Conformational and functional analysis of the lipid
binding protein Ag-NPA-1 from the parasitic nematode
Ascaridia galli
Rositsa Jordanova
1
, Georgi Radoslavov
1
, Peter Fischer
2
, Eva Liebau
2
, Rolf D. Walter
2
, Ilia Bankov
1
and Raina Boteva
3
1 Institute of Experimental Pathology and Parasitology, Sofia, Bulgaria
2 Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
3 National Center of Radiobiology and Radiation Protection, Sofia, Bulgaria
Lipid-binding proteins (LBPs) regulate the physiological
activity, metabolism and disposition of essential hydro-
phobic compounds like fatty acids, phospholipids,
eicosanoids and retinoids. Fatty acids and phospho-
lipids are the major energy reserves and components of
the cell membranes, whereas eicosanoids and retinoids
are important signaling molecules involved in several
cellular processes including gene transcription,
cell growth and differentiation, tissue repair, inflamma-
tion and immune responses. Conjugated with LBPs,
Keywords

0
-dimethyl-N-(iodoacetyl)-N
0
-
(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by
MALDI-TOF analysis showed that only Cys66 was labeled. The observed
similar affinities for fatty acids of the modified and native Ag-NPA-1 sug-
gest that Cys66 is not a part of the protein binding pocket but is located
close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and
it is distributed extracellularly mainly as shown by immunohistology and
immunogold electron microscopy. This suggests that Ag-NPA-1 plays an
important role in the transport of fatty acids and retinoids.
Abbreviations
DAUDA, 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid; FRET, fluorescence resonance energy transfer; IAEDANS,
5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid; IANBD, N,N
0
-dimethyl-N-(iodoacetyl)-N
0
-(7-nitrobenz-2-oxa-1,3-diazol-
4-yl)ethylenediamine; LBP, lipid binding protein; NPA, nematode polyprotein allergens/antigen.
180 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS
these compounds are solubilized, protected from
chemical damage and delivered to the correct destina-
tion [1–4].
LBPs from parasitic nematodes are of special inter-
est because these organisms typically exhibit limited
lipid metabolism and have to import complex lipids
from the host [5]. Nematodes possess two classes of
structurally novel types of helix-rich LBPs [6–8]. The
first class consists of small 15 kDa fatty acid and reti-

conformation and of the thiol reactive probe 5-({[(2-
iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic
acid (1,5-IAEDANS), covalently attached to Cys66.
Results
Conformational and oligomeric properties
of Ag-NPA-1
After gel filtration, natural or recombinant Ag-NPA-1
was eluted in a single protein peak of  24 kDa sug-
gesting a dimer formation. In several species, the units
of the nematode polyproteins differ from each other
in their amino acid sequences [7]. This, however, is
probably not true for the group of nematodes to which
A. galli belongs as suggested by the molecular homo-
geneity of native Ag-NPA-1 proved by N-terminal
sequencing followed by MS analysis (data not shown).
Native gel electrophoresis in the presence and absence
of palmitate, which is one of the preferred ligands of
Ag-NPA-1 [12], showed that the binding did not cause
any changes in the protein oligomeric state (data not
shown). The pI of Ag-NPA-1 was determined by 2D
gel electrophoresis and compared with the value calcu-
lated from the protein amino acid sequence. A good
correspondence of the experimentally determined pI of
6.1 and the theoretically deduced pI value of 6.22 was
found.
A theoretical prediction of the secondary structural
organization of Ag-NPA-1 performed on the basis of
the amino acid sequence [12] showed up to 80%
a-helical content. The model of the backbone folding
[17] suggests that the protein molecule is organized

-1
Fig. 1. CD spectrum of Ag-NPA-1 in the far UV-range (190–
260 nm).
R. Jordanova et al. Conformation, ligand binding and distribution of nematode protein Ag-NPA-1
FEBS Journal 272 (2005) 180–189 ª 2004 FEBS 181
The theoretical modelling of the protein backbone
folding predicts that these two residues are localized
on two neighbouring helices and the separation
between their C
a
atoms approaches 1 nm, a distance,
suitable for a disulfide (S-S) bridge formation. The
electrophoretic analysis of the native protein by gra-
dient SDS/PAGE, performed in the presence and
absence of the reducing agent 2-mercaptoethanol,
showed no changes in the migration of the protein
when free or conjugated with palmitate. This suggests
that even if Cys66 and Cys122 formed a disulfide
bridge, it is not important for the structural integrity
and stability of the Ag-NPA-1 molecule.
This was further tested by chemical modification
of Ag-NPA-1 with two fluorescent iodacetamides,
IAEDANS and IANBD, characterized by high specifi-
city and reactivity to free sulfhydryl groups [18]. The
covalent binding of the dyes to either native or recom-
binant Ag-NPA-1 was confirmed by denaturation of
the labeled proteins with 6 m guanidium chloride [19].
This procedure did not cause any release of the markers
as both emission and absorbance bands specific to
IAEDANS or IANBD could be registered. The quantity

Q
of 14.8 m
)1
for the free dye, indi-
cated a partial accessibility of the marker to external
solvent molecules.
Binding of palmitate, identified as one of the preferred
ligands [12], caused an additional 25–30 nm blue-shift of
the emission maximum position of the dye accompanied
by almost twofold emission intensity enhancement.
These changes indicated significant conformational rear-
rangements in Ag-NPA-1 molecules upon ligand bind-
ing which strongly affected the surrounding of Cys66
and increased its hydrophobicity. The fluorescence
changes allowed calculation of the apparent dissociation
constant K
d
of the protein–palmitate complex. A value
of 0.25 ± 0.10 lm, similar to that reported in [12] for
the native protein was obtained. The similar affinities of
the native and modified proteins suggest that Cys66 is
not a part of the binding site, however, it is located close
to the binding pocket as the ligand binding strongly
influences the fluorescence properties of the dansyl chro-
mophore, covalently attached to this Cys.
Intrinsic fluorescence properties of Ag-NPA-1
The protein fluorescence emission spectrum obtained
upon 275 nm excitation (where both Tyr and Trp
chromophores absorb) shows a maximum at 318 nm
(Fig. 2). Upon excitation at 300 nm, where only the

for the K
Q
con-
stant which indicated a poor accessibility of the sin-
gle Trp residue to external solvent molecules by
pointing out a position in the hydrophobic interior
of the protein molecules.
As the Trp absorption spectrum overlaps the Tyr
emission, a radiation-less energy transfer from Tyr to
Trp chromophores could take place. We studied this
process and found a relatively high efficiency of
 65% (Fig. 4) which suggests a high degree of Tyr to
Trp dipole-dipole coupling.
Binding activities of Ag-NPA-1 determined
by changes in Trp fluorescence
Binding of retinol, oleic and arachidonic acids caused
a slight (£ 11%) increase of the emission of the single
Trp residue of Ag-NPA-1. Saturation of the binding
sites followed a hyperbolic trend (Fig. 5) and the Trp
emission maximum remained at 325 nm. From the Trp
fluorescence enhancement, we calculated values for K
d
of 0.30 ± 0.04 lm for retinol, 0.23 ± 0.10 lm for
oleic and 0.15 ± 0.01 lm for arachidonic acid. These
values were similar to those reported in [12] which
were calculated from fluorescence displacement experi-
ments with DAUDA where the fatty acids, retinoids
and DAUDA competed for the single binding site of
the Ag-NPA-1 monomers.
FRET between Trp17 and DAUDA in Ag-NPA-1

site were estimated. This supports previous observa-
tions (A Timanova, EPP, Sofia, Bulgaria, unpublished
data) and shows that like the Trp residue of the
0.00 0.05 0.10 0.15
1.0
1.1
1.2
1.3
[acrylamide] M
F
0
/F
Fig. 3. Quenching of Trp fluorescence of Ag-NPA-1 by acrylamide.
A value of 1.3
M
)1
was calculated for the K
Q
constant.
260 270 280 290 300
0.8
0.9
1.0
Φ
trp
/Φ
300
e=0.5
e=0.65
e=0.8

emission of the single Trp residue, similar to that regis-
tered after binding of oleic and arachidonic acids,
indicating no dipole-dipole interactions between the
two types of chromophores. FRET is exponentially
dependent on the distance between the donor-acceptor
pair [22]. Therefore, the process should be most effi-
cient within the Fo
¨
rster’s radius of 1.5 nm which we
calculated for the Trp-retinol couple in Ag-NPA-1. As
no energy transfer could be detected after the forma-
tion of the Ag-NPA-1–retinol complexes, either the
distance between the Trp residue and retinol is signifi-
cantly longer than 1.5 nm or there is an unfavourable
mutual orientation of the chromophores for dipole-
dipole interactions.
Immunohistology and immunogold TEM
Using the antiserum raised against native Ag-NPA-1,
worms fixed either with ethanol or formalin were
stained. In general, the labeling was more intense in eth-
anol fixed specimens compared to the formalin fixed
ones. The preimmune serum, used as a negative control,
showed absence of unspecific reactions (Figs 6A and
7A). A significant staining of the fluid of the pseudocoe-
lomatic cavity was observed (Fig. 6B). In A. galli the
inner hypodermis (Fig. 6D), the lateral and the median
chord were mainly stained. Furthermore, sperm that
were attached to the uterus tissue and the oviduct, were
intensively labeled in contrast to the ovary and the
uterus which were not stained. No staining was also

to 1 : 4000 in A. galli (Fig. 6).
Ultrastructural localization of Ag-NPA-1 in A. galli
by immunogold electron microscopy confirmed the
results from the light microscopy. Sections through the
contractile portion of the somatic musculature revealed
that the interstitial space between the striate muscula-
ture, which is filled with pseudocoelomatic fluid, was
also strongly labeled by gold particles (Fig. 7B). These
observations suggest that Ag-NPA-1 is localized
mainly in cells of the inner hypodermis and the epithe-
lium of the oviduct as well as extracellularly in the
pseudocoelomic cavity of the worms.
Discussion
A bundle of four a-helices constitutes the secondary
structure organization of Ag-NPA-1 and of other
homologous NPAs as suggested by theoretical predic-
tions of the protein backbone folding. These helices
might shape the hydrophobic binding pocket which
was shown to bind fatty acids, retinoids and arachi-
donic acid with high affinity. CD analysis confirmed
the predicted helical structure of Ag-NPA-1 and
showed 66% a-helical content for both native and
recombinant proteins. It increased 10–12% upon lig-
and binding, suggesting additional conformational sta-
bilization of the protein in the complexes.
The single Trp and the two Cys residues are highly
conserved in all amino acid sequences of NPAs from
parasitic nematodes [12] suggesting important struc-
tural or functional roles of these residues. According
to secondary structure predictions, Trp17, Cys66 and

molecule and poorly accessible to the solvent as sugges-
ted by the very low value of the quenching constant (K
Q
1.3 m
)1
) obtained with acrylamide as external quencher.
The contribution of Tyr chromophores to the overall
protein fluorescence was  45% and the efficiency of
Tyr to Trp energy transfer 65%, suggesting a high
degree of Tyr-Trp dipole-dipole coupling.
The Trp fluorescence increased up to 11% upon
binding of fatty acids, retinol and DAUDA that
allowed studies on the protein binding affinities. The
values of the dissociation constants calculated by chan-
ges in Trp emission were close to those determined in
displacement experiments with the fluorescent fatty acid
analogue DAUDA [12]. Thus, in contrast to the
homologous Trp chromophore of ABA-1, the single
Trp of Ag-NPA-1 is a sensitive marker of the protein
conformation and its emission reflects conformational
changes after ligand binding. Analysis of FRET from
the singlet excited state of Trp17 to DAUDA, noncova-
lently bound to Ag-NPA-1, allowed calculation of the
Fig. 7. Immunogold electron microscopic localization of Ag-NPA-1
in a male A. galli worm. (A) Section of the striate musculature (mu)
and the interstitial space (is) stained with the preimmune serum as
primary antibody. (B) Consecutive section to A using the antiserum
raised against native Ag-NPA-1 showing strong accumulation of
gold particles in the pseudocoelomatic fluid of the interstitial space
(arrows). Bar size is 0.5 lm (A–B).

possible. Interestingly, the localization of Ag-NPA-1 to
the hypodermis is similar to that of the lipid binding
proteins in the filarial parasites of humans [24,25]
which lends them to in situ iodination in the whole
living worms. EM experiments localized the protein
mainly extracellularly, in the interstitial space, but also
in some of the muscle cells. Although a specific cell
surface receptor could exist it is possible that this small
protein interacts directly with the cell membranes, as
reported for ABA-1 [26], and acts as a shuttle for
delivering lipids to the place of their metabolic trans-
formation. In summary, the general distribution of Ag-
NPA-1, which is one of the main proteins in A. galli
cytosol, suggests important functions of the protein in
the internal lipid transport, which might be essential
for the parasite survival as these organisms exhibit lim-
ited ability to synthesize long chain fatty acids de novo.
Experimental procedures
Protein purification
Natural and recombinant Ag-NPA-1 from A. galli used in
the experiments were purified as described previously
[12,16]. The protein concentration was determined spectro-
photometrically using a molar extinction coefficient of
9.5 · 10
3
m
)1
Æcm
)1
at 280 nm as calculated on the basis of

N-Ac-Trp-NH
2
normalized to the same absorbance at 300
nm. A value of 0.13 was used for the quantum yield of the
standard [28]. In order to minimize inner filter and self-
absorption effects, the sample absorbance at the excitation
wavelength (k
exc
) was always lower than 0.05. The effi-
ciency of Tyr to Trp energy transfer was calculated by a
procedure described in [29].
The Tyr contribution to the total protein fluorescence was
estimated by subtraction of the Trp emission spectrum (k
exc
300 nm) from that obtained at k
exc
275 nm, after normalizing
the two spectra above 380 nm, where the Tyr emission is neg-
ligible. Quenching of Trp fluorescence and of the emission of
the bound IAEDANS was performed with acrylamide as
external quencher. The data were analyzed according to the
Stern–Volmer equation [28]: F
o
/F ¼ 1+K
Q
[X], where, F
o
and F are the fluorescence emission intensities in the absence
and presence of acrylamide, [X] is the molar concentration of
acrylamide and K

removed by gel filtration with Bio-Spin30 Tris columns
(Bio-Rad). The extent of labeling and the protein to dye
ratio were determined spectrophotometrically, from the
protein absorbance at 280 nm (e
M,280
9.5 · 10
3
M
)1
Æcm
)1
for Ag-NPA-1) and the dye absorbances at 337 nm for
IAEDANS (e
M,337
6 · 10
3
M
)1
Æcm
)1
) and at 472 nm for
IANBD (e
M,472
23 · 10
3
M
)1
Æcm
)1
). The covalent attach-

)
after a full saturation of the protein binding sites.
Changes in the specific emission of the fluorescent probe
IAEDANS (k
exc
360 nm), covalently attached to Cys66 of
Ag-NPA-1 upon binding of palmitate, were also examined.
Fluorescence resonance energy transfer
Intramolecular fluorescence resonance energy transfer
(FRET) from the single Trp residue of Ag-NPA-1 (donor) to
the dansyl group of DAUDA (acceptor) was studied by the
decrease in the Trp fluorescence after saturation of the pro-
tein binding sites with the fluorescent probe. This allowed
calculation of the average distance r between the energy
donor-acceptor pair: r ¼ R
o
[(1 ) E)/E]
1/6
, where, E is the
efficiency of the energy transfer process, calculated from the
decrease of the donor quantum yield (Q
Trp
) in the presence
of the acceptor (Q
Trp-A
): E ¼ 1–Q
Trp-A
/Q
Trp
, where, R

tial orientation of the transition dipole moments of the donor
and acceptor. As no data on the spatial orientation of the
transition dipole moments of the chromophores are avail-
able, a random orientation of the donor–acceptor pair was
assumed (K
2
0.667 [30]). A value of 1.36 was taken for the
refractive index n [31].
MALDI-TOF analysis
MALDI-TOF analysis of the peptides obtained after tryptic
digestion of the labeled and nonlabeled Ag-NPA-1 was per-
formed as described in [20,32]. The data were analyzed with
peptide mass software (us.expasy.org/tools). The chemically
modified Cys residue was identified indirectly, upon com-
parative analysis of the peptide patterns obtained after
proteolytic cleveage of the Ag-NPA-1 with and without
treatment by the sulfhydryl reagents.
Data analysis and structure predictions
Sequence analysis and secondary structure predictions were
performed with programs available on the ExPaSy mole-
cular biology server (us.expasy.org/tools/); the molecular
mass and isoelectric point (pI) of the protein were estimated
with the protparam program; goriv and jpred programs
were used for secondary structure predictions and 3d-pssm
software for backbone fold recognition [17].
Immunohistology and immunogold TEM
An antiserum against Ag-NPA-1 was raised in a rabbit
using a standard immunization protocol (Eurogentec, Sera-
ing, Belgium). The preimmune serum was used as a con-
trol. Immunolocalization on the light and on the electron

tions were treated with Protein A Gold 10 nm (University
of Utrecht, School of Medicine, Department Cell Biology,
NL) at a dilution of 1 : 70. Later, the sections were fixed in
2% glutardialdehyde and counterstained as described
above. For negative controls, the primary antibody was
replaced by the corresponding preimmune sera.
Acknowledgements
R.J. was supported by Deutscher Academischer
Austauschdienst (DAAD) and Deutsche Forschungs-
gemeinschaft (DFG). Special thanks to Elisabeth Wey-
her-Stingl (MPI, Martinsried) for the help with CD
measurements and data interpretation, Joachim Clos
(BNI, Hamburg) for preparing the MALDI-TOF
experiments, Insa Bonow and Christel Schmetz (BNI,
Hamburg) for the technical assistance with immunohis-
tology and immunogold electron microscopy, Christina
Mertens and Manfred Uphoff (Intervet Innovation
GmbH) for A. galli samples and Paul Tucker (EMBL-
Hamburg) for the critical reading of the manuscript.
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