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D-enantiomer peptide of the TCR transmembrane domain inhibits T-cell activation in vitro and in vivo

Doron Gerber^*,1, Francisco J. Quintana^,1, Itai Bloch^*, Irun R. Cohen and Yechiel Shai^*,2

^* Department of Biological Chemistry and
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel

² Correspondence: Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: Yechiel.Shai{at}weizmann.ac.il

SPECIFIC AIMS

Intermolecular interactions are sterically constrained. Accordingly, sequence-specific interactions between D and L-stereoisomers were ruled out. This teaching was challenged recently in studies of intramembrane protein assembly. Here, we studied the ability of a TCR transmembrane domain (TMD) D-stereoisomer peptide (D-CP), compared with the L-stereoisomer peptide (L-CP), to interact with native intramembrane protein domains and inhibit T-cell activation.

PRINCIPAL FINDINGS

1. D-CP is a structural mirror image of L-CP
To test the role of chirality in the recognition process of the TCR transmembrane domain (TMD), we chemically synthesized three CP peptides: wild-type L-CP, which has been shown to inhibit antigen-specific T cell activation; D-CP, which is a mirror image of the first; and 2G CP, an inactive mutated peptide.

Circular dichroism experiments were performed to ensure that the secondary structure of the D-CP was indeed a mirror image of the L-CP. Experiments were performed in a zwitterionic detergent (1% LPC in H₂O) to simulate a membrane environment. The spectrum of the D-CP was found to be the exact mirror image of L-CP (Fig. 1 ); both are partially helical. The two peptides are 9 aa long, hence their structure is likely to be less stable than that in the context of the full-length protein.

View larger version (18K): [in this window] [in a new window]   Figure 1. L-CP and D-CP peptides manifest mirror image structures. Far-UV circular dichroism spectra of L-CP () and D-CP () were collected in a membrane mimetic environment (1% lysophosphatitylcholine). The structures are not canonical -helices, as can be expected for such short peptides (a population with random coil conformation is likely). However, the spectra of the L-CP and D-CP are exact mirror images.

2. Both D-CP and L-CP inhibit T-cell activation
We studied the T-cell response of lymph node cells (LNC) from rats immunized with Mycobacterium tuberculosis (Mt) or of T clone A2b to the PPD antigen or to the Mt176-90 peptide; the T-cell responses to these antigens are known to be involved in causing adjuvant arthritis (AA) in rats. We found that both L-CP and D-CP inhibited the T-cell proliferative responses to PPD and to Mt176-90 in a dose-dependent manner. The inhibition mediated by D-CP was consistently higher than that of L-CP at the lower concentrations. The inhibition was specific; the control 2G CP caused no inhibitory effect. This suggests that critical molecular interactions were mediated by the two positive charges by which both L-CP and D-CP differ from 2G CP. L-CP, D-CP, and 2G CP manifested no cytotoxicity, excluding the possible role of toxicity as a cause for inhibition.

3. Both D-CP and L-CP inhibit T-cell-mediated inflammation in vivo
To test the inhibitory effects of CP on the activation of specific T cells in vivo, we used the AA model. Immunization of Lewis rats with Mt in oil triggers AA, an experimental autoimmune disease driven by Mt-specific T cells cross-reactive with self-antigens. We found that administering D-CP or L-CP with the Mt antigen at the time of AA induction led to a significantly milder arthritis, in terms of clinical score (Fig. 2 ) and of ankle swelling. The control peptide 2G CP did not inhibit AA. The mean maximum score was 12 ± 0.3 in the control-treated rats, compared with 6 ± 0.7 in the D-CP-treated rats and 7.3 ± 0.7 in the L-CP-treated rats (P<0.05 for the L and D-CP groups compared with control groups).

View larger version (14K): [in this window] [in a new window]   Figure 2. Inhibition of T cell activation by L-CP and D-CP. AA was induced by immunization to Mt in oil, mixed with L-CP, D-CP, 2G CP, or PBS (6 rats per group). Arthritis was scored every 2–3 days, starting at day 10. A) Time course of the AA disease. B) Leg swelling scores measured at day 26 after AA induction. The results are presented as the mean ± SE of the difference between the values for hind limb diameter taken at days 0 and 26. The presence of either L-CP or D-CP significantly reduced the severity of AA compared with the control groups (P<0.05).

The activity of the T cells that mediate AA can also be detected in vivo by studying the delayed type hypersensitivity (DTH) response to PPD. Administration of D-CP reduced DTH by 48%; L-CP produced only a 39% reduction in the DTH response; and inhibition by treatment with the 2G CP peptide was < 10%. Thus, in vivo, as in vitro, the effect of D-CP was greater than that of L-CP.

4. Colocalization of L-CP and D-CP with the TCR
The CP peptides are thought to block T-cell activation by uncoupling signaling between the TCR and the CD3 complex. To test this idea, we incubated the T cells with FITC-labeled antibodies to the TCR and either L-CP or D-CP labeled with rhodamine. The labeled T cells demonstrated the capping of the TCR, characteristic of responding T cells. There was almost complete overlap between TCR and either L-CP or D-CP labeled peptides. These results suggest that the CP analogs bind to the T-cell membrane and colocalize with the TCR within the capping regions. The labels themselves did not affect TCR localization, since similar results were obtained with a different set of labels (data not shown).

The colocalization results were confirmed in a series of bleaching experiments that demonstrated fluorescence energy transfer between the CP peptides and the TCR. We bleached a point on the membrane of the cells, which exhibited high intensity of both rhodamine and FITC, using the 543 nm laser. The CP rhodamine-labeled peptides were bleached while the TCR-FITC was unaffected. This procedure resulted in a significant increase in the fluorescence of the TCR-FITC. Thus, we could conclude that fluorescence energy transfer occurs between the TCR and the L-CP or D-CP peptides.

CONCLUSIONS AND SIGNIFICANCE

These findings demonstrate that D-CP can inhibit T-cell function in vivo and in vitro, at least as well as L-CP. These CP molecules function by interacting with the transmembrane domains (TMD) of the CD3 complex to uncouple signal transduction between the TCR and CD3 molecules. Thus, a structural adaptation of D-CP probably compensates for the change in aa chirality and allows interactions with L molecules. Nevertheless, the double mutant 2G CP demonstrated that the mechanism of action of the D-CP is sequence specific.

We propose that the structural adaptation of TMD is a general phenomenon that can be accounted for by a common mechanism; the rotation of the tilt angle may compensate for the replacement of an L chain with a D chain. The TCR-CD3 complex has an intricate network of TMD interactions. The model in Fig. 3 A describes the main TMD interactions between TCR and CD3 ,. The TCR TMD point outward while the two CD3 TMDs point inward in a classic dimer orientation, as found for example in the glycophorin A homodimer. Salt bridges form between the positive charges within the TCR TMD and the negative charges within the CD3 TMDs. When we replace TCR with its D enantiomer, an adjustment of the tilt angle is required to allow the formation of these salt bridges (Fig. 3B ). This model demonstrates that adjusting the tilt angle can compensate for replacing a TMD with its mirror image enantiomer, even in a multi-TMD complex.

View larger version (35K): [in this window] [in a new window]   Figure 3. A general model for the assembly of an L helix with its D enantiomer. The TCR complex consists of several TMDs; nevertheless, it seems to follow the same rules as for simple homodimer forming TMDs. The TCR is known to interact with CD3 and CD3 through salt bridges. A) Model of the 3 -helices, oriented in a manner that allows the formation of salt bridges. The L TCR helix is tilted with respect to the CD3 helices. B) Tests of our hypothesis; we replaced the L TCR with D TCR. We reoriented the D TCR helix by changing the tilt angle until the Arg and Lys side chains were placed at similar positions in space as the original L TCR side chains. The direction of movement is illustrated by the black arrows.

The energy barrier associated with shifting the angle between two TMDs may be extensive. However, it is reasonable to assume that once two helices pass this barrier they will remain in contact. Thus, the assembly of TMDs with opposite chirality may differ from the wild-type L homodimer in kinetics. Short TMD peptides are not restricted by any intra- or extracellular interactions. Thus, D-CP may be able to compete with the wild-type TCR on its interaction with the CD3 TMDs. In turn, this interaction uncouples the physical transmission of signal from the TCR (ligand binding-extracellular) to the CD3 (signaling unit-intracellular).

In this work we demonstrate that a D peptide corresponding to the TCR TMD can interfere with the TCR complex in a sequence specific manner and interfere with T-cell activation. The sequence-specific interaction between D and L polypeptides seems to apply to several membrane complexes, including both prokaryotic (the E. coli aspartate receptor) and eukaryotic proteins (GPA and TCR). This phenomenon may serve as a useful tool for investigating the physical transfer of signals through the cell membrane. Peptide displacement strategies based on the use of D peptides may allow the formulation of immunotherapeutic peptides with increased resistance to protease degradation useful clinically for inhibiting T cell activation in immune-mediated disorders.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3498fje; doi: 10.1096/fj.04-3498fje

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