Báo cáo khoa học: Increased flexibility and liposome-binding capacity of CD1e at endosomal pH ppt

Increased flexibility and liposome-binding capacity of
CD1e at endosomal pH
Natalia Bushmarina
1,2
, Sylvie Tourne
1,2
, Gae
¨
lle Giacometti
1,2
, Franc¸ois Signorino-Gelo
1,2
,
Luis F. Garcia-Alles
3,4
, Jean-Pierre Cazenave
2,5
, Daniel Hanau
1,2
and Henri de la Salle
1,2
1 INSERM, UMR-S725, INSERM-Universite
´
de Strasbourg, France
2 Etablissement Franc¸ais du Sang-Alsace, Strasbourg, France
3 CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France
4 Universite
´
de Toulouse, UPS, IPBS, France
5 INSERM, UMR-S949, Etablissement Franc¸ ais du Sang-Alsace, Strasbourg, France
Introduction

CD1 molecules are nonclassic major histocompatibility
complex class I molecules that are mainly expressed by
dendritic cells, the professional antigen-presenting cells
of the immune system. CD1 proteins are heterodimers
composed of a poorly polymorphic membrane-anchored
a-chain and b
2
-microglobulin (b2m). In humans, four
Keywords
CD1e; conformational changes; lipid binding;
structure; surface plasmon resonance
Correspondence
H. de la Salle or N. Bushmarina,
Etablissement Franc¸ais du Sang-Alsace,
10 rue Spielmann, 67065 Strasbourg, France
Fax: +33 388 212 544
Tel: +33 388 212 525
E-mail: ;

(Received 25 January 2011, revised 30
March 2011, accepted 4 April 2011)
doi:10.1111/j.1742-4658.2011.08118.x
The plasma membrane proteins CD1a, CD1b and CD1c are expressed by
human dendritic cells, the professional antigen-presenting cells of the
immune system, and present lipid antigens to T lymphocytes. CD1e
belongs to the same family of molecules, but accumulates as a membrane-
associated form in the Golgi compartments of immature dendritic cells and
as a soluble cleaved form in the lysosomes of mature dendritic cells. In
lysosomes, the N-terminal propeptide of CD1e is also cleaved, but the
functional consequences of this step are unknown. Here, we investigated

how the pH changes encountered during transport to lysosomes affect the
structure of CD1e and its ligand-binding properties. Circular dichroism
studies demonstrated that the secondary and tertiary structures of recombi-
nant CD1e were barely altered by pH changes. Nevertheless, at acidic pH,
guanidium chloride-induced unfolding of CD1e molecules required lower
concentrations of denaturing agent. The nonfunctional L194P allelic vari-
ant was found to be structurally less stable at acidic pH than the functional
forms, providing an explanation for the lack of its detection in lysosomes.
The number of water-exposed hydrophobic patches that bind 8-anilino-
naphthalene-1-sulfonate was higher in acidic conditions, especially for the
L194P variant. CD1e molecules interacted with lipid surfaces enriched in
anionic lipids, such as bis(monoacylglycero)phosphate, a late endoso-
mal ⁄ lysosomal lipid, especially at acidic pH, or when the propeptide was
present. Altogether, these data indicate that, in the late endosomes ⁄ lyso-
somes of DCs, the acid pH promotes the binding of lipid antigens to CD1e
through increased hydrophobic and ionic interactions.
Abbreviations
ANS, 8-anilinonaphthalene-1-sulfonate; bis-ANS, 4,4¢-bis(1-anilinonaphthalene-8-sulfonate); b2m, b
2
-microglobulin; BMP,
bis(monoacylglycero)phosphate; ER, endoplasmic reticulum; NBD, nitrobenzoxadiazole; PtdCho, phosphatidylcholine; PtdSer,
phosphatidylserine; PtdIns, phosphatidylinositol; PtdInsM
6
, hexamannosylated phosphatidylinositol; rsCD1e, recombinant soluble CD1e;
rsCD1b, recombinant soluble CD1b; rsCD1e2, recombinant soluble CD1e2; rsCD1e4, recombinant soluble CD1e4; sCD1e, soluble CD1e;
TMP, transition midpoint.
2022 FEBS Journal 278 (2011) 2022–2033 ª 2011 The Authors Journal compilation ª 2011 FEBS
forms (CD1a–CD1d) are directly involved in the presen-
tation of lipid antigens to T cells. These proteins are
internalized from the plasma membrane, and then traffic

through the endocytic pathway, where they capture anti-
genic ligands, before returning to the plasma membrane
to stimulate antigen-specific T cells. In contrast, newly
assembled CD1e molecules are transported from the
endoplasmic reticulum (ER) to the endocytic pathway
without passing through the plasma membrane [1]. In
late endosomal compartments, CD1e undergoes double
processing and becomes functional; it is cleaved into a
soluble form [soluble CD1e (sCD1e)], and a nonpolar
12-residue N-terminal propeptide (APQALQSYHLAA)
is removed [2,3]. This propeptide is unique to CD1e
among classic and nonclassic human leukocyte-associ-
ated class I molecules. It facilitates the assembly of
a-chain–b2m complexes in the ER, but has no other
ascribed function [3]. Soluble lysosomal CD1e molecules
participate in the processing of antigenic glycolipids pre-
sented by CD1b [4,5]. Thus, coexpression of both CD1b
and CD1e by antigen-presenting cells is indispensable
for the activation of specific T-cell clones by hexaman-
nosylated phosphatidylinositol (PtdInsM
6
), a structur-
ally complex mycobacterial glycolipid. The use of
antigen-presenting cells deficient in lysosomal a-man-
nosidase and of recombinant sCD1e (rsCD1e) produced
in Drosophila cells has allowed us to demonstrate that
sCD1e molecules bind PtdInsM
6
and facilitate the com-
plete processing of its four a-mannoses by lysosomal

a-mannosidase into dimannosylated PtdIns, a CD1e-
independent CD1b-restricted antigen [5]. Additional
investigations showed that, among the six natural vari-
ants of CD1e, only one is unable to sustain PtdInsM
6
presentation. This molecule, CD1e4, is characterized by
the replacement of Leu194 by proline, as compared with
the common CD1e1 variant. In human cells, only small
amounts of CD1e4 reach late endosomal compartments,
and soluble lysosomal forms are not detected. Never-
theless, recombinant soluble CD1e4 (rsCD1e4) can be
normally expressed in insect cells and, like other natural
variants, assists in vitro digestion of PtdInsM
6
by
a-mannosidase [6].
CD1b, CD1c, CD1d and CD1e transit through
acidic endosomal compartments and CD1b and CD1d,
at least, are subject to pH-dependent conformational
changes. Acidification of late endosomal compartments
is required for the presentation of several CD1b-
restricted and CD1d-restricted antigens, for at least
two reasons. First, as shown for CD1b and CD1d,
lipid loading appears to be mediated by lysosomal
lipid transfer proteins [7–9], which are optimally func-
tional at acidic pH [10]. Second, acid-induced confor-
mational changes allow CD1b and CD1d to adopt a
conformation with partially unfolded a-helices, thereby
facilitating the access of hydrophobic ligands to their
antigen-binding pockets [11–13].

The aim of this study was to determine how acidifi-
cation modifies different structural features of CD1e
and affects its interaction with lipid membranes and
ligands. The role played by the propeptide in these
processes was also investigated. Finally, we looked at
how these properties are modified in the immunologi-
cally nonfunctional CD1e4 variant.
Results
The secondary structure of rsCD1e is stable at
physiological pH
In this work, recombinant soluble CD1e2 (rsCD1e2)
molecules including or not including the propeptide
(rsCD1e2
+
or rsCD1e2
)
) were produced in Drosoph-
ila melanogaster S2 cells. First, we investigated how
pH variations similar to those occurring during
transport from neutral Golgi to acidic lysosomal com-
partments influence the secondary structure of the
active lysosomal form, rsCD1e2
)
. The alteration of
the secondary structure was followed with far-UV
(190–240 nm) CD spectroscopy. Similar experiments
were performed with rsCD1e2
+
, in order to determine
the impact of the propeptide on the stability of CD1e.

As shown in Fig. 1, the circular dichroism spectra of
rsCD1e2
)
and rsCD1e2
+
did not differ significantly,
and remained unaltered over pH values ranging from
4 to 7 (Fig. 1), being affected only at pH < 3.5 (data
not shown). The pronounced minimum at 219 nm and
the maximum at 195–196 nm are characteristic features
of a ⁄ b class proteins with a major b-sheet content. The
percentages of different secondary structures calculated
from these spectra, namely 16 ± 1% a-helices,
37±1% b-strands, and 47 ± 1% other structures
[14,15], are in full agreement with the content deduced
from a homology model derived from the crystal struc-
ture of recombinant soluble CD1b (rsCD1b) [5].
Physiological pH changes induce minor
perturbations in the tertiary structure of CD1e
We next examined the changes in the tertiary structure
of rsCD1e2
)
and rsCD1e2
+
induced by acidification,
by near-UV (250–320 nm) circular dichroism spectros-
copy. This method allows characterization of the envi-
ronment of the aromatic amino acid side chains in
proteins, and thus gives information about the com-
pactness of the tertiary structure. It is widely used to

study the conformational changes caused by physico-
N. Bushmarina et al. Structural changes underlying CD1e activity
FEBS Journal 278 (2011) 2022–2033 ª 2011 The Authors Journal compilation ª 2011 FEBS 2023
chemical perturbations [16]. At pH 7, the near-UV
spectra of rsCD1e proteins displayed several pro-
nounced peaks characteristic of native proteins with a
compact tertiary structure, with no significant differ-
ences between the two types of rsCD1e2 molecule
(Fig. 2A,B). Shifting the pH to 4.8 only slightly
decreased the ellipticity, revealing a subtle increase in
the flexibility of the tertiary structure at lysosomal pH
(Fig. 2A,B).
Enhanced interaction between rsCD1e2 molecules
and hydrophobic probes at endosomal pH
As the overall structure of rsCD1e2 molecules under-
went no major pH-induced alterations, we decided to
investigate whether pH variations influence solvent
accessibility to the hydrophobic interfaces of CD1e,
which include the lipid-binding groove. Hence, we mea-
sured the fluorescence resulting from binding to CD1e
of 8-anilinonaphthalene-1-sulfonate (ANS) and 4,4¢-
bis(1-anilinonaphthalene-8-sulfonate) (bis-ANS). These
probes are widely used to study the solvent-accessible
hydrophobic surfaces of proteins [17]. ANS binds
strongly to hydrophobic clusters associated with loose
tertiary contacts or hydrophobic binding sites. Bis-ANS
is a superior molecular probe for nonpolar cavities in
proteins, and allows the determination of saturation
curves.
At a given pH and probe concentration, rsCD1e2

+
and rsCD1e2
)
bound similar amounts of ANS. A
shift from neutral to acidic pH caused a significant
increase in fluorescence intensity in the two forms of
CD1e, with values that were a function of the ANS
concentration (Fig. 3A). Experiments with bis-ANS
demonstrated a similar dependence on bis-ANS con-
centration for both rsCD1e2
+
and rsCD1e2
)
. How-
ever, bis-ANS binding saturation could only be attained
at pH 4.5 and not at neutral pH, regardless of the
CD1e2 molecule studied. It is also noteworthy that a
nearly two-fold higher maximal fluorescence was
obtained for rsCD1e2
+
than for rsCD1e2
)
(Fig. 3B).
At pH 4.5 and substoichiometric bis-ANS⁄ CD1e
Fig. 1. pH dependence of the secondary structure of rsCD1e molecules. rsCD1e2
+
or rsCD1e2
)
were diluted to 4 lM in 5 mM monoso-
dium ⁄ disodium phosphate (pH 7) or 5 m

M sodium phosphate ⁄ citrate (pH 4) buffer containing 150 mM sodium sulfate. The solutions were
incubated overnight, and the far-UV circular dichroism spectra were recorded in 1-mm cuvettes. MRW, mean residue weight; res, residue.
Fig. 2. pH dependence of the tertiary structure of rsCD1e molecules. rsCD1e proteins were diluted to 20 lM in 5 mM sodium phosphate
buffer (pH 7 or pH 4.8) containing 150 m
M sodium sulfate. (A, B) The near-UV circular dichroism spectra of CD1e2
)
(A) and CD1e2
+
(B) were
recorded at pH 7 and pH 4.8. (C) Comparison of the near-UV circular dichroism spectra of rsCD1e2
)
and rsCD1e4
)
at pH 7. MRW, mean
residue weight; res, residue.
Structural changes underlying CD1e activity N. Bushmarina et al.
2024 FEBS Journal 278 (2011) 2022–2033 ª 2011 The Authors Journal compilation ª 2011 FEBS
ratios, the fluorescence was proportional to the
probe concentration (Fig. 3C). This allowed us to draw
a Scatchard plot of the data obtained in three indepen-
dent experiments (one representative experiment is
shown in Fig. 3C), and to deduce that rsCD1e2
)
and
rsCD1e2
+
bound, respectively, 12 ± 0.3 and 24 ± 0.7
bis-ANS molecules, with an apparent K
d
of

16 ± 0.2 lm for the two proteins. In conclusion, experi-
ments with ANS and bis-ANS confirmed a beneficial
effect of lysosomal pH on the binding of hydro-
phobic ligands to both rsCD1e2
+
and rsCD1e2
)
.In
addition, bis-ANS fluorescence data indicated that
the CD1e propeptide influences the number of binding
sites.
A
B
C
D
Fig. 3. Binding of ANS and bis-ANS to
rsCD1e molecules. rsCD1e proteins were
diluted to 2 l
M in pH 7 and pH 4.5 buffers
as described in Fig. 1, and incubated in the
presence of different concentrations of ANS
(A) or bis-ANS (B, C, D) for 30 min. The fluo-
rescence intensity of the solutions was then
measured [k
ex
= 370 nm and k
em
= 480 nm
(ANS); k
ex

= 390 nm and k
em
= 490 nm
(bis-ANS)] in a FlexStation automate. Each
condition in each row (B and D) was tested
in triplicate on a same day. However each
of these rows corresponds to a set of
experiments performed on an different day.
Mean values of triplicate analyses with their
respective standard deviations are shown.
The binding of bis-ANS to CD1e proteins
was studied at low [bis-ANS] ⁄ [CD1e] ratios
(< 1), or in the presence of an excess of
bis-ANS (up to 25-fold molar excess). The
left panel shows the bis-ANS fluorescence
as a function of [bis-ANS], for [bis-ANS] ⁄
[CD1e2] < 1. Scatchard representations of
the binding of bis-ANS to rsCD1e2
)
and
rsCD1e2
+
in one representative experiment
at pH 4.5 are shown in the middle and right
panels. (D) Comparison of the binding of
bis-ANS to rsCD1e2 and rsCD1e4, with and
without the propeptide.
N. Bushmarina et al. Structural changes underlying CD1e activity
FEBS Journal 278 (2011) 2022–2033 ª 2011 The Authors Journal compilation ª 2011 FEBS 2025
Unfolding of rsCD1e is facilitated at acidic pH

To determine the influence of pH on the structural
robustness of rsCD1e2 molecules, we examined the
effect of the denaturing agent guanidium chloride at
pH 7 and pH 4.8. First, the stability of the secondary
structure of the molecules was analyzed by far-UV cir-
cular dichroism spectroscopy. The values of the
normalized ellipticity at 219 nm (h
219 nm
) for rsCD1e2
)
and rsCD1e2
+
are plotted in Fig. 4 as a function of
guanidium chloride concentration. The differences
between rsCD1e2
)
and rsCD1e2
+
proved to be rather
subtle, as the transition midpoint (TMP) of the unfold-
ing curve of the secondary structure of rsCD1e2
+
(Table 1) was slightly higher than that of rsCD1e2
)
at
pH 7 (4 m versus 3.7 m guanidium chloride, respec-
tively), whereas the two TMPs were equal at pH 4.8
(3.8 m).
The resistance of tertiary contacts in rsCD1e2 mole-
cules to guanidium chloride-induced denaturation was

then explored by measuring the intrinsic fluorescence
of the proteins and the extent of ANS binding. A com-
parison of the intrinsic fluorescence of rsCD1e2
+
and
rsCD1e2
)
, at different guanidium chloride concentra-
tions and pH values, revealed that the two CD1e mole-
cules were more susceptible to guanidium chloride-
induced denaturation at acidic pH (Fig. 5A and
Table 1). Although the two forms of rsCD1e2 bound
equivalent amounts of ANS, the concentration of gua-
nidium chloride required to induce maximal ANS
binding decreased from 3–3.2 m at neutral pH to 2.3 m
at acidic pH (Fig. 5C); these values are close to the
TMPs of the denaturation curves of the tertiary struc-
tures (Table 1).
Altogether, these data suggest that, upon arrival in
the lysosomes, CD1e could become less stable through
exposure of hydrophobic surfaces, including, presum-
ably, the lipid-binding pocket.
The propeptide moderately influences lipid
binding to rsCD1e molecules
The binding of lipids to CD1e proteins was investi-
gated by using phosphatidylserine (PtdSer) with a fluo-
rescent nitrobenzoxadiazole (NBD) moiety linked to
the terminus of one of the fatty acid chains. The bind-
ing of PtdSer–NBD to rsCD1e was analyzed by mea-
suring the increase in the fluorescence of the NBD

group upon its insertion into the hydrophobic lipid-
binding pocket of CD1e. The kinetics of binding to the
different rsCD1e molecules were compared at pH 7
and pH 4.5. Three independent experiments were per-
formed, resulting in similar profiles for the different
protein and experimental conditions. The reproducibil-
ity of the experiments was validated by determining
the fluorescence intensities at the end of the assays,
and the times required to reach half these values. The
standard deviation of these parameters deduced from
the three experiments fell between 2% and 5% (data
not shown); representative curves for each condition
are shown in Fig. 6. Regardless of the pH, the binding
of PtdSer–NBD to CD1e reached a plateau for all pro-
teins, except for rsCD1e2
)
, at pH 7. For a given
rsCD1e molecule, the maximal fluorescence intensity
was barely affected by the pH. The time required to
reach half the maximal lipid binding was significantly
less at acidic pH for rsCD1e2
+
but not for rsCD1e2
)
(data not shown). These results indicate that the two
CD1e2 proteins, with and without the propeptide, are
fairly equivalent in terms of PtdSer binding.
Enhanced interaction of rsCD1e with anionic
lipids at endosomal pH
The interaction of rsCD1e molecules with liposomes

immobilized on sensor chips was studied by surface
plasmon resonance. The liposomes were composed
of mixtures of neutral phosphatidylcholine (PtdCho)
(70 molÆ%) and either PtdSer, phosphatidylinositol
(PtdIns) or sulfatide (30 molÆ%), or PtdSer plus
bis(monoacylglycero)phosphate (BMP) (15 molÆ%of
each) for BMP-enriched liposomes. RsCD1e2
+
and
Fig. 4. Guanidium chloride-induced unfolding of the secondary struc-
ture of rsCD1e molecules. rsCD1e2
+
(h, j), rsCD1e2
)
(4,m) and
rsCD1e4
)
(), ¤) were diluted to 20 lM in 10 mM monosodium ⁄
disodium phosphate buffer containing 150 m
M sodium sulfate at
pH 7 (open symbols) or pH 4.8 (closed symbols). The ellipticity at
219 nm at a given molar concentration of guanidium chloride
(h[219;
M]) was normalized with the following formula: (h[219;M]–
h[219;6]) ⁄ h[219;0].
Structural changes underlying CD1e activity N. Bushmarina et al.
2026 FEBS Journal 278 (2011) 2022–2033 ª 2011 The Authors Journal compilation ª 2011 FEBS
rsCD1e2
)
were injected over the lipid surfaces for

300 s, after which the surfaces were rinsed with buffer
for the same period of time. For comparison, rsCD1b
was also analyzed.
Irrespective of the pH, in terms of resonance units,
the interaction of rsCD1e2
)
with liposomes was con-
siderably higher than that of rsCD1b molecules
(Fig. 7). Moreover, at acidic pH, the interaction of
rsCD1e2
)
with liposomes increased significantly, the
intensity of the signal being three times higher. The
protein–liposome interactions were also dependent on
the liposome composition. Thus, the order of preference
was sulfatide > BMP + PtdSer > PtdIns @ PtdSer
for rsCD1e2
)
at pH 7 or pH 4.8, whereas for rsCD1b it
was sulfatide @ PtdSer > PtdIns @ BMP + PtdSer at
pH 7, and sulfatide > PtdSer @ PtdIns @ BMP + Ptd-
Ser at pH 4.8. The behavior of rsCD1e2
+
in the
presence of liposomes was qualitatively comparable to
that of rsCD1e2
)
, although the interaction appeared
to be stronger, with a two-fold higher resonance
signal (Fig. 7, right panel), demonstrating that the

ability of CD1e to interact with membranes at neutral
or acidic pH does not rely on cleavage of the propep-
tide.
Table 1. TMPs of guanidium chloride-induced denaturation transitions in the secondary and tertiary structures of rsCD1e proteins. Equations
fitting the data in Fig. 4 or in Fig. 5A,B were used to deduce the guanidium chloride-induced denaturation transitions in the secondary and
tertiary structures of rsCD1e proteins, respectively. The form of the equations was Y =1⁄ (1 + 10^((logEC50 – [guanidium chloride])*HillS-
lope)), proposed by
GRAPHPAD PRISM, where Y is the normalized fluorescence intensity at 340 nm or normalized ellipticity at 219 nm, logEC50
is the guanidium chloride concentration at which the ellipticity is half its initial value in the absence of guanidium chloride (TMP), [guanidium
chloride] is the molar concentration of guanidium chloride, and HillSlope is the Hill constant or slope factor defining the steepness of the
curve. ss, secondary structure; ts, tertiary structure; ANS, [guanidium chloride] inducing the maximal ANS fluorescence.
CD1e2
+
, pH 7 CD1e2
)
, pH 7 CD1e4, pH 7 CD1e2
+
, pH 4.8 CD1e2
)
, pH 4.8 CD1e4
)
, pH 4.8
TMP for ss 4.0 ± 0.1 3.7 ± 0.1 3.6 ± 0.1 3.8 ± 0.1 3.8 ± 0.1 3.5 ± 0.1
TMP for ts 2.8 ± 0.1 2.7 ± 0.1 1.6 ± 0.1 2.1 ± 0.1 2.4 ± 0.1 1.2 ± 0.1
ANS 3.2 ± 0.3 3.0 ± 0.1 2.0 ± 0.3 2.3 ± 0.1 2.3 ± 0.3 2.0 ± 0.3
Fig. 5. Guanidium chloride-induced changes in the fluorescence of rsCD1e proteins. rsCD1e2
+
or rsCD1e2
)
and rsCD1e4

)
were diluted to
2 l
M in 10 mM monosodium ⁄ disodium phosphate buffer containing 150 mM sodium sulfate at pH 7 (open symbols) or pH 4.8 (closed sym-
bols). Symbols are the same as in Fig. 4. (A, B) Normalized tryptophan fluorescence at 340 nm. (C, D) Normalized ANS fluorescence at
490 nm (molar ANS ⁄ protein ratio is 40 : 1). The fluorescence intensities were normalized by dividing by the maximal intensity of the spec-
trum of the native protein. For each condition, the curves represent the means of three independent experiments. Because of their values,
the bars corresponding to the standard deviations are smaller than the symbols, and are therefore not represented. (A, C) Comparison of
rsCD1e2
)
and rsCD1e2
+
. (B, D) Comparison of rsCD1e2
)
and rsCD1e4
)
. Normalized I, normalized intensity.
N. Bushmarina et al. Structural changes underlying CD1e activity
FEBS Journal 278 (2011) 2022–2033 ª 2011 The Authors Journal compilation ª 2011 FEBS 2027
Compromised stability of the CD1e4 variant
We previously reported that the CD1e4 allelic variant
does not facilitate the presentation of PtdInsM
6
anti-
gens to CD1b-restricted T cells, and that this was
attributable to inefficient transport of this variant to
CD1b
+
compartments and the absence of a detectable
soluble lysosomal form. Here, we compared the con-

formational properties of rsCD1e4
)
with those of
rsCD1e2
)
, from which it differs by the replacement of
Leu194 with a potentially helix-destructuring proline.
At pH 7, the far-UV (data not shown) and near-UV
(Fig. 2C) circular dichroism spectra showed similar
secondary and tertiary structures of rsCD1e2
)
and
rsCD1e4
)
. Shifting the pH to 4.8 resulted in only
minor changes in the tertiary structure of rsCD1e4
)
as
compared with rsCD1e2
)
(data not shown). At pH 7
and guanidium chloride concentrations below 3 m, the
normalized ellipticity of rsCD1e4
)
was nevertheless sig-
nificantly weaker than that of rsCD1e2
)
. In addition,
at least 1 m lower concentrations of guanidium chlo-
ride were required for rsCD1e4

)
than for rsCD1e2
)
to induce a similar decrease in ellipticity, at acidic or
neutral pH (Fig. 4), to reduce the intrinsic protein flu-
orescence (Fig. 5B), or to reach maximal ANS binding
(Fig. 5D). Thus, although the L194P substitution
seems to only weakly affect the secondary structure of
CD1e, it appears to have a profound effect on the sta-
bility of its tertiary structure.
Under nondenaturing conditions, rsCD1e4
)
and
rsCD1e2
)
bound comparable amounts of ANS at neu-
tral pH. Conversely, incubation at pH 4.5 resulted in a
dramatic increase in the binding of ANS to rsCD1e4
)
,
but not to rsCD1e2
)
(Fig. 3A). In experiments with
bis-ANS, rsCD1e4
)
and rsCD1e2
)
behaved compara-
bly at pH 4.5 when [bis-ANS] ⁄ [rsCD1e] ratios were
below 20. At higher stoichiometries, bis-ANS binding

to rsCD1e4
)
appeared to be unsaturable (Fig. 3D). At
acidic pH, with rsCD1e4
+
the fluorescence of bound
bis-ANS reached a plateau, although at a higher value
of fluorescence intensity than for CD1e2
+
. At low
[bis-ANS] ⁄ [rsCD1e4
+
or rsCD1e4
)
] ratios (i.e. < 1),
the fluorescence was proportional to the concentration
of bis-ANS. Calculations indicated that the apparent
number of bis-ANS-binding sites on CD1e4
+
was
12.4 ± 0.3, i.e. half that on CD1e2
+
.
Fig. 6. Interaction of rsCD1e molecules with PtdSer–NBD. The fluorescence of NBD in reaction mixtures containing rsCD1e2
+
, rsCD1e2
)
,
rsCD1e4
+

or rsCD1e4
)
and PtdSer–NBD (both 2 lM) at pH 7 (left) or pH 4.5 (right) was recorded for 6000 s. Curve C represents the fluores-
cence of PtdSer–NBD alone in the buffer.
Fig. 7. Interaction of rsCD1e2 and rsCD1b molecules with liposomes. Liposomes containing 70 molÆ% PtdCho and 30 molÆ% PtdSer, PtdIns
or sulfatide, or 70 molÆ% PtdCho, 15 molÆ% PtsSer and 15 molÆ% BMP, were adsorbed onto L1 chips in a Biacore 3000 system. RsCD1e2
)
and rsCD1b proteins (0.2 lM)in10mM monosodium ⁄ disodium phosphate buffer containing 150 mM NaCl at pH 7 (left) or pH 4.8 (middle)
were injected over the chips for 300 s, after which the surfaces were rinsed with the same buffer for the same period of time. The interac-
tions of rsCD1e2
+
with liposomes at pH 7 and pH 4.8 were compared (right) in a similar manner.
Structural changes underlying CD1e activity N. Bushmarina et al.
2028 FEBS Journal 278 (2011) 2022–2033 ª 2011 The Authors Journal compilation ª 2011 FEBS
At neutral pH, the fluorescence of PtdSer–NBD
bound to CD1e4
+
reached a plateau with a higher
value than for CD1e2 molecules at acidic pH (Fig. 6).
These observations suggest that, at neutral pH,
CD1e4
+
molecules are more receptive to lipids, or
form more stable complexes with PtdSer–NBD.
CD1e4
)
behavior appears to be the opposite of that of
CD1e4
+
. Indeed, at acidic pH, CD1e4

+
and CD1e2
+
displayed similar PtdSer–NBD binding curves. In con-
trast, rsCD1e4
)
bound almost two times less PtdSer–
NBD than rsCD1e2
)
.
Discussion
CD1 molecules and human leukocyte antigen class I
and class II molecules are structurally related proteins.
In particular, their antigen-binding pockets are formed
of a-helices lying on b-sheets, and include hydrophobic
residues pointing to the groove, in an optimal architec-
ture for lipid binding [18]. The 3D structure of CD1e
is not known, and no rigorous structural study of this
protein has been reported to date. With the intention
of filling this gap and gaining a more precise picture of
the role played by the CD1e propeptide sequence,
we performed structural investigations on different
forms of CD1e, using complementary biophysical
approaches.
Prediction of the secondary structure on the basis of
far-UV circular dichroism spectra indicated that the
a-helix and b-sheet contents of rsCD1e2
+
or rsCD1e2
)

and rsCD1e4
)
were almost identical to those of CD1b
and major histocompatibility complex class I and clas-
s II molecules [11,19]. We also analyzed rsCD1b pro-
duced in S2 cells by circular dichroism spectroscopy,
and the results obtained were in excellent agreement
with the structural contents deduced from crystallo-
graphic CD1b structures (data not shown), which vali-
dates our experimental approach. In subsequent
studies, the secondary structure of rsCD1e was found
to remain unaltered when the pH was shifted from
neutral to acidic (Fig. 1A), and structural changes only
became evident under nonphysiological conditions
(pH < 3.5, data not shown). A comparison with liter-
ature data suggested that the a1-helix and a2-helix of
CD1e are less sensitive to pH changes than those of
CD1b or CD1d [11,12].
In contrast, several lines of evidence presented in
this work indicate that CD1e could gain flexibility in
its tertiary structure while transiting from the ER to
acidic CD1b
+
late endosomes⁄ lysosomes. First, the
exposure of hydrophobic surfaces to ANS increased
considerably at acidic pH in both rsCD1e2
)
and
rsCD1e2
+

(Fig. 3A). On the other hand, circular
dichroism studies (Fig. 4) and analyses of intrinsic flu-
orescence (Fig. 5A) and ANS binding (Fig. 5C) in the
presence of various concentrations of guanidium chlo-
ride revealed increased structural instability at acidic
pH. These data suggest that acidification could gener-
ate stable conformational intermediates with loose ter-
tiary contacts and improved access to the lipid-binding
groove. An enhancement of ANS binding at lysosomal
pH has been reported for CD1b and CD1d molecules,
and found to correlate with an accompanying
increased capacity to bind lipids [11,12].
A second important point addressed in this study
was whether the CD1e propeptide, which is present on
the membrane-associated but not on the soluble lyso-
somal form of the molecule, influences protein struc-
ture and stability. Circular dichroism data (Figs 1 and
2), ANS binding experiments (Fig. 3A) and unfolding
experiments with guanidium chloride (Figs 4 and 5)
failed to reveal any significant differences between
rsCD1e2
+
and rsCD1e2
)
. In contrast, rsCD1e2
+
was
found to bind twice as many bis-ANS molecules as
rsCD1e2
)

(Fig. 3B,C). This intriguing observation is
difficult to explain, as the estimated stoichiometries of
interaction (24 and 12 bis-ANS molecules for one
rsCD1e2
+
and one rsCD1e2
)
molecule, respectively)
are largely in excess of the number of bis-ANS mole-
cules that could be expected to interact directly with
the CD1e lipid-binding groove or the 12-residue pro-
peptide. Nonetheless, our observation is supported by
the fact that the propeptide also triggered an almost
two-fold higher response in surface plasmon resonance
experiments on the interaction of CD1e with surfaces
containing anionic lipids (Fig. 7C). These interactions
may be partly driven by electrostatic forces. Indeed,
theoretical estimations with a 3D homology model of
CD1e and the propka algorithm [20] indicate that the
overall charge of CD1e could shift from ) 3to+18
as the pH drops from 7 to 4.5, whereas these parame-
ters are ) 7 and + 2 for CD1b. Such a strong positive
charge on CD1e proteins could partially explain why
saturation was only attained at acidic pH with high
bis-ANS binding stoichiometries. Similarly, this prop-
erty would explain why CD1e interacted better with
surfaces enriched in anionic lipids when the pH was
acidic (Fig. 7), and would strongly suggest that this
effect could control its interaction with lysosomal sur-
faces rich in negatively charged lipids such as BMP

[10]. It nevertheless remains challenging to elucidate
why the presence of the propeptide leads to an almost
two-fold increase in bis-ANS binding stoichiometry
and in surface plasmon resonance signals.
This study was also intended to shed light on the
structural behavior of the natural variant CD1e4,
N. Bushmarina et al. Structural changes underlying CD1e activity
FEBS Journal 278 (2011) 2022–2033 ª 2011 The Authors Journal compilation ª 2011 FEBS 2029
which is inefficiently transported from the ER to lyso-
somes, with the result that a soluble lysosomal form
cannot be detected [6]. Although rsCD1e4 molecules
can be efficiently expressed in S2 cells, and the circular
dichroism data presented here demonstrate that the
protein is folded adequately, there is cumulative evi-
dence for lower intrinsic stability and higher structural
sensitivity in acidic environments than for rsCD1e2.
Thus, the ANS-binding data revealed a remarkable
exposure of hydrophobic patches at pH 4.5 (Fig. 3A).
Moreover, significantly lower guanidium chloride con-
centrations were required to cause a similar destabili-
zation or exposure to ANS as in rsCD1e2 molecules
(Figs 4 and 5). The instability of rsCD1e4
)
at acidic
pH might explain why CD1e4 could be immunolocal-
ized in CD63
+
compartments, whereas no soluble
form could be detected. Our data strongly suggest that,
once they are in acidic late endosomal ⁄ lysosomal com-

partments, CD1e4 molecules probably adopt a more
water-exposed conformation, which would render the
protein susceptible to proteolysis, thus preventing its
accumulation.
Overall, the results presented in this study suggest
that the conformational behavior of CD1e is optimized
to facilitate its interaction with and binding of lipids in
a pH-regulated manner. Thus, the arrival of CD1e in
acidic late endosomal and lysosomal compartments
would be synchronized with proteolytic events permit-
ting release of the soluble domain from the mem-
branes, cleavage of the propeptide, and interaction
with anionic lipids, possibly the anionic lipid domains
present in CD1b
+
compartments. The propeptide
would have no influence on the conformational struc-
ture of CD1e or the properties of the lipid-binding
groove. This short N-terminal oligopeptide might, on
the contrary, interact with key residues to prevent the
occurrence of intermolecular or intramolecular con-
tacts. How these properties combine to optimize the
repertoire of CD1e ligands and their subsequent pre-
sentation by CD1b molecules in dendritic cells remains
to be investigated.
Experimental procedures
Reagents and recombinant proteins
Recombinant soluble sCD1 (rsCD1) molecules were
expressed in transfected Drosophila melanogaster S2 cells
and purified as previously described [5,6]. Briefly, rsCD1e

molecules with (CD1e2
+
and CD1e4
+
) or without
(CD1e2
)
and CD1e4
)
) the propeptide, i.e. amino acids 20–
305 or 32–305 of the pre-a-chain of the corresponding vari-
ant, were expressed fused to the signal peptide of heat-
shock 70-KD protein 5 (HSPA5) and a C-terminal tag
including a tandem WSHPQFEK(streptag II)-His8 peptide
tag. In the case of CD1b, amino acids 17–300 of the pre-a-
chain were expressed by use of the same vector. The
a-chains were coexpressed in S2 cells with human b2m.
Recombinant proteins were purified by metal chelate chro-
matography followed by affinity purification on immobi-
lized Strep-Tactin (Qiagen, Courtaboeuf, France). Eluted
proteins were concentrated to 15 mgÆmL
)1
(0.32 mm)in
10 mm sodium phosphate buffer containing 150 mm NaCl
(NaCl ⁄ P
i
). Protein purity was checked with the Experion
system (BioRad, Marne la Coquette, France), and found to
be 95–99%, depending on the CD1 preparation. Purified
rsCD1e preparations were biologically active in vitro in

PtdInsM
6
digestion assays [5]. All CD1e variants and CD1b
were able to bind lipids in vitro [15] (this article and data not
shown). The concentrations of purified rsCD1 molecules
were determined by measurement of the absorbance at
280 nm, using extinction coefficients of 1.8 and 1.7 mgÆ
mL
)1
Æcm
)1
for rsCD1e and rsCD1b, respectively (protpa-
ram; Lipids
were purchased from Sigma (Saint Quentin Fallavier,
France) and Avanti Polar Lipids (Alabaster, AL, USA).
When necessary, stock protein solutions were diluted
directly in buffer at the desired pH, which was checked
with a microelectrode (Inlab 423; Mettler-Toledo GmbH,
Giessen, Germany). For reversibility measurements, small
quantities (1–10% of the volume) of disodium phosphate
(pH 9.9) or 100 mm NaOH were added to the solution to
obtain a pH of 7 or 4.8, and the results were corrected for
protein dilution.
Liposome preparation
Lipids solubilized in chloroform or chloroform ⁄ methanol
(2 : 1) were dried under a gentle stream of nitrogen, and
then placed under vacuum for at least 2 h to remove any
traces of the organic solvents. The thin lipid film was resus-
pended in 10 mm NaCl ⁄ P
i

(pH 7) by vortex mixing, and
hydrated for 1 h at room temperature (10 mgÆmL
)1
). Large
unilamellar vesicles were prepared with a Mini-extruder
(Avanti Polar Lipids), according to the manufacturer’s
instructions. Briefly, the lipid suspension was subjected to
seven freeze–thaw–vortex cycles, consisting of freezing on
dry ice for 10 min, immersion in a water bath at 37 °C for
10 min, and thorough vortexing of the sample. The suspen-
sion was then passed through a 100-nm-pore membrane at
room temperature, stored at 4 °C, and used within 1 week.
Liposomes of different composition were prepared: neu-
tral liposomes containing 100% PtdCho; negatively
charged liposomes containing 70% PtdCho and 30% Ptd-
Ser, PtdIns or sulfatide (w ⁄ w); and BMP-enriched lipo-
somes containing 70% PtdCho, 15% PtdSer, and 15%
BMP. The vesicle size was checked by electron microscopy
Structural changes underlying CD1e activity N. Bushmarina et al.
2030 FEBS Journal 278 (2011) 2022–2033 ª 2011 The Authors Journal compilation ª 2011 FEBS
(negative staining) and dynamic light scattering, and found
to be 100 ± 10 nm.
Fluorescence measurements (ANS and bis-ANS)
For measurement of the fluorescence of ANS or bis-ANS
(Invitrogen, Cergy-Pontoise, France) in the presence or
absence of protein, the concentration of ANS or bis-ANS
was determined from the absorbance at 350 or 390 nm,
with an extinction coefficient of 5000 or 16 790 m
)1
Æcm

)1
,
respectively. Measurements were performed with 200 lLof
reagents in 96-well polystyrene plates (Becton-Dickinson,
Meylan, France), using a FlexStation3 (Molecular Devices,
Saint Gre
´
goire, France). As this robot provides relative
data that cannot be directly compared from one day to
another, each comparison of conditions corresponds to a
set of experiments performed in triplicate on a same day.
The excitation and emission wavelengths were k
ex
=
370 nm and k
em
= 480 nm for ANS, and k
ex
= 390 nm
and k
em
= 490 nm for bis-ANS. In experiments with bis-
ANS, the fluorescence was corrected for the inner filter
effect, with the equation F
corr
= F
obs
antilog[(A
ex
=

A
em
) ⁄ 2], where F
corr
and F
obs
are the corrected and
observed fluorescence intensities at the emission and excita-
tion wavelengths, respectively [21]. Data were analyzed as
described previously [22]. Briefly, the measured fluorescence
of bis-ANS (F) was confirmed to be proportional to [bis-
ANS], when [bis-ANS] ⁄ [CD1e] < 1 (F = B · [bis-ANS]).
At higher [bis-ANS] ⁄ [CD1e] ratios, the number of bis-ANS
molecules bound to rsCD1e molecules was calculated to be
F ⁄ B. This allowed calculatation of r = [bis-ANS
bound] ⁄ [rsCD1e]. The Scatchard representation of r versus
r ⁄ [bis-ANS] = n ⁄ K
d
) r ⁄ K
d
, where n is the number of
binding sites and K
d
the apparent dissociation constant,
then enables determination of n and K
d
.
Fluorescence measurements (PtdSer–NBD)
To quantify the binding of PtdSer–NBD to CD1e molecules,
100 lg of acyl-labeled PtdSer–NBD (1-oleoyl-2-{12-[(7-

nitro-2-1,3-benzoxadiazole-4-yl)amino]lauroyl}-sn-glycero-3-
phosphoserine) (Avanti Polar Lipids) was dissolved in
100 lL of ethanol ⁄ binding buffer (150 mm NaCl, 10 mm
monosodium ⁄ disodium phosphate, pH 7 or 4.5) (50 : 50,
v ⁄ v), diluted in 1 mL of buffer, and sonicated for 10 min
(final PtdSer–NBD concentration of 200 lm). A 2-lL ali-
quot of diluted PtdSer–NBD was then added to 200 lLof
2 lm CD1e in binding buffer. To standardize the beginning
of the experiments, all measurements were started 12 s
after the addition of PtdSer–NBD to the protein solution.
The fluorescence was recorded in a PTI spectrofluorimeter
(PTI, Birmingham, NJ, USA), using a 1-cm path length
and 100-lL minimal volume quartz cuvettes, with slit
widths of 3 nm for excitation (k
ex
= 460 nm) and 4 nm for
emission (k
em
= 525 nm). The kinetic curves were fitted by
use of a two-phase association fitting curve (graphpad
prism). The fluorescence at 6000 s and the time required to
reach half this fluorescence intensity were deduced from
these curves.
Circular dichroism spectroscopy
Protein samples were diluted to 2 or 21 lm in the appropri-
ate buffer containing 0.15 m sodium sulfate and the indi-
cated concentration of guanidium chloride. Sodium
phosphate ⁄ citrate buffers (5 mm) and monosodium ⁄ disodi-
um phosphate buffers (5 mm) were used for the pH ranges
2.5–4.8 and 5–9, respectively. Samples were incubated over-

night at room temperature to permit the denaturation reac-
tion to reach equilibrium before measurement of the
circular dichroism. In the case of kinetic and reversibility
experiments, the incubation time is indicated. Spectra were
acquired with a Jasco J-810 spectropolarimeter (Jasco,
Tokyo, Japan) with a PTC-423S temperature controller and
a Peltier cell holder. The far-UV spectra were recorded with
a 0.02-cm path length rectangular ‘sandwich’ cuvette
(closed) or a 0.1-cm cuvette. The spectra were acquired with
a scan rate of 50–100 nmÆmin
)1
, a response time of 2–4 s,
and a step and band width of 1 nm, and were averaged
over three to five acquisitions. The near-UV spectra were
recorded at 20 °C with a 1-cm cuvette and a protein con-
centration of 21 lm. The spectra were acquired with a scan
rate of 50 nmÆmin
)1
, a response time of 1 s, a step of
0.2 nm, and a band width of 1 nm, and were averaged over
10 acquisitions. Buffer spectra were subtracted from the
sample spectra. The ellipticity was converted to the mean
residue weight ellipticity, using the path length of the cuv-
ette, the protein concentration, and mean residue weights
of 113 for rsCD1e and 112.6 for rsCD1b. The secondary
structure content was calculated with the program cdsstr
of the DichroWeb Site ( />html/home.shtml) [23], reference set SP175 [24].
Surface plasmon resonance
Surface plasmon resonance experiments were performed in
a Biacore 3000 system (GE Healthcare Biacore AB,

Uppsala, Sweden). An L1 chip was used for liposome
immobilization, and the running buffer was 25 mm mono-
sodium ⁄ disodium phosphate at pH 7 or 4.8, containing
150 mm NaCl. The chip was first washed with three
injections of isopropanol ⁄ NaOH solution (2 : 3, v ⁄ v) at
30 lLÆmin
)1
, and then rinsed thoroughly with buffer. Dif-
ferent liposome mixtures (2 lm) were injected separately at
1 lLÆmin
)1
through each of the four L1 chip flow cells,
until the surfaces were saturated (generally 20–30 min). A
resonance level of 7000–10 000 RU was usually achieved.
The chips covered with liposomes were then washed with
three 1-min pulses of 100 mm NaOH at 30 lLÆmin
)1
to
remove loosely bound lipids and stabilize the surfaces. The
N. Bushmarina et al. Structural changes underlying CD1e activity
FEBS Journal 278 (2011) 2022–2033 ª 2011 The Authors Journal compilation ª 2011 FEBS 2031
surface saturation was checked by injecting liposomes again
for 1 min at 1 lLÆmin
)1
. BSA (1 mgÆmL
)1
, pH 7; Eurome-
dex, Souffelweyersheim, France) was also injected to check
the chip saturation. Protein solutions at the indicated con-
centrations were then injected over the chips, allowing real-

time monitoring of the interaction of the proteins with lipo-
somes of different composition. The liposome surfaces were
regenerated with two 1-min injections of 100 mm NaOH.
Acknowledgements
This work was supported by the Etablissement Franc¸ -
ais du Sang (EFS)-Alsace, the Institut National de la
Sante
´
et de la Recherche Me
´
dicale (INSERM), the
Centre National de la Recherche Scientifique, and a
grant from the Agence Nationale de la Recherche
(ANR-05-MIME-006). N. Bushmarina was supported
by the Association de Recherche et de De
´
veloppement
en Me
´
decine et Sante
´
Publique (ARMESA). We thank
B. Lorber (UPR 9002, Cristallogene
`
se des Macromole
´
-
cules Biologiques, IBMC-CNRS) for making available
the Zetasizer Nano S apparatus and for his helpful
advice. We are also indebted to the INSERM U 949

team for valuable advice and Biacore training. We
thank J. Mulvihill for excellent editorial assistance.
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