of the α5β1-integrin
headpiece (Protein Data Bank ID: 3VI3) (Nagae et al., 2012) as a template.
Expression and immunoprecipitation of the soluble αIIbβ3 integrin

For the expression of soluble ectodomain fragments of αIIbβ3 integrin,
HEK293T cells that had been plated on 6-well plates were transiently
transfected with plasmid DNAs coding for soluble αIIb and β3 subunits(2 μg/subunit/well) using X-tremeGENE HP DNA Transfection Reagent(Roche). Culture supernatants were harvested ∼72 h after the transfection
and incubated with either 2H11–Sepharose or NZ-1–Sepharose for ∼2 h
at 4°C. After washing three times with TBS containing 1 mM CaCl2 and1 mM MgCl2, the resin-bound αIIbβ3 integrin was eluted by using ×2 SDS
sample buffer, subjected to 7.5% SDS-PAGE and stained with Oriole
fluorescent dye (Bio-Rad). For some experiments, integrins were
immunoprecipitated from the culture supernatants with purified IgG (antiβ3-integrin antibody 7E3, 2 µg/ml; anti-PA-tag antibody NZ-1, 2 µg/ml;
anti-FLAG antibody M2, 2 µg/ml; and anti-Myc antibody 9E10, 2 µg/ml)
and immobilized Protein G (GE Healthcare), followed by western blotting
using biotinylated anti-velcro polyclonal antibody. For the detection,
membranes were incubated with streptavidin conjugated to horseradish
peroxidase (HRP) (VECTOR), developed with ECL™ Prime reagent, andanalyzed using ImageQuant LAS 4000mini (GE Healthcare). Expression
and immunoprecipitation of the α6β1-integrin headpiece and Sema3A were
performed with similar protocols, with the exception of using HEK293S
GnT1 cells for α6β1 integrin. As the positive control experiment for the
immunoprecipitation, either GoH3–Sepharose (for α6β1 integrin) or NiNTA agarose (for His6–Sema3A) were used.
Biolayer interferometry

For the large-scale production of soluble αIIbβ3 integrin, transient transfectionof Expi293F cells (Life Technologies) using ExpiFectamine 293
Transfection Kit (Life Technologies) was utilized. Briefly, 20-ml cell
cultures were transfected with 13.3 μg each of DNAs coding for various PAtagged αIIb-integrin and wild-type β3-integrin chains, and the culture medium
was collected after 96 h. Each mutant integrin was purified with NZ-1–
Sepharose column chromatography, followed by further gel filtration
purification on a Superdex 200 10/300 GL column. After concentration to∼0.7 mg/ml, these PA-bearing integrins were subjected to binding evaluation
with BLI using Octet RED system (Pall ForteBio), as described previously(Fujii et al., 2014). Binding curves were analyzed with Octet Data Analysis
software (Pall ForteBio) and curve-fitting using a 1:1 binding model.

Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685

fragments described above. Cells were harvested after ∼48 h of transfection,
washed twice with fresh serum-containing medium and resuspended in
Tyrode’s buffer (137 mM NaCl, 12 mM NaHCO3, 2.6 mM KCl, 5 mM
HEPES, 2 mM CaCl2, 2 mM MgCl2, 1 mg/ml BSA, 1 mg/ml dextrose, pH
7.4). For the integrin expression analysis, cells were incubated with the 10E5
antibody (1 μg/ml) and NZ-1 (0.5 μg/ml) for 30 min at 20°C, followed bystaining with Alexa-Fluor-488-conjugated goat anti-mouse IgG (1:400) andphycoerythrin-conjugated goat anti-rat IgG polyclonal antibody (1:1000).
Flow cytometry was performed on an EC800 system (SONY), and the data
were analyzed with FlowJo software (Tomy Digital Biology). For theevaluation of epitope exposure, cells were incubated with a mixtureof DyLight680-conjugated AP3 (5 μg/ml) and FITC-conjugated NZ-1
(0.5 μg/ml) antibodies in the absence or presence of the activation mixture,comprising 0.5 mM MnCl2 and 1 mM RGD peptide. Evaluation of the
LIBS exposure on mouse β1 integrin was performed in a similar way, using
the 293T-PA− cells transiently transfected with human α4 integrin
and mouse β1 integrin. Cells were incubated with the NZ-1 (1 μg/ml)
or 9EG7 (5 μg/ml) antibodies for 30 min at 20°C, followed by staining
with the Alexa-Fluor-488-conjugated goat anti-rat IgG polyclonal
antibody (1:400).
Ligand-binding assay for cell surface αIIbβ3

The ligand-binding activity of αIIbβ3 integrin on the cell surface was evaluatedby the flow cytometry analysis of PAC-1 binding. PAC-1 is a ligand-mimetic
anti-αIIbβ3-integrin antibody whose binding is solely dependent on thehigh-affinity state of integrin, serving as a surrogate for the physiologicalligand fibrinogen (Tomiyama et al., 1992). The 293T-PA− cells that had
been transiently transfected with various full-length αIIbβ3-integrin constructs
were incubated with Tyrode’s buffer containing FITC–PAC-1 (1:20 dilution)
and DyLight680-AP3 (5 μg/ml) for 30 min at 20°C, washed once andresuspended in Tyrode’s buffer, and analyzed on an EC800 flow cytometer.
When activation of the integrin was required, cells were incubated with
15 mM dithiothreitol (DTT) for 10 min at 37°C, which is known to activate
resting (i.e. low affinity) αIIbβ3 integrin (Kouns et al., 1994).
Acknowledgements
We would like to thank the staff of the beamlines at Photon Factory and National
Synchrotron Radiation Research Center for their help with X-ray data collection;
Emiko Mihara and Keiko Tamura-Kawakami for excellent technical assistance; andMie Sakai for preparation of the manuscript.

Competing interests
The authors declare no competing or financial interests.

Author contributions

A CRISPR/Cas plasmid (Sigma-Aldrich), which targets the sequence
5′-GACACTGAGACTACAGGTTTGG-3′ of human podoplanin, wastransfected into HEK293T cells using a Gene Pulser Xcell electroporation
system (Bio-Rad Laboratories). Single-cell cloning was performed bylimiting dilution in 96-well plates using 10% FBS in Dulbecco’s modified
Eagle’s medium (DMEM), and podoplanin expression was checked withflow cytometry using NZ-1. The transfection–cloning cycles were repeateduntil a complete lack of NZ-1 reactivity was attained. The stable nature of theknockout phenotype was confirmed by the lack of NZ-1 reactivity after morethan ten passages. The established podoplanin-knockout cell line (293TPA−) showed similar morphology to that of the parent HEK293T cells bothunder a microscope and in the flow cytometry analysis (i.e. forward- andside- scattering characteristics), but exhibited some phenotypic differences,including a slight reduction in the growth rate (doubling time of ∼17 h) anda reduced (by ∼50%) level of protein secretion after transient transfection.
These variations, however, can be seen among different laboratory strains of
HEK293T cells, and it is unclear whether any of the changes are caused bypodoplanin depletion.
Expression and flow cytometry analyses of full-length integrins
on the cell surface

Transient transfection of full-length αIIbβ3-integrin into 293T-PA− cells was
conducted under the same conditions utilized for the soluble ectodomain

Y.F. and Y.M. designed and performed experiments, and wrote the manuscript. T.A.,
Y.K., S.O. and M.K.K. performed experiments and analyzed the data. Y.K.
supervised the experiments and analyzed the data. J.T. conceived the experimental
design, analyzed the data and wrote the manuscript. All authors contributed to the
preparation of the manuscript.

Funding
This work was supported in part by the Grant-in-Aid for Scientific Research on
Innovative Areas (Analysis and Synthesis of Multidimensional Immune Organ Network
to J.T.) from the Ministry of Education, Culture, Sports, Science and Technology ofJapan (MEXT); by the ‘Platform for Drug Discovery, Informatics, and Structural Life
Science’ grant from the MEXT; by the Japan Agency for Medical Research andDevelopment (AMED) (to J.T. and Y.K.); by the ‘X-ray Free Electron Laser Priority
Strategy Program’ grant (to J.T.) from the MEXT; by the Basic Science and Platform
Technology Program for Innovative Biological Medicine from the MEXT and AMED
(to Y.K.); and by the Regional Innovation Strategy Support Program from the MEXT
(to Y.K.).

Data availability
Atomic coordinates and structural factors have been deposited in the Protein Data
Bank under accession numbers 4YNY (ligand-free NZ-1 Fab) and 4YO0 (NZ-1 Fabin complex with the PA14 peptide).

Supplementary information
Supplementary information available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.176685/-/DC1

1521

Journal of Cell Science

Establishment of podoplanin-knockout cell lineReferences
Adams, P. D., Afonine, P. V., Bunkó czi, G., Chen, V. B., Davis, I. W., Echols, N.,
Headd, J. J., Hung, L.-W., Kapral, G. J., Grosse-Kunstleve, R. W. et al. (2010).
PHENIX: a comprehensive Python-based system for macromolecular structure
solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213-221.
Bazzoni, G., Shih, D.-T., Buck, C. A. and Hemler, M. E. (1995). Monoclonal
antibody 9eg7 defines a novel beta(1) integrin epitope induced by soluble ligand
and manganese, but inhibited by calcium. J. Biol. Chem. 270, 25570-25577.
Carman, C. V. and Springer, T. A. (2003). Integrin avidity regulation: are changes inaffinity and conformation underemphasized? Curr. Opin. Cell Biol. 15, 547-556.
Chang, H. C., Bao, Z., Yao, Y., Tse, A. G., Goyarts, E. C., Madsen, M., Kawasaki,E., Brauer, P. P., Sacchettini, J. C., Nathenson, S. G. et al. (1994). A general
method for facilitating heterodimeric pairing between two proteins: application toexpression of alpha and beta T-cell receptor extracellular segments. Proc. Natl.
Acad. Sci. USA 91, 11408-11412.
Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M.,
Kapral, G. J., Murray, L. W., Richardson, J. S. and Richardson, D. C. (2010).
MolProbity: all-atom structure validation for macromolecular crystallography. Acta
Crystallogr. D Biol. Crystallogr. 66, 12-21.
Coller, B. S. (1985). A new murine monoclonal antibody reports an activationdependent change in the conformation and/or microenvironment of the platelet
glycoprotein IIb/IIIa complex. J. Clin. Invest. 76, 101-108.
Dennison, S. M., Anasti, K. M., Jaeger, F. H., Stewart, S. M., Pollara, J., Liu, P.,
Kunz, E. L., Zhang, R., Vandergrift, N., Permar, S. et al. (2014). Vaccineinduced HIV-1 envelope gp120 constant region 1-specific antibodies expose a
CD4-inducible epitope and block the interaction of HIV-1 gp140 withgalactosylceramide. J. Virol. 88, 9406-9417.
Dinculescu, A., McDowell, J. H., Amici, S. A., Dugger, D. R., Richards, N.,
Hargrave, P. A. and Smith, W. C. (2002). Insertional mutagenesis andimmunochemical analysis of visual arrestin interaction with rhodopsin. J. Biol.
Chem. 277, 11703-11708.
Dyson, H. J., Lerner, R. A. and Wright, P. E. (1988). The physical basis for
induction of protein-reactive antipeptide antibodies. Annu. Rev. Biophys. Biophys.
Chem. 17, 305-324.
Emsley, P., Lohkamp, B., Scott, W. G. and Cowtan, K. (2010). Features anddevelopment of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486-501.
Eswar, N., Eramian, D., Webb, B., Shen, M.-Y. and Sali, A. (2008). Protein
structure modeling with MODELLER. Methods Mol. Biol. 426, 145-159.
Facey, S. J. and Kuhn, A. (2003). The sensor protein KdpD inserts into the
Escherichia coli membrane independent of the Sec translocase and YidC.
Eur. J. Biochem. 270, 1724-1734.
Fujii, Y., Kaneko, M., Neyazaki, M., Nogi, T., Kato, Y. and Takagi, J. (2014). PA tag: aversatile protein tagging system using a super high affinity antibody against adodecapeptide derived from human podoplanin. Protein Expres Purif. 95, 240-247.
Guruprasad, K. and Rajkumar, S. (2000). Beta-and gamma-turns in proteinsrevisited: a new set of amino acid turn-type dependent positional preferences andpotentials. J. Biosci. 25, 143-156.
Humphries, M. J. (2004). Monoclonal antibodies as probes of integrin priming andactivation. Biochem. Soc. Trans. 32, 407-411.
Humphries, M. J., Symonds, E. J. and Mould, A. P. (2003). Mapping functional
residues onto integrin crystal structures. Curr. Opin. Struct. Biol. 13, 236-243.
Irannejad, R., Tomshine, J. C., Tomshine, J. R., Chevalier, M., Mahoney, J. P.,
Steyaert, J., Rasmussen, S. G. F., Sunahara, R. K., El-Samad, H., Huang, B.
et al. (2013). Conformational biosensors reveal GPCR signalling fromendosomes. Nature 495, 534-538.
Johnson, J. L., Entzminger, K. C., Hyun, J., Kalyoncu, S., Heaner, D. P., Jr,
Morales, I. A., Sheppard, A., Gumbart, J. C., Maynard, J. A. and Lieberman,
R. L. (2015). Structural and biophysical characterization of an epitope-specificengineered Fab fragment and complexation with membrane proteins: implications
for co-crystallization. Acta Crystallogr. D Biol. Crystallogr. 71, 896-906.
Kabsch, W. (2010). Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125-132.
Kendall, R. T. and Senogles, S. E. (2006). Investigation of the alternatively spliced
insert region of the D2L dopamine receptor by epitope substitution. Neurosci. Lett.
393, 155-159.
Koide, S. (2009). Engineering of recombinant crystallization chaperones. Curr.
Opin. Struct. Biol. 19, 449-457.
Kouns, W. C., Steiner, B., Kunicki, T. J., Moog, S., Jutzi, J., Jennings, L. K.,
Cazenave, J. P. and Lanza, F. (1994). Activation of the fibrinogen binding site onplatelets isolated from a patient with the Strasbourg I variant of Glanzmann’s
thrombasthenia. Blood 84, 1108-1115.
Krauss, N., Wessner, H., Welfle, K., Welfle, H., Scholz, C., Seifert, M., Zubow, K.,
Aÿ, J., Hahn, M., Scheerer, P. et al. (2008). The structure of the anti-c-myc
antibody 9E10 Fab fragment/epitope peptide complex reveals a novel binding
mode dominated by the heavy chain hypervariable loops. Proteins 73, 552-565.

1522

Journal of Cell Science (2016) 129, 1512-1522 doi:10.1242/jcs.176685

Luo, B.-H., Springer, T. A. and Takagi, J. (2004). A specific interface between
integrin transmembrane helices and affinity for ligand. PLoS Biol. 2, e153.
McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C.
and Read, R. J. (2007). Phaser crystallographic software. J. Appl. Crystallogr. 40,
658-674.
Mitchell, W. B., Li, J. H., Singh, F., Michelson, A. D., Bussel, J., Coller, B. S. and
French, D. L. (2003). Two novel mutations in the alpha IIb calcium-binding
domains identify hydrophobic regions essential for alpha IIbbeta 3 biogenesis.
Blood 101, 2268-2276.
Morlacchi, S., Sciandra, F., Bigotti, M. G., Bozzi, M., Hü bner, W., Galtieri, A.,
Giardina, B. and Brancaccio, A. (2012). Insertion of a myc-tag within alphadystroglycan domains improves its biochemical and microscopic detection. BMC
Biochem. 13, 14.
Mould, A. P., Barton, S. J., Askari, J. A., McEwan, P. A., Buckley, P. A., Craig,
S. E. and Humphries, M. J. (2003). Conformational changes in the integrin beta A
domain provide a mechanism for signal transduction via hybrid domain
movement. J. Biol. Chem. 278, 17028-17035.
Murshudov, G. N., Skubá k, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A.,
Nicholls, R. A., Winn, M. D., Long, F. and Vagin, A. A. (2011). REFMAC 5 for therefinement of macromolecular crystal structures. Acta Crystallogr. D Biol.
Crystallogr. 67, 355-367.
Nagae, M., Re, S., Mihara, E., Nogi, T., Sugita, Y. and Takagi, J. (2012). Crystal
structure of alpha5beta1 integrin ectodomain: atomic details of the fibronectin
receptor. J. Cell Biol. 197, 131-140.
Newman, P. J., Allen, R. W., Kahn, R. A. and Kunicki, T. J. (1985). Quantitation of
membrane glycoprotein IIIa on intact human platelets using the monoclonal
antibody, AP-3. Blood 65, 227-232.
O’shea, E. K., Lumb, K. J. and Kim, P. S. (1993). Peptide Velcro - design of aheterodimeric coiled coil. Curr. Biol. 3, 658-667.
Otwinowski, Z. and Minor, W. (1997). Processing of X-ray diffraction data collectedin oscillation mode. Methods Enzymol. 276, 307-326.
Peerschke, E. I. and Coller, B. S. (1984). A murine monoclonal antibody that blocks
fibrinogen binding to normal platelets also inhibits fibrinogen interactions withchymotrypsin-treated platelets. Blood 64, 59-63.
Smith, W. C., Dinculescu, A., Peterson, J. J. and McDowell, J. H. (2004). The
surface of visual arrestin that binds to rhodopsin. Mol. Vis. 10, 392-398.
Spangler, B. D. (1991). Binding to native proteins by antipeptide monoclonal
antibodies. J. Immunol. 146, 1591-1595.
Springer, T. A. (1998). An extracellular beta-propeller module predicted inlipoprotein and scavenger receptors, tyrosine kinases, epidermal growth factor
precursor, and extracellular matrix components. J. Mol. Biol. 283, 837-862.
Tainer, J. A., Getzoff, E. D., Alexander, H., Houghten, R. A., Olson, A. J., Lerner,
R. A. and Hendrickson, W. A. (1984). The reactivity of anti-peptide antibodies isa function of the atomic mobility of sites in a protein. Nature 312, 127-134.
Takagi, J. and Springer, T. A. (2002). Integrin activation and structural
rearrangement. Immunol. Rev. 186, 141-163.
Takagi, J., Isobe, T., Takada, Y. and Saito, Y. (1997). Structural interlock between
ligand-binding site and stalk-like region of beta1 integrin revealed by a monoclonal
antibody recognizing conformation-dependent epitope. J. Biochem. 121, 914-921.
Takagi, J., Erickson, H. P. and Springer, T. A. (2001). C-terminal opening mimics“inside-out” activation of integrin a5b1. Nat. Struct. Biol. 8, 412-416.
Takagi, J., Petre, B. M., Walz, T. and Springer, T. A. (2002). Global conformational
rearrangements in integrin extracellular domains in outside-in and inside-out
signaling. Cell 110, 599-611.
Tomiyama, Y., Brojer, E., Ruggeri, Z. M., Shattil, S. J., Smiltneck, J., Gorski, J.,
Kumar, A., Kieber-Emmons, T. and Kunicki, T. J. (1992). A molecular model of
RGD ligands. Antibody D gene segments that direct specificity for the integrinalpha IIb beta 3. J. Biol. Chem. 267, 18085-18092.
Walker, F., Orchard, S. G., Jorissen, R. N., Hall, N. E., Zhang, H.-H., Hoyne, P. A.,
Adams, T. E., Johns, T. G., Ward, C., Garrett, T. P. et al. (2004). CR1/CR2
interactions modulate the functions of the cell surface epidermal growth factor
receptor. J. Biol. Chem. 279, 22387-22398.
Wallace, A. C., Laskowski, R. A. and Thornton, J. M. (1995). LIGPLOT: a program togenerate schematic diagrams of protein-ligand interactions. Protein Eng. 8, 127-134.
Wood, D. W. (2014). New trends and affinity tag designs for recombinant protein
purification. Curr. Opin. Struct. Biol. 26, 54-61.
Xiao, T., Takagi, J., Coller, B. S., Wang, J.-H. and Springer, T. A. (2004).
Structural basis for allostery in integrins and binding to fibrinogen-mimetic
therapeutics. Nature 432, 59-67.
Zhu, J., Luo, B.-H., Xiao, T., Zhang, C., Nishida, N. and Springer, T. A. (2008).
Structure of a complete integrin ectodomain in a physiologic resting state andactivation and deactivation by applied forces. Mol. Cell 32, 849-861.

Journal of Cell Science

TOOLS AND TECHNIQUES