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Originally published In Press as doi:10.1074/jbc.M404575200 on June 15, 2004

J. Biol. Chem., Vol. 279, Issue 34, 35320-35325, August 20, 2004
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The Importance of Lys-352 of Human Immunoglobulin E in Fc{epsilon}RII/CD23 Recognition*

Ian Sayers{ddagger}§, Jonathan E. M. Housden§, Alan C. Spivey||, and Birgit A. Helm¶**

From the Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, the {ddagger}Division of Therapeutics and Molecular Medicine, Queens Medical Centre, Nottingham NG7 2UH, and the ||Department of Chemistry, Imperial College, South Kensington Campus, London SW7 2AZ, United Kingdom

Received for publication, April 26, 2004 , and in revised form, June 14, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The interaction of immunoglobulin E (IgE) with its low affinity receptor (Fc{epsilon}RII/CD23) plays a central role in the initiation and regulation of type I hypersensitivity responses. We have previously identified the importance of amino acid residues in the A-B loop of the C{epsilon}3 domain of human IgE and implicated a region close to the glycosylation site at asparagine 371 as contributing to IgE-CD23 interaction. These residues were now targeted by site-directed mutagenesis. The IgE-CD23 interaction was assessed by semiquantitative flow cytometry. Replacement of the entire C{epsilon}3 A-B loop (residues 341–356) with the homologous rat IgE sequence resulted in complete loss of human CD23 recognition, as did replacement of residues 346–353, indicating that class-specific effector residue(s) are contained within these eight amino acids. Lysine 352 within the A-B loop was identified as contributing directly to human CD23 interaction. Mutation to the rodent homologue glycine or glutamate resulted in a significant reduction in binding compared with native IgE, whereas conservative substitution with arginine effected a small, but statistically significant, enhancement of CD23 binding. Mutation of the C{epsilon}3 glycosylation site at asparagine 371 to threonine or glutamine did not significantly affect CD23 recognition. Our results yield new insights into the structural basis of the hIgE-CD23 interaction and hold promise for the rational design of drugs that can manipulate IgE-mediated regulation of the allergic response.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies of the immunoglobulin E (IgE)1 isotype have a central role in the initiation and regulation of allergic disorders (1). IgE mediates these functions via the interaction of the Fc region of the molecule with its Fc receptors. The crystal structure of the high affinity receptor (Fc{epsilon}RI, KA ~1010 M–1) complexed to IgE-Fc has been determined, identifying critical residues involved in the interaction (2). Fc{epsilon}RII/CD23 has been identified as a low affinity receptor for human (h) IgE (KA ~107 M–1) (3). Structurally, CD23 is not a member of the Ig superfamily but a type II transmembrane receptor expressed on a variety of cells of the immune system (4). In the absence of a high resolution structure, a composite molecular model has been proposed based on homology of the lectin head with the rat mannose-binding protein (5, 6). The model predicts that five heptadic repeats of hydrophobic Leu/Ile residues form a trimeric {alpha}-helical stalk linking the N-terminal extracellular domain to the transmembrane and C-terminal cytoplasmic domain of the receptor (7).

Previous studies based on the interaction of hIgE-Fc with a soluble 16-kDa form of the lectin homology domain expressed in NS-O cells identified this region as containing the IgE binding site(s) (8). More recent studies indicate the necessity of the stalk region for IgE interaction (9). Using site-specific mutagenesis, the IgE binding region of CD23 has been mapped to two discontinuous segments between residues 165–190 and 224–256 (10). Although several other ligands for CD23 have been identified (11), the role of the receptor and receptor-derived fragments in the regulation of the IgE response has attracted particular interest (12, 13). A greater understanding of the IgE-CD23 interaction, based on the identification of complementary binding regions on each molecule, could assist the development of strategies for the manipulation of the allergic response via this regulatory mechanism.

The interaction of IgE with CD23 is species-specific. Human IgE does not bind to the rodent receptor, and rat and mouse IgE will not engage the human homologue, although rat IgE recognizes the mouse receptor (14). Previous studies have identified the hC{epsilon}3 domain as the site of interaction with CD23 (15, 16), although monoclonal antibodies that map to epitopes in the C{epsilon}2 and C{epsilon}4 domains have been described and are thought to inhibit receptor interaction by steric hindrance (15, 17). Our own studies (18) and those by Nissim et al. (16) highlighted the importance of residues in the A-B loop region of hC{epsilon}3 for binding to CD23.

The present investigation aimed to identify class-specific effector residue(s) in the A-B loop influencing the hIgE-CD23 interaction. The A-B loop was targeted by site-directed mutagenesis. Substitutions involved the introduction of highly conservative changes, e.g. Leu to Ile or Asp to Glu in order to minimize potential structural perturbations caused by the mutagenesis. Residues targeted were also exchanged for rodent homologues in order to investigate the species specificity of the CD23 interaction (19).

The use of a eukaryotic expression system (20) facilitated an investigation of the role of hIgE glycosylation. IgE is heavily glycosylated (12–15%), but only N-linked glycosylation has been detected to date. Glycosylation of IgE is not essential for CD23 recognition, in contrast with the interaction of this receptor with CD21 or CD11b/c, which involves carbohydrate residues (21, 22). The role of N-linked glycosylation in IgE is still unresolved, although it has been demonstrated that enzymatically deglycosylated IgE (PS) and hIgE-Fc fragments expressed in Escherichia coli engage both Fc receptors, apparently enhancing the affinity of the CD23 interaction by ~10-fold (15). To clarify the role of glycosylation, the solvent-exposed C{epsilon}3 glycosylation site at Asn-371 was targeted and assessed for a role in the folding, processing, and secretion of IgE variants in mammalian cells.

The functional consequences of site-specific mutagenesis were assessed by examining the binding of hIgE mutants to CD23 on the surface of the RPMI-8866 cell line employing a semiquantitative flow cytometry assay. We show that substitution of C{epsilon}3 A-B loop residues in hIgE with rodent homologues is associated with loss of human CD23 recognition. Site-directed mutagenesis of specific residues within the A-B loop highlighted Lys-352 as an effector determinant residue in the human IgE-CD23 interaction.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Gene Constructs and Site-directed Mutagenesis—Human epsilon ({epsilon}) gene variants were constructed by overlap extension mutagenesis PCR and subcloned into the mammalian expression vector pSV-VNP-H{epsilon} as described (20). The numbering scheme for the h{epsilon}-chain has been maintained as in previous publications (18). IgE variants were generated by transfection of expression vectors into the J558L cell line. Positive clones were selected, and IgE was purified from cell culture supernatant by affinity purification on NIP-CAP-Sepharose columns (19). All pSV-VNPH{epsilon} variants were sequenced (DyeDeoxy sequencing; Applied Biosystems, Warrington, UK) to ensure the desired mutation had been engineered into the {epsilon} gene and spurious errors had not been introduced during the PCR reaction. The IgE variants were quantified using the BIAcore biosensor (Amersham Biosciences) as described (19).

Assessment of Cell Surface Receptor Interactions—Binding of IgE to cell surface receptors was assessed using flow cytometry. Three cell lines expressing different IgE receptors were used in this investigation: RPMI-8866 cells expressing human CD23 (23), Chinese hamster ovary (CHO) cells transfected with mouse CD23 (Fc1.7 cells; Ref. 24) (a gift from D. Conrad), and rat basophilic leukemia (RBL) cells transfected with human Fc{epsilon}RI {alpha}-chain (25). Cells were maintained in culture as described previously (23–25). For RPMI-8866 cell analysis, cells were washed twice in wash buffer (phosphate-buffered saline, 1% fetal calf serum, 0.1% sodium azide) and resuspended at 5 x 106 cells/ml. 100 µl of cell suspension was added to 20 µl of recombinant IgE (0.125–5 µg). After incubation on ice for 30 min, cells were washed twice with 1 ml of wash buffer and then incubated with 100 µl of biotinylated anti-IgE (Vector Laboratories, Ltd., Peterborough, UK) diluted 1/400 in wash buffer. Following a 30-min incubation on ice, cells were washed and incubated on ice with 100 µl of streptavidin R-phycoerythrin (PE) (1/20 dilution) (Sigma) for 30 min. Before analysis by flow cytometry, cells were washed twice and resuspended in 200 µl of wash buffer. To determine background levels of fluorescence, phosphate-buffered saline or control antibody (mouse IgE SPE7; Sigma) was used in place of the recombinant IgE. Fc1.7 cells or RBL cells transfected with human Fc{epsilon}RI{alpha} cells were first detached from the culture dish using cell dissociation solution. Substitution of anti-IgE with biotinylated anti-{lambda} antibody (Amersham Biosciences) allowed direct comparison between mouse and recombinant human IgE binding using the Fc1.7 cells. Data were collected using a Coulter Etics Elite flow cytometer and analyzed using Datamate (Dako, Ely, Cambridgeshire, UK).

Data Analysis—Each assay was carried out using chimeric IgE with wild type (WT) human Fc{epsilon} sequence (Serotec, Kidlington, Oxford, UK) as a reference control. This commercial preparation of recombinant IgE was prepared using the pSV-VNPH{epsilon} expression plasmid and J558L host cell line, identical to that used in the current study. The median log channel number was used for the quantification of WT and variant IgE receptor binding interactions. The mean values from at least three independent experiments were calculated and compared by analysis of variance assay and two sample t tests. Statistical analysis was completed using Minitab (Minitab Inc., State College, PA); a p value <0.05 was considered significant. To facilitate discussion, results were expressed as percentages of WT binding (100%).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
IgE variants generated, together with the rationale explaining the mutations, are shown in Table I. The positions of C{epsilon}3 A-B loop residues within the crystal structure of IgE-Fc' are shown (Fig. 1) The interaction of IgE with cell surface high and low affinity Fc receptors was observed employing a semiquantitative flow cytometry assay. Measurement of fluorescence over a range of IgE concentrations allowed the construction of binding curves (Figs. 2 and 3) and comparison of IgE variants with wild type IgE (Table I). WT IgE binding to RPMI-8866 cells reached saturation at 2.5 µg/assay.


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  		TABLE I
Summary of IgE-CD23 interaction data

IgE variants generated along with the rationale for construction are listed together with an evaluation of the effect of receptor interaction. IgE binding was compared to WT using median channel numbers at 2.5 µg/assay and expressed as a percentage of the WT binding (designated 100%). The median channel numbers from three independent experiments were compared by t test. Fc{epsilon}RI interactions for some variants were not determined by flow cytometry (ND) but have previously been shown to be as wild type.

 



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  		FIG. 1.
The position of C{epsilon}3 A-B loop residues within IgE-Fc. Upper panel, solid three-dimensional rendering of IgE C{epsilon}3 and -4 in open conformation (2). C{epsilon}4 domains are shown a shade darker than C{epsilon}3 domains. A-B loop residues within C{epsilon}3 are highlighted close to the C{epsilon}4. Lower panel, arrangement of A-B loop residues within the closed conformation crystal structure (26). Other residues of the C{epsilon}3 and -4 domains are in solid rendering with C{epsilon}4 in darker gray. Residues 341–356 were targeted by mutagenesis. Lys-352, an exposed residue, was identified as a key effector residue of the human IgE-CD23 interaction.

 



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  		FIG. 2.
Interactions of human IgE variants with human CD23 and Fc{epsilon}RI. Cells bearing CD23 (RPMI-8866 cells) and cells bearing Fc{epsilon}RI{alpha} (transfected RBL) were harvested and washed three times in cold wash buffer (phosphate-buffered saline with 0.1% NaN3, 1% fetal calf serum) and then resuspended in the same at 5 x 106/ml. 100 µl of cell suspension was added to 20 µl of IgE (0.125–5 µg) incubated on ice for 30 min and then washed (2 x 1 ml of wash buffer). Cells were incubated with 100 µl of biotinylated anti-IgE antibody (1/400) for a further 30 min, washed as before, incubated for 30 min with 100 µl of streptavidin R-phycoerythrin (1/25 dilution), and washed again. Cells were resuspended in 200 µl of wash buffer and analyzed by flow cytometry. For a positive control, chimeric human IgE (Serotec) was used and the background fluorescence determined in the absence of IgE, using mouse IgE (SPE7) on RPMI-8866 cells or parental RBLs. Median channel numbers show the mean ± S.E. of three separate determinations.

 



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  		FIG. 3.
Interactions of human IgE variants with rodent CD23. CHO cells bearing mouse CD23 (Fc1.7 cells) were harvested and washed three times in cold wash buffer (phosphate-buffered saline with 0.1% NaN3, 1% fetal calf serum) and resuspended in the same at 5 x 106/ml. 100 µl of cell suspension was added to 20 µl of IgE (0.125–5 µg), incubated on ice for 30 min, and then washed (2 x 1 ml of wash buffer). Cells were incubated with 100 µl of biotinylated anti-{lambda} antibody (1/400) for a further 30 min, washed as before, incubated for 30 min with 100 µl of streptavidin R-phycoerythrin (1/25 dilution), and washed again. Cells were resuspended in 200 µl of wash buffer and analyzed by flow cytometry. For a positive control, mouse IgE (SPE7) was used and the background fluorescence determined in the absence of IgE. Control experiments using parental CHO cells are also shown. Median channel numbers show the mean ± S.E. of three separate determinations.

 
Replacement of the entire C{epsilon}3 A-B loop (residues 341–356) in the hIgE-Fc for the homologous rat IgE residues (variant R16) resulted in complete loss of hCD23 binding. Binding of the R16 variant was found to be not statistically different from the background level fluorescence (mean value 3% of wild type fluorescence at 2.5 µg of IgE) and significantly reduced compared with wild type binding (p = 0.02, Table I). Throughout this study, Fc{epsilon}RI interactions were assessed in parallel as an indicator of potential structural perturbations that might have occurred as a consequence of mutagenesis. Changes in Fc{epsilon}RI interaction observed for this variant (Fig. 2A) were not statistically different from the wild type molecule (88.7%, p = 0.13, Table I).

The R16 variant was constructed prior to crystal structure determination of receptor-bound IgE. When the structural coordinates for IgE/Fc{epsilon}RI became available (2), it could not be ruled out that mutation of residues within the {beta}-strands of the immunoglobulin fold (Ser-341, Arg-342, Thr-355, and Ile-356) could induce significant structural perturbations affecting receptor recognition. To address this, a further IgE variant (R8) was generated with the more limited substitution of residues 346–353 of the C{epsilon}3 A-B loop with the homologous rat residues. The binding characteristics of the R8 variant confirmed earlier results obtained with the R16 chimera: the interaction with CD23 was completely inhibited (97% reduction (versus WT, p = 0.02), identical to background, p = 0.08). The Fc{epsilon}RI interaction (Fig. 2A) was modestly reduced with a marginal statistical significance for this variant (91%, p = 0.03, Table I).

Previous observations suggested Lys-352 within the A-B loop may be a key effector residue, with mutation to Gly, the homologous residue in rodent IgE, resulting in a considerable reduction in receptor interaction (50% decrease (versus WT, p = 0.0007), Table I) (11). We interpret the effect of this substitution as identifying Lys-352 as a class-specific effector residue contributing directly to the binding of hIgE to CD23, possibly facilitating docking via an electrostatic interaction. The elimination of this interaction appeared to account, at least in part, for the observed loss of CD23 binding associated with the R16 and R8 variants, which contained this substitution. Of all the A-B loop variants generated within this study, the most prominent change in the IgE-CD23 interaction was observed for the K352G substitution. Point mutations involving D347N, D347E, L348I, or R351K had no significant effect on the IgE-CD23 interaction (Table I).

Mutation of Pro-345 to Ala resulted in an apparent increase in CD23 binding compared with the wild type molecule, although statistical analysis shows this is of marginal significance (12.5% increase, p = 0.09, Table I). The molecular basis of this observation is currently uncertain but could be because of a potential role of Pro-345 in the restriction of structure and maintenance of the rigid conformation of the A-B loop. An increase in loop flexibility may account for the apparent enhancement of the docking process of hIgE and CD23, at the same time suggesting that the Pro-345 residue is not critical for the overall maintenance of the A-B loop CD23 binding conformation.

To substantiate the identification of Lys-352 within the A-B loop as a key effector residue involved in the IgE-CD23 interaction, further point mutations were generated (Fig. 2B). Conservative substitution of Lys-352 with Arg resulted in a small, but statistically significant, increase in the CD23 receptor interaction (14% increase, p = 0.0006). Substitution with the negatively charged Glu residue effected the complete loss of CD23 receptor interaction (98% decrease (versus WT, p = 0.0019), not significantly different to background, p = 0.61, Table I).

These results substantiate a role for residue Lys-352 in the interaction of hIgE with CD23 and suggest the possibility of an electrostatic interaction between Lys-352 of IgE and the receptor. Mutation of Lys-352 to Arg or Glu maintained the Fc{epsilon}RI interaction, and a binding level similar to the wild type molecule was observed (Table I).

Variant R16/K352 (Lys-352 of hIgE restored within the R16 variant) was generated to evaluate the contribution of residue 352 to the loss of interaction seen with replacement of the entire A-B loop. This variant showed a limited but significant degree of restoration of CD23 recognition (binding level 39% (versus WT, p = 0.043), significantly different from background, p = 0.025, Table I). The specificity of the interaction was confirmed by the finding that this interaction could be inhibited using anti-CD23 monoclonal antibody (MHM6) (data not shown), which is known to inhibit hIgE/CD23 binding. As observed for the R16 variant, the Fc{epsilon}RI binding levels for the R16/K352 were similar to WT binding, supporting a localized effect of mutagenesis (Table I, Fig. 2).

To evaluate whether substitution with homologous rat A-B residues conferred the ability to interact with the rodent receptor, IgE variants were screened by flow cytometry analysis using CHO cells transfected with mouse CD23 (Fc1.7 cells).

Neither the R16 chimera or hIgE-Gly-352 variants conferred binding to rodent CD23; however, the R8 variant was found to have an increased interaction with Fc1.7 cells (20% compared with WT mouse IgE; Fig. 3). Although these data indicated that the R8 variant was able to interact with rodent CD23, further analysis showed that this interaction was observed with parental CHO cells (Fig. 3), indicating that this interaction was not specific to CD23.

Two additional variants were generated to assess the effect of Asn-371-linked glycosylation on the IgE-CD23 interaction (Fig. 2C). Thr-371 and Gln-371 aglycosylated variants both demonstrated a modest decrease in CD23 binding (84%, p = 0.024, and 88%, p = 0.15, respectively, Table I) indicating that enhanced binding reported for deglycosylated IgE cannot be attributed to a lack of glycosylation at this residue.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have confirmed the role of the C{epsilon}3 A-B loop of human IgE in the IgE-CD23 interaction and identify Lys-352 as an important determinant in CD23 receptor recognition, although the scope of flow cytometry is limited by the fact that it does not yield kinetic data for the interaction studied. These data are in agreement with our previous study (18) and the study of Nissim et al., who replaced residues within the N-terminal of the hIgE C{epsilon}3 domain with the homologous mouse residues (16). Replacement of residues 330–346 (variant C3BX) maintained the interaction. However, replacement of residues 330–356 (variant C3HD) dramatically reduced activity, suggesting a role for this region in the CD23 interaction (16). It is unlikely that the substitutions generated within the R16 variant have effected the overall C{epsilon}3 domain conformation with concomitant loss of CD23 recognition because Fc{epsilon}RI interaction, which is dependent on residues in hC{epsilon}3, was fully maintained in this study (Table I, Fig. 2) and in a previous study that used surface plasmon resonance to assess the interaction of IgE variants with soluble truncated Fc{epsilon}RI{alpha} (19).

A significant role for the A-B loop in IgE function has previously been proposed by Wurzburg et al. (26). Based on crystal structures of IgE-Fc fragments, Wurzburg et al. describe how interactions of A-B loop residues facilitate transition between an open conformation when bound to Fc{epsilon}RI and a closed conformation when uncomplexed. Wurzburg proposes that this conformational flexibility may allow IgE to form optimal interactions with both Fc{epsilon}RI and CD23.

It could be argued that substitution with homologous rat residues in the R16 and R8 constructs has effectively constricted domain movement and locked the molecule in a non-binding conformation with respect to CD23. However, maintenance of normal Fc{epsilon}RI interactions would argue in favor of a functionally active molecule. In addition, the absence of noteworthy contacts made by Lys-352 within the available IgE structures and the considerable loss of receptor interaction observed with the Gly-352 variant point to direct involvement in the interaction with CD23. We propose therefore that residues within CD23 bind directly to Lys-352 in hIgE and probably involve electrostatic interaction and concomitant conformational changes to provide optimal docking.

The IgE-CD23 interaction has been proposed to have a 2:1 binding stoichiometry, with two lectin heads of the trimeric receptor interacting with the two {epsilon}-chains of an individual IgE molecule (27), and dimerization of the hIgE molecule is required (15). Dual kinetics have been reported for IgE binding to CD23 (24). The most straightforward explanation for this phenomenon in the context of this model is that one lectin head would initially dock with low affinity, providing the basis for a second interaction of higher affinity. This could involve two sets of distinct interactions, possibly involving different points of contact in the IgE molecule and/or within the receptor. The IgE binding site on CD23 has been mapped to two discontinuous segments between residues 165–190 and 224–256 (10). Our observation demonstrating that mutation of a single residue has a profound effect on receptor recognition suggests that Lys-352 makes a definitive contribution, either by establishing an initial point in the docking with CD23 and/or by contributing to the stability of the interaction. Although these data strongly suggest a dominant role for Lys-352 (e.g. mutation to Glu resulted in an equivalent loss of CD23 binding as observed for the replacement of the entire A-B loop in the R16 and R8 variants), we cannot exclude the contribution of other residues in the A-B loop, including residues at positions 346, 349, and 353 that were not evaluated directly in the current study.

Abrogation of the higher affinity interaction using an anti-CD23 stalk region antibody has been shown to result in cells that appear negative for IgE binding as determined by flow cytometry, whereas low affinity binding is still detectable using 125I-labeled IgE (28). The use of flow cytometry to assess IgE-CD23 interaction does not allow us to conclude whether loss of binding observed with mutants in the present study results from abrogation of one or both of these interactions.

The localization of Asn-371 on the same face as Lys-352 and our preliminary data regarding the interaction of mutants inhibiting glycosylation led us to propose a role of glycosylation at Asn-371 in the inhibition of hIgE-CD23 interaction. Our present study shows, however, that Asn-371 glycosylation is not involved in this process. Others have suggested that glycosylation of the C{epsilon}2 domain accounts for this effect. Deglycosylation of IgE has been shown to expose epitopes on the C{epsilon}2 domain (29), and a role for Asn-265 has been postulated (30) to which our results would lend some tentative support.

At present, limited structural information is available concerning the interaction of hIgE and CD23. In the absence of high resolution structural data, we identify key regions of the hIgE molecule involved in CD23 recognition that provide new insights into the structural nature of this interaction. The A-B loop in C{epsilon}3 was identified as a critical region for the interaction, with Lys-352 as a class-specific residue. Acquiring information concerning the structure of complementary binding sites on IgE and its receptors holds potential for the rational design of drugs that could modulate the allergic response.


   FOOTNOTES
 
* This work was supported by grants from the Biotechnology and Biological Sciences Research Council, the Medical Research Council, and The Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

** To whom correspondence should be addressed. Tel.: 44-114-2824375; Fax: 44-114-2795495; E-mail: B.Helm{at}sheffield.ac.uk.

1 The abbreviations used are: IgE, immunoglobulin E; hIgE, human IgE; CHO, Chinese hamster ovary; RBL, rat basophilic leukemia; WT, wild type. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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