Colocalization of ULBP1, 2, and 3 and MICA with Cross-linked Lipid Rafts.
To study the distribution of ULBP1, 2, and 3 and MICA with respect to lipid rafts on Daudi/Class I+ transfectants, we cross-linked the lipid raft resident GM1 ganglioside with CT-B to create distinct patches visible by fluorescence microscopy ( a). After patching, the cells were fixed and stained to show the distribution of NKG2D ligands. As negative controls, we looked at the distribution of CD45 and TfR that do not accumulate in GM1 patches. Strikingly, the majority of ULBP and MICA staining ( a, red) accumulated at the same areas on the cell surface as the GM1 patches ( a, green), whereas CD45 remained even around the plasma membrane.
Figure 1. NKG2D ligands colocalize with cross-linked patches of CT-B in Daudi transfectants. (a) After cross-linking GM1, Daudi/Class I+ cells transfected with ULBP1, 2, 3, or MICA were fixed and stained for CT-B (second column) and the appropriate NKG2D ligand (more ...)
We developed software to calculate the correlation coefficient between the intensity corresponding to CT-B and each of the NKG2D ligands, similar to a method described previously (21
). A practical upper limit of the calculated correlation coefficient was obtained by targeting anti–CT-B with two different secondary antibodies. The distribution of calculated correlation coefficients was quite broad. Nevertheless, our analysis shows clearly that ULBP and MICA colocalize within the GM1 patches ( b). Thus, ULBP and MICA are preferentially concentrated in glycosphingolipid-rich lipid rafts.
ULBP3 But Not MICA Associates with DRMs.
GPI-linked proteins have a propensity for robust association with biochemically isolated DRMs as a result of their Triton X-100 insolubility at 4°C and their low density (22
). At the same time, other membrane proteins, such as TfR, do not copurify with DRMs (23
). Thus, we set out to investigate whether ULBP3 and MICA associate with DRMs; the mAbs for ULBP1 and ULBP2 were found to be unsuitable for Western blotting.
DRMs were isolated by fractionating cell lysates and analyzed by Western blotting. HRP-conjugated CT-B demonstrated that GM1 ganglioside accumulated in DRMs as expected (, a and b). Western blotting revealed that ULBP3 accumulated in the DRM fractions from both Daudi/Class I+/ULBP3 transfectants and Namalwa, a B cell line endogenously expressing ULBP3 ( a). Thus, ULBP3 is constitutively associated with DRMs.
Figure 2. Probing the cell surface organization of NKG2D ligands in DRMs and by electron microscopy. DRMs were prepared from (a) Daudi/Class I+/ULBP3 and Namalwa or (b) Daudi/Class I+/MICA cells. 10 fractions were collected from the top of the gradient (fraction (more ...)
MICA was absent from the DRM fractions as were TfR and MHC class I proteins ( b). Thus, although MICA clearly is expressed in particular membrane microdomains (), it may have a weaker association to lipid rafts that is not detected by the stringent technique used to isolate DRMs. This is reminiscent of T cell receptor colocalizing with patches of cell surface GM1, yet not routinely in biochemically isolated DRMs (25
It may be important to note that Western blotting of gels in reducing conditions identified MICA with an apparent molecular weight approximately twice that predicted by its amino acid sequence. By cleaving N-linked oligosaccharides in lysates of cells expressing MICA, a band corresponding to a molecular mass of ~45 kD can be detected by Western blotting (unpublished data). Thus the high apparent molecular weight of MICA in b is likely due to extensive glycosylation.
Electron Microscopy Reveals Clusters of ULBP at the Cell Surface.
For immunoelectron microscopy, cells were fixed rapidly at 5°C, cryoprotected, and snap frozen in liquid nitrogen; therefore, movement and redistribution during subsequent labeling procedures should not occur. CD45 was distributed evenly over the cell surface ( c). However, immunogold labels for ULBP1 tended to concentrate in distinct clusters of 2–5 particles ( d). The cell surface between these clusters was free of label, suggesting that ULBP1 is expressed in distinct “islands” within the plasma membrane. ULBP-rich membrane domains spanned ~30–100 nm, which is consistent with estimates of the size of lipid rafts. Similar observations were seen for ULBP3 (unpublished data). This suggests that ULBPs are constitutively expressed in lipid rafts, and not just upon cross-linking GM1 as shown in .
Accumulation of ULBP3, MICA, and Target Cell Lipid Rafts at the Activating NK Cell Immune Synapse.
To assess the distribution of NKG2D ligands at human NK cell immune synapses (26
), NK cells were coincubated with Daudi/Class I+
/MICA for 15 min before conjugates were fixed and stained. The peripheral blood NK cell line efficiently lysed Daudi/Class I+
/MICA (unpublished data). MICA was found to accumulate at 50% of intercellular contacts (n
= 301; a) and was even seen to accumulate at two immune synapses formed by a single NK cell ( a, first row). Thus, MICA accumulates at activating NK cell immune synapses.
Figure 3. Recruitment of ULBP3 and MICA to the NK cell immune synapse. (a) CD56+ CD3− human peripheral blood NK cells were coincubated with Daudi/Class I+/MICA cells for 15 min, fixed, and stained with anti-MICA mAb, and the distribution at the immunological (more ...)
To assess the role of target cell lipid rafts at the activating NK cell immune synapse, we transfected Namalwa with a construct encoding GPI-anchored GFP (GPI-GFP). Western blotting of DRMs confirmed that this transfectant expressed GFP in the GM1-rich membrane compartment (unpublished data). We imaged conjugates formed between this transfectant and an NK clone that efficiently lysed Namalwa. b clearly demonstrates the simultaneous accumulation of ULBP3 and GPI-GFP (i.e., target cell lipid rafts) at the activating NK cell immune synapse.
Due to the lower level of expression of ULBP3 in this cell line compared with the Daudi transfectants, it was difficult to image ULBP3 at these synapses. Nevertheless, even though we could clearly see accumulation of ULBP3 at the immune synapse in only 25% of conjugates, in nearly all of these cases (13 out of 14), GPI-GFP simultaneously accumulated at the synapse. Thus, these data provide the first evidence that target cell lipid rafts containing NKG2D ligands are important in NK cell activation.
MICA Is S-acylated.
Many proteins that associate with lipid rafts are attached to saturated lipid groups likely to prefer the ordered environment of rafts. S-acylated proteins are often targeted to lipid rafts; however, one exception is TfR. Thus, we set out to determine if MICA is lipid modified. The fact that MICA contains a dicysteine motif at positions 331 and 332 juxtaposed to its putative transmembrane sequence suggested that the protein might be S-acylated. To test this, MICA was immunoprecipitated from lysates of cells preincubated with radiolabeled palmitic acid. Immunoprecipitated MICA was radiolabeled, demonstrating that MICA is indeed S-acylated ( a).
Figure 4. MICA is S-acylated, and a truncated form of MICA is unable to activate NK cells. (a) Daudi/Class I+/MICA cells were labeled for 5 h with either [35S]methionine (35S MET) or with [3H]palmitate (3H PAL), lysed, immunoprecipitated with anti-MICA or anti-TfR (more ...)
A Truncated Form of MICA Is Unable to Activate NK Cells.
Toward demonstrating functional significance for the transmembrane and/or cytoplasmic portions of MICA, we generated a transfectant of Daudi expressing a truncated form of MICA in which the cysteine at position 331 was replaced with a stop codon (Daudi/Class I+
/MICA/C331*). The level of cell surface staining of this transfectant with a MICA mAb was similar to that of wild-type MICA in Daudi/Class I+
/MICA (). We tested the susceptibility of these transfectants to NK cell cytotoxicity ( d). A peripheral blood NK cell line was efficiently able to kill untransfected Daudi, which lacks endogenous expression of MHC class I protein, but was inhibited from killing Daudi-expressing MHC class I protein (Daudi/Class I+
). In agreement with previous data (5
), Daudi-expressing MHC class I protein and MICA was efficiently killed, demonstrating how activation via NKG2D recognition can overcome inhibitory signaling. However, Daudi transfectants expressing MHC class I protein and the truncated form of MICA were not efficiently killed. Thus, removal of the putative site of S-acylation and the cytoplasmic tail of MICA abrogates cytolysis by human NK cells. From this first study, it is possible that either S-acylation or another, currently unknown, function of the cytoplasmic portion of MICA could account for the loss of function of the truncated MICA. Thus, we are currently establishing and testing the effect of numerous point mutations in MICA to determine precisely which amino acid residues control the localization and function of MICA.
In summary, we demonstrated by fluorescence imaging, Western blotting of DRMs, and electron microscopy that the stress-inducible ligands ULBP1, 2, 3, and MICA are expressed in specific membrane microdomains ( and ). In addition, we show that these proteins accumulate at activating NK cell immune synapses (). The molecular mechanism by which these proteins are organized at the cell surface likely involves attachment to specific lipids, which may be important in triggering NK immune responses (). It has been demonstrated previously that viruses can prevent cell surface expression of NKG2D ligands to evade NK cell activation. In light of our analysis, it might be interesting to investigate if some viruses or tumors have evolved mechanisms to subvert NK activation by disrupting the cell surface organization of NKG2D ligands.