Báo cáo khoa học: Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli potx

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

NPA, Trp and IAEDANS fluorescence, FRET,
immunohistology
Correspondence
R. Boteva, National Center of Radiobiology
and Radiation Protection, Sofia 1756,
Bulgaria
Fax: +359 28621059
Tel: +359 28626036/210
E-mail:
(Received 27 July 2004, revised 17
September 2004, accepted 20 September
2004)
doi:10.1111/j.1432-1033.2004.04398.x
Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascari-
dia galli, is a member of the nematode polyprotein allergen/antigen (NPA)
family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered,
a-helical protein and that ligand binding slightly increases the ordered sec-
ondary structure content. The conserved, single Trp residue (Trp17) and
three Tyr residues determine the fluorescence properties of Ag-NPA-1.
Analysis of the efficiency of the energy transfer between these chromo-
phores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of
fatty acids and retinol was accompanied by enhancement of the Trp emis-
sion, which allowed calculation of the affinity constants of the binary
complexes. The distance between the single Trp of Ag-NPA-1 and the
fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfo-
nyl)amino]undecanoic acid (DAUDA) from the protein binding site is
1.41 nm as estimated by fluorescence resonance energy transfer. A chem-
ical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122)
with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naph-
thalene-1-sulfonic acid (IAEDANS) and N,N

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-

noid binding proteins, characterized by extremely non-
polar binding sites. They are synthesized as large
precursor polypeptides and subsequently cleaved into
functional units. Based on this peculiarity and on
their allergenicity, these LBPs are named nematode
polyprotein allergens/antigens (NPAs) [6,9,10]. The
second class of fatty acid and retinoid binding proteins
(FAR proteins) are slightly larger, 20 kDa, with stron-
ger affinity for retinol than for fatty acids. They nota-
bly differ in their amino acid sequence from NPAs
[11].
Ag-NPA-1 from the parasitic nematode Ascarida
galli is a member of the NPAs family [12]. Its fatty
acid and retinoid binding activities have been studied
indirectly in displacement experiments with the fluores-
cent substrate 11-[(5-dimethylaminonaphthalene-1-
sulfonyl)amino]undecanoic acid (DAUDA), a dansyl-
ated undecanoic acid. The fluorescence properties of
DAUDA strongly depend on the polarity and accessi-
bility of the protein binding sites and this has been
widely used in studies on the binding properties of
parasitic as well as of mammalian LBPs [9,12–16]. The
changes in the dye emission upon binding characterize
the binding sites of NPAs as highly nonpolar and com-
pletely isolated from the solvent.
Here we analyze the conformational and functional
properties of Ag-NPA-1 as well as the tissue and cellular
distribution of the protein. The binding affinities are
characterized by changes in the fluorescence of the single
Trp chromophore that is used as a marker of the protein

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

in four helices. The CD measurements in the far
UV-region (190–260 nm) confirmed the theoretical
prediction and showed that Ag-NPA-1 was a typical
a-helical protein containing 66% a-helices and 12%
b-turns (Fig. 1). Incubation of the protein with dif-
ferent ligands such as palmitate, caprylic and arachi-
donic acids and retinol in concentrations sufficient to
saturate the protein binding sites, caused similar
effects; slight enhancement of the helical content at
the expense of random coil mainly. Helical content
reached 78% in the presence of caprylic acid, indica-
ting additional stabilization of the Ag-NPA-1 confor-
mation upon ligand binding.
Role of Cys residues for Ag-NPA-1 conformation
and function
According to the amino acid sequence, Ag-NPA-1 con-
tains two Cys residues at positions 66 and 122 [12].
200 225 250
-5000
0
5000
10000
λ nm
[
θ
]
R
deg cm
2
dmol

-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

of the bound dye was determined spectrophoto-
metrically and showed binding of one molecule of either
IAEDANS or IANBD to a protein monomer.
The labeled Cys residue was identified by MALDI-
TOF analysis of the trypsin digested Ag-NPA-1 [20].
Cys66 and Cys122 were found in the peptide fragments
of the nontreated, natural protein with theoretically
calculated masses of 1033.5459 Da (AKESLIGGCR)
and 1236.6292 Da (ELIKDYGPACK). However, in
the mass spectrum of Ag-NPA-1 covalently labeled
with IAEDANS or IANBD, the 1033.5459 Da frag-
ment containing Cys66 could not be detected. This
could be explained by the chemical modification of
Cys66, leading to a change in the properties of the
dye-carrying fragment which prevented its detection.
Thus, Cys66 is the reactive and accessible to the bulky
dye molecules residue.
The fluorescence properties of IAEDANS are
strongly dependent on the polarity of its environment.
The emission maximum of the dye bound to Ag-NPA-
1 is at 460 nm, typical for a chromophore in a highly
hydrophobic environment [19]. The exposure and
accessibility of the dye attached to Cys66 were further
characterized by acrylamide quenching. We calculated
a Stern–Volmer constant (K
Q
) of 6.7 m
)1
which, when
compared to the K

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

Trp chromophore absorbs, the emission maximum
was registered at 325 nm, a position, indicative of a
highly hydrophobic environment of the single Trp
residue. The contribution of Tyr fluorescence to the
overall protein emission was calculated to amount to
45% and the Trp emission quantum yield 0.015. The
low value of the quantum yield indicates a strong
conformational quenching of the Trp chromophore.
To further characterize the location and accessibility
of Trp17, quenching experiments with acrylamide as
290 310 330 350 370 390
0
5
10
15
a
b
c
λ nm
fluorescence (a.u.)
Fig. 2. Fluorescence emission spectra of Ag-NPA-1. Spectra ‘a’ and
‘b’ are obtained with 275 and 300 nm excitation, respectively.
Spectrum ‘c’ represents the Tyr contribution and is calculated as a
difference between spectra ‘a’ and ‘b’ after their normalization
above 380 nm.
Conformation, ligand binding and distribution of nematode protein Ag-NPA-1 R. Jordanova et al.
182 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS
an external quencher were performed (Fig. 3). We
calculated a low value of 1.3 m
)1

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

The absorption spectrum of DAUDA (maximum at
335 nm) largely overlaps the protein Trp emission.
Hence, a fluorescence radiation-less energy transfer
(FRET) from the singlet excited state of Trp residues
to the dansyl group of the bound fluorescent fatty acid
could be envisaged. This process would result in
quenching of protein Trp fluorescence and in an
increase of the specific DAUDA emission after the
noncovalent incorporation of the ligand. Binding of
DAUDA to Ag-NPA-1 caused  20% quenching of
the emission of Trp17 indicating FRET between the
single Trp residue and the dansyl group. We calculated
a value of 3.74 · 10
)15
cm
3
Æm
)1
for the spectral integ-
ral J
AD
and of 0.012 for the Trp emission quantum
yield (Q
Trp-A
) of the protein–DAUDA complexes.
Hence a critical distance (R
o
) of 1.05 nm and an aver-
age intramolecular distance (r) of 1.41 nm, between
Trp17 and the dansyl chromophore from the binding

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

e=1
λ nm
Fig. 4. Tyr to Trp energy transfer efficiency in Ag-NPA-1. The
curves are theoretical and are obtained for different values of trans-
fer efficiency e. The experimental data are represented by empty
triangles (n). They follow best the theoretical curve with e ¼ 65%.
0.00 0.25 0.50 0.75 1.00
0.0
2.5
5.0
7.5
10.0
oleate [µM]
∆
F %
Fig. 5. Binding of oleic acid, followed by the enhancement of the
Trp fluorescence (DF). The curve fits best the experimental data,
obtained with a K
d
constant of 0.23 ± 0.10 lM, calculated by a non-
linear regression for a single binding site.
R. Jordanova et al. Conformation, ligand binding and distribution of nematode protein Ag-NPA-1
FEBS Journal 272 (2005) 180–189 ª 2004 FEBS 183
homologous ABA-1 [21], the single Trp of Ag-NPA-1
is not a part of the lipid binding pocket.
Like DAUDA, the retinol absorption spectrum lar-
gely overlaps Trp emission and FRET from the Trp
residue, buried in the interior of the protein molecule,
to the ionone ring of retinol would be expected. Inter-
estingly, binding of retinol caused an increase of the

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

observed in the cuticle, the muscle syncytia or the intes-
tine (Fig. 6B). Besides, the antibody, raised against Ag-
NPA-1 gave cross-reactions with similar proteins from
other ascaridis. When the localization of the protein in
A. galli was compared to that in Ascaridia suum, a sim-
ilar staining pattern was found (Fig. 6C,E,F). However,
Fig. 6. Immunohistological localization of
Ag-NPA-1 in adult A. galli and comparison
with that in A. suum using a polyclonal anti-
serum raised against native Ag-NPA-1.
(A) Section of A. galli showing the ovary (ov)
the uterus (ut) the intestine (i) and the parts
of the pseudocoel (ps) stained with the pre-
immune serum as control. (B) Consecutive
section of A showing intense staining of the
pseudocoel (ps) especially in the vicinity
of the uterus (ut) with developing eggs.
(C) Strong labeling of the oviduct (ovi) next
to the unstained ovary (ov) in A. suum.
(D) Section of the body wall of a female
A. galli showing an intense labeling of the
inner hypodermis (ihy, arrow). (E) Staining of
the labyrinth of the lateral chord (lc, arrow)
in A. suum. (F) Staining of the median chord
(mc, arrow) in A. suum. Bar size is 50 lm
(A–F).
Conformation, ligand binding and distribution of nematode protein Ag-NPA-1 R. Jordanova et al.
184 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS
an equal intensity of staining was obtained in A. suum
with a primary antiserum dilution of 1 : 1000 compared

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

Cys122 are part of three neighboring helices and the
distance between the two Cys residues is suitable for
S-S bridge formation. The chemical modification of
Cys66 by two different, highly specific to free sulfhyd-
ryl groups fluorescence dyes, IAEDANS and IANBD,
shows that this Cys residue is not involved in disulfide
bonding. According to the emission characteristics of
the covalently attached dyes, Cys66 is located in a
hydrophobic environment and is partially accessible to
external solvent molecules. In spite of its high reactiv-
ity Cys66 is not a part of the protein binding pocket as
K
d
similar for the palmitate binding by the native and
by the chemically modified Ag-NPA-1 were found
[12]. The ligand binding significantly increased the
IAEDANS emission and caused 20 nm blue-shift of the
emission maximum, changes indicating significant rear-
rangements in the protein molecule which increased the
hydrophobicity of the Cys66 environment.
In contrast to the homologous ABA-1 protein whose
emission spectrum was strongly dominated by Tyr fluor-
escence and the Trp contribution was registered as a
shoulder [21], only the Trp fluorescence of Ag-NPA-1
showed a peak with a maximum at 325 nm, typical for a
chromophore in a highly nonpolar environment. These
differences in the emission properties of the proteins
probably reflect local conformational differences
between the two homologous NPAs. Trp17 is deeply
buried in the hydrophobic interior of the Ag-NPA-1

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).

R. Jordanova et al. Conformation, ligand binding and distribution of nematode protein Ag-NPA-1
FEBS Journal 272 (2005) 180–189 ª 2004 FEBS 185
average distance between these chromophores. The dis-
tance approaches 1.41 nm which suggests that Trp17 of
Ag-NPA-1, like the single Trp residue of ABA-1 [21], is
not involved directly in the protein binding pocket.
The first described NPA was ABA-1 from A. suum.
It is an allergen from the excretory-secretory (ES)
products of the nematode and has a high affinity for
fatty acids and retinoids [8]. This finding applies to all
the representatives of the family and suggests an
important role of these proteins in importing essential
lipids from the host. Furthermore, worms could use
this mechanism to export hydrophobic signalling mole-
cules, including retinoids, in order to modulate the
host response. By reporter-gene assays in Caenorhabdi-
tis elegans [7] and by Northern blotting in Ascaris [23],
the cells of the gut were identified as a place of the
NPA synthesis. The immunohistological analysis in
this study suggests that Ag-NPA-1 is distributed
mainly in the pseudocoelom of A. galli. This indicates
an additional function of the protein as an internal
transporter of lipid metabolites in the parasitic tissues.
Cross reactions of the Ab used for the comparative
localization in A. suum and Anisakis larvae confirmed
this distribution. Besides, in Anisakis a secretory cell
was specifically labeled indicating a possible mechan-
ism of protein excretion in the host tissues (data not
shown). However, as no similar structure could be
identified in A. galli and A. suum, no comparison was

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

the aromatic amino acid content of one Trp and three Tyr
residues per protein monomer [27].
Fluorescence measurements and reagents
The fluorescent fatty acid analogue 11-[(5-dimethylamino-
naphthalene-1-sulfonyl) amino] undecanoic acid (DAUDA)
and the thiol reactive probes 5-({[(2-iodoacetyl) amino]}eth-
ylamino) naphthalene-1-sulfonic acid (1,5-IAEDANS) and
N,N
0
-dimethyl-N-(iodoacetyl)-N
0
-(7-nitrobenz-2-oxa-1,3-dia-
zol-4-yl)ethylenediamine (IANBD) were obtained from
Molecular Probes (Eugene, OR, USA). Retinol and fatty
acids were obtained from Sigma (St Louis, MO, USA).
IAEDANS and IANBD were dissolved in N,N-dimethyl
formamide, the fatty acids and retinol in ethanol at concen-
trations of 1 or 0.1 mm. The concentrations of DAUDA,
IAEDANS, IANBD and retinol were calculated from their
absorption spectra using the corresponding molar extinc-
tion coefficients. The concentration of the organic solvents
in the final reaction did not exceed 2%.
Steady-state fluorescence was measured with a Shimadzu
model RF5000 spectrofluorometer and a Kontron SMF 25,
both equipped with thermostatically controlled cell holders.
The relative Trp emission quantum yield (Q
Trp
) was deter-
mined by comparing the integrated fluorescence spectrum
of the protein excited at 300 nm with that of the standard

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

Q
the overall quenching constant.
Circular dichroism measurements
CD spectra were recorded in 10 mm Tris, pH 7.5, 20 °C,
using a Jasco Model 715 automatic recording circular
dichroism spectrophotometer with a thermostatically con-
trolled cell holder. A fused quartz cell with a path-length of
0.1 cm was used. The protein concentration was 0.82 mm.
The spectra measured in the far UV-region 190–260 nm
Conformation, ligand binding and distribution of nematode protein Ag-NPA-1 R. Jordanova et al.
186 FEBS Journal 272 (2005) 180–189 ª 2004 FEBS
were averages of four scans and were corrected by subtract-
ing the baseline of the buffer. They are reported as mean
residue molar ellipticity ([h]
R
) in degrees cm
2
Ædmol
)1
. Spec-
tra subtraction, normalization and smoothing were per-
formed by using jasco cd j-715 data manipulation
software and the analysis of the data was carried out with
the programs contin and selcon.
Protein labeling
Ag-NPA-1 was covalently labeled with the thiol reactive
probes 1,5-IAEDANS and IANBD. Labeling was per-
formed with the native and the recombinant proteins in
10 mm Tris, pH 7.4 after incubation for 4 h at 4 °C with a
20-fold molar excess of the dyes. The unbound dye was

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-

ment of the dyes was confirmed after denaturation of the
labeled protein with 6 m guanidine hydrochloride followed
by dialysis against 3 m guanidine hydrochloride in 10 mm
Tris buffer, pH 7.5. Then, both the absorption and emis-
sion spectra were recorded. In addition to the protein
peaks, they also contained IAEDANS or IANBD bands,
indicative of covalent binding of the dyes [19].
Binding activities of Ag-NPA-1
Binding of fatty acids, retinol and DAUDA were studied
by changes in the intrinsic Trp fluorescence of Ag-NPA-1
after excitation at 300 nm (k
exc
300 nm). Ag-NPA-1
(0.2 lm) was incubated with increasing ligand concentra-
tions overnight, 4 °C, in the dark, in order to prevent
chemical changes of the light-sensitive ligands. As DAUDA
and retinol absorb at the excitation (k
exc
300 nm) and emis-
sion (k
em
325 nm) wavelengths, the emission spectra were
corrected for inner filter effects and background fluores-
cence [19]. A least-square analysis of the transformed data
was carried out by Graphpad prism computer program.
This allowed calculation of the apparent dissociation con-
stants (K
d
) and the maximal fluorescence change (DF
max

)
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

o
is
the Fo
¨
rster radius or the critical distance for a 50% probabil-
ity of the energy transfer process: R
o
¼ (9.79 · 10
3
) · (J
AD
n
)4
K
2
Q
Trp
)
1/6
A
˚
, where J
AD
is the overlap integral between
the decadic molar absorbance of the acceptor and the correc-
ted emission spectrum of the donor on a wavenumber scale
normalized to unity [30], n the refractive index of the medium
and K
2
the orientation factor, determined by the mutual spa-

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

microscopical level was performed as described previously
[33].
For light microscopic immunohistology, adult A. galli
worms were either fixed in 4% (v/v) buffered formaldehyde
or in 80% (v/v) ethanol and embedded in paraffin. For
R. Jordanova et al. Conformation, ligand binding and distribution of nematode protein Ag-NPA-1
FEBS Journal 272 (2005) 180–189 ª 2004 FEBS 187
comparison with other ascaridis sections of female A. suum
were also used. The alkaline phosphatase-antialkaline phos-
phatase (APAAP) technique was used for immunostaining
according to the manufacturer’s recommendations (Dako
Diagnostika, Hamburg, Germany). As primary antibodies,
the rabbit antitserum against Ag-NPA-1 or the preimmune
serum were used at dilutions of 1 : 500 to 1 : 4000. As sec-
ondary antibodies, anti-rabbit, mouse immunoglobulins
(Dako) were applied and Fast Red TR salt (Sigma, Dei-
senhofen, Germany) was used as chromogen. Hematoxylin
functioned as the counterstain.
For immunogold TEM, adult A. galli worms were fixed
for 4 h in a solution of 1% paraformaldehyde and 0.025%
glutardialdehyde. Then samples were preserved in 0.2 m
sodium cacodylate buffer (pH 7.2) and stored at 4 °C.
Dehydrated specimens were embedded in medium-hardness
LR white acrylic resin (Polysciences, Warrington, USA).
Ultrathin sections were collected on filmed hexagonal nickel
grids (200 mesh). Following incubation in NaCl/P
i
, samples
were blocked in 10% BSA and incubated with the primary
antiserum at a dilution 1: 10000. After washing, the sec-

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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