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: henri.delasalle@efs-alsace.fr; natalia.bushmarina@gmail.com (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; http://www.expasy.ch/tools/protparam.html). 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 (http://dichroweb.cryst.bbk.ac.uk/ 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. 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B, Hanau D & de La Salle H (2000) Characterization of CD1e, a third type of CD1 molecule expressed in dendritic cells J Biol Chem 275, 37757–37764 3 Maitre B, Angenieux C, Wurtz V, Layre E, Gilleron M, Collmann A, Mariotti S, Mori L, Fricker D, Cazenave JP et al (2009) The assembly of CD1e is controlled by an N-terminal propeptide which is processed in endosomal compartments Biochem J 419, 661–668 . 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. 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. 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