The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[5-dimethylaminonaphthalene-1- sulfo-nylamino]undecanoic acid DAUDA from the protein binding sit
Trang 1binding protein Ag-NPA-1 from the parasitic nematode
Ascaridia galli
Rositsa Jordanova1, Georgi Radoslavov1, Peter Fischer2, Eva Liebau2, Rolf D Walter2, Ilia Bankov1 and Raina Boteva3
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: r.boteva@ncrrp.org
(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,N0-dimethyl-N-(iodoacetyl)-N0 -(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.
Trang 2these 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]
-5000 0 5000 10000
λ nm
]R
2 dmol
Fig 1 CD spectrum of Ag-NPA-1 in the far UV-range (190–
260 nm).
Trang 3The theoretical modelling of the protein backbone
folding predicts that these two residues are localized
on two neighbouring helices and the separation
between their Ca 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 (KQ) of 6.7 m)1 which, when
compared to the KQof 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 surroundbind-ing of Cys66 and increased its hydrophobicity The fluorescence changes allowed calculation of the apparent dissociation constant Kdof 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
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.
Trang 4an external quencher were performed (Fig 3) We
calculated a low value of 1.3 m)1 for the KQ
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 Kd
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 cm3Æm)1 for the spectral integ-ral JAD and of 0.012 for the Trp emission quantum yield (QTrp-A) of the protein–DAUDA complexes Hence a critical distance (Ro) 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
1.0
1.1
1.2
1.3
[acrylamide] M
F 0
Fig 3 Quenching of Trp fluorescence of Ag-NPA-1 by acrylamide.
A value of 1.3 M )1was calculated for the K
Q constant.
0.8 0.9
1.0
Φtrp /Φ300
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 e¼ 65%.
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 l M , calculated by a non-linear regression for a single binding site.
Trang 5homologous 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).
Trang 6an 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
Kdsimilar 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 (KQ 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).
Trang 7average 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· 103m)1Æcm)1at 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,N0-dimethyl-N-(iodoacetyl)-N0 -(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 concenconcen-trations of DAUDA, IAEDANS, IANBD and retinol were calculated from their absorption spectra using the corresponding molar extinc-tion coefficients The concentraextinc-tion 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 (QTrp) was deter-mined by comparing the integrated fluorescence spectrum
of the protein excited at 300 nm with that of the standard N-Ac-Trp-NH2normalized 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 (kexc) 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 (kexc
300 nm) from that obtained at kexc275 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]: Fo/F¼ 1 + KQ[X], where, Fo and F are the fluorescence emission intensities in the absence and presence of acrylamide, [X] is the molar concentration of acrylamide and KQthe overall quenching constant
Circular dichroism measurements
CD spectra were recorded in 10 mm Tris, pH 7.5, 20C, 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
Trang 8were 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 cm2Ædmol)1
Spec-tra subSpec-traction, 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 4C 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 (eM,280 9.5· 103
M)1Æcm)1 for Ag-NPA-1) and the dye absorbances at 337 nm for
IAEDANS (eM,337 6· 103M)1Æcm)1) and at 472 nm for
IANBD (eM,472 23· 103M)1Æcm)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 (kexc 300 nm) Ag-NPA-1
(0.2 lm) was incubated with increasing ligand
concentra-tions overnight, 4C, in the dark, in order to prevent
chemical changes of the light-sensitive ligands As DAUDA
and retinol absorb at the excitation (kexc300 nm) and
emis-sion (kem 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 (Kd) and the maximal fluorescence change (DFmax)
after a full saturation of the protein binding sites
Changes in the specific emission of the fluorescent probe
IAEDANS (kexc 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: r¼ Ro[(1) E)/E]1/6
, where, E is the efficiency of the energy transfer process, calculated from the decrease of the donor quantum yield (QTrp) in the presence
of the acceptor (QTrp-A): E¼ 1 – QTrp-A/QTrp, where, Rois the Fo¨rster radius or the critical distance for a 50% probabil-ity of the energy transfer process: Ro¼ (9.79 · 103)· (JAD
n)4K2QTrp)1/6A˚, where JADis 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 K2the 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 (K20.667 [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 masssoftware (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 molemole-cular 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
Trang 9comparison 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 4C
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/Pi, 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
References
1 Veerkamp JH, Peeters RA & Maatman RGHJ (1991)
Structural and functional features of different types of
cytoplasmic fatty acid-binding proteins Biochim Biophys
Acta 1081, 1–24
2 Noy N (2000) Retinoid-binding proteins: mediators of
retinoid action Biochem J 348, 481–495
3 Zimmerman AW & Veerkamp JH (2002) New insights
into the structure and function of fatty acid-binding
proteins Cell Mol Life Sci 59, 1096–1116
4 Tielnes AGM (1994) Energy generation in parasitic helminths Parasitol Today 10, 346–352
5 Kennedy MW (2000) The polyprotein lipid binding proteins of nematodes Biochim Biophys Acta 1476, 149–164
6 Kennedy MW (2001) Structurally novel lipid-binding proteins In Parasitic Nematodes: Molecular Biology, Biochemistry and Immunology (Kennedy MW & Har-nett W, eds), pp 309–330 CABI Publishing, Walling-ford, UK
7 Garofalo A, Rowlinson MC, Ngwa A, Hughes JM, Kelly SM, Price NC, Cooper A, Watson DG, Kennedy
MW & Bradley JE (2003) The FAR protein family of the nematode Caenorhabditis elegans Differential lipid binding properties, structural characteristics, and devel-opmental regulation J Biol Chem 278, 8065–8074
8 Kennedy MW, Brass A, McCruden AS, Price NC, Kelly
SM & Cooper A (1995) The ABA-1 allergen of the parasitic nematode Ascaris suum: fatty acid and retinoid binding function and structural characterization Biochemistry 34, 6700–6710
9 Moore J, McDermott NC, Price SM, Kelly A, Cooper
MW & Kennedy MW (1999) Sequence-divergent units
of the ABA-1 polyprotein array of the nematode Ascaris suumhave similar fatty-acid- and retinol-binding prop-erties but different binding-site environments Biochem J
340, 337–343
10 McDermott L, Cooper A & Kennedy MW (1999) Novel classes of fatty acid and retinol binding protein from nematodes Mol Cell Biochem 192, 69–75
11 Kennedy MW, Garside LH, Goodrick LE, McDermott
L, Brass A, Price SM, Kelly A, Cooper MW & Bradly
JE (1997) The Ov20 protein of the parasitic nematode Onchocerca volvulus A structurally novel class of small helix-rich retinol-binding proteins J Biol Chem 272, 29442–29448
12 Timanova A, Mueller S, Marti T, Bankov I & Walter
RD (1999) Ascaridia galli fatty acid-binding protein, a member of the nematode polyprotein allergens family Eur J Biochem 261, 569–576
13 Wilton DC (1990) The fatty acid analogue 11-(dansyl-amino) undecanoic acid is a fluorescent probe for the bilirubin-binding sites of albumin and not for the high-affinity fatty acid-binding sites Biochem J 270, 163–166
14 Barrett J, Saghir N, Timanova A, Clarke K & Brophy
PM (1997) Characterisation and properties of an intra-cellular lipid-binding protein from the tapeworm Monie-zia expansa Eur J Biochem 250, 269–275
15 Wilkinson TC & Wilton DC (1986) Studies on fatty acid-binding proteins The detection and quantification
of the protein from rat liver by using a fluorescent fatty acid analogue Biochem J 238, 419–424
16 Timanova A, Marti T, Walter RD & Bankov I (1997) Isolation and partial characterization of a
Trang 10fatty-acid-binding protein from Ascaris suum reproductive tissue.
Parasitol Res 83, 518–521
17 Kelley LA, MacCallum RM & Sternberg MJE (2000)
Enhanced genome annotation using structural profiles
in the program 3D-PSSM J Mol Biol 299, 501–522
18 Hudson EN & Weber G (1973) Synthesis and
character-ization of two fluorescent sulfhydryl reagents
Biochem-istry 12, 4154–4161
19 Boteva R., Ricchelli F, Sartor G & Decker H (1993)
Labeling of tarantula hemocyanin (Eurypelma
californi-cum) with dansyl-type fluorescent tags: identification of
the dye-binding site by fluorescence spectroscopy
J Photochem Photobiol B Biol 17, 155–161
20 Stone KL & Williams KR (1986) High-performance
liquid chromatographic peptide mapping and amino
acid analysis in the sub-nanomole range J Chromatogr
359, 205–212
21 McDermott L, Moore J, Brass A, Price NC, Kelly SM,
Cooper A & Kennedy MW (2001) Mutagenic and
che-mical modification of the ABA-1 allergen of the
nema-tode Ascaris: consequences for structure and lipid
binding properties Biochemistry 40, 9918–9926
22 Fo¨rster T (1959) Intermolecular energy migration and
fluorescence Ann Phys (Leipzig) 2, 55–75
23 Xia Y, Spence HJ, Moore J, Heaney N, McDermott L,
Cooper A, Watson DG, Mei B, Komuniecki R &
Kennedy MW (2000) The ABA-1 allergen of Ascaris
lumbricoides: sequence polymorphism, stage and
tissue-specific expression, lipid binding function, and protein
biophysical properties Parasitology 120, 211–224
24 Poole CB, Grandea AG, Maina CV, Jenkis RE, Selkirk
ME & McReynolds LA (1992) Cloning of a cuticular
antigen that contains multiple tandem repeats from the
filarial parasite Dirofolaria immitis Proc Natl Acad Sci
USA 89, 5986–5990
25 Tweedie S, Paxton WA, Ingram L, Maizels RM,
McReynolds LA & Selkirk ME (1993) Brugia pahangi
and Brugia malayi: a surface associated glycoprotein
(gp15/400) is composed of multiple tandemly repeated units and processed from a 400-kDa precursor Exp Parasitol 76, 156–164
26 McDermott L, Kennedy MW, McManus DP, Bradley
JE, Cooper A & Storch J (2002) How helminth lipid-binding proteins offload their ligands to membranes: differential mechanisms of fatty acid transfer by the ABA-1 polyprotein allergen and Ov-FAR-1 proteins of nematodes and Sj-FABPc of schistosomes Biochemistry
41, 6706–6713
27 Beaven GH & Holiday ER (1952) Ultraviolet absorp-tion spectra of proteins and amino acids Adv Protein Chem 7, 319–325
28 Lehrer SS (1971) Solute perturbation of protein fluores-cence The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion Biochemistry 10, 3254–3263
29 Eisinger J (1969) Intramolecular energy transfer in adrenocorticotropin Biochemistry 8, 3902–3908
30 Genov N & Boteva R (1986) Fluorescence technique for comparative studies of substrate-binding subsites in ser-ine proteinases Application to subtilisins Biochem J
238, 923–926
31 Horrocks WW & Collier WE (1981) Lanthanide ion luminescence probes Measurement of distance between intrinstic protein fluorophores and bound metal ions: quantitation of energy transfer between tryptophan and terbium (III) or Europium (III) in the calcium-binding protein parvalbumin J Am Chem Soc 103, 2856–2862
32 Clos J, Westwood JT, Becker PB, Wilson S, Lambert K
& Wu K (1990) Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to nega-tive regulation Cell 63, 1085–1097
33 Fischer P, Schmez C, Bandi C, Bonow I, Mand S, Fischer K & Buttner DW (2002) Tunga penetrans: mole-cular identification of Wolbachia endobacteria and their recognition by antibodies against proteins of endobac-teria from filarial parasites Exp Parasitol 102, 201–211