Specificity Analysis of S25-Nb10 and stx-Nb6

Although both nanobodies were shown to bind their target with a high affinity using purified antigen, it also needed to be confirmed that they do not cross-react with other proteins on cells. First, I tested if the nanobodies also bind to other SNARE proteins of the same family. Equimolar amounts of native recombinant protein (purchased from Origene™ Technologies, see also section 2.1.5) were spotted on a nitrocellulose membrane without adding reducing or denaturating agents (see Figure 21). Three members of the SNAP and syntaxin protein family were chosen out of which two variants share the highest level of conservation with SNAP-25 and syntaxin 1A.

After detection with my fluorescently labeled nanobodies, I could confirm that S25-Nb10 detected SNAP-25 but not the related proteins SNAP-23, SNAP-29 and SNAP-47 as sown in Figure 21. Similarly, stx-Nb6 bound to syntaxin 1A as expected, but not to syntaxin 1B and syntaxin 2. Notably, the nanobody seems to have a weak affinity to syntaxin 3, which shares a certain sequence identity with syntaxin 1A. The observed binding rate to syntaxin 3 was in the range of 7 % compared to the syntaxin 1A binding and therefore can be considered as a negligible interaction, especially in IF methods for microscopy.

After confirming the binding specificity under native conditions, I tested if the nanobodies are also capable of detecting the antigen in a Western blot after denaturating SDS-PAGE. As nanobodies have the tendency to prefer structural epitopes (refer to section 1.4.4), they might not detect a denatured form of the antigen [51]. To my surprise, both S25-Nb10 and stx-Nb6 were able to detect their antigen also after denaturation and transfer to a membrane.

This feature was used to test if the nanobodies are specific for protein lysates and tissue samples not tested during the validation. For this, I performed a Western blot using neuronal and cell-line lysates as well as tissue samples isolated from an adult mouse. Equal amounts of total protein were loaded on a denaturing SDS-PAGE gel and blotted on a nitrocellulose membrane for detection with fluorescently labeled nanobody. As shown in Figure 20, both S25-Nb10 and stx-Nb6 did not reveal any bands in tissue samples of cell lysates lacking the expression of SNAP-25 and syntaxin 1A, respectively. I therefore conclude that my novel nanobodies S25-Nb10 and stx-Nb6 bind their antigens with both high affinity and high specificity also in complex protein lysates even after separation with denaturating SDS-PAGE.

To confirm the specificity of my nanobodies in IF microscopy, I once more used COS-7 cells expressing the antigen fused to EGFP detecting it with directly labeled nanobody. I used cells transfected with the contrary EGFP fusion construct to rule out binding of the nanobody to EGFP, which has not been tested so far.

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Figure 20: Specificity analysis of S25-Nb10 and stx-Nb6 by Western blotting. A: 5 µg of purified recombinant protein, 20 µg of HEK-cell lysate containing overexpressed antigen an 20 µg lysate from primary cultured neurons were loaded on a denaturing SDS-PAGE gel and subsequently blotted onto a nitrocellulose membrane.

Protein concentration was determined using BCA-assay. B: 20 µg of isolated tissue sample (determined via BCA assay) were loaded on a denaturing SDS-PAGE gel and blotted as previously. After blocking, all membranes were incubated with nanobodies conjugated to atto647N for detection of the target antigen. Since recombinant proteins expressed in bacteria or mammalian cell lines possess additional tags, the expected molecular weight differs from the native protein as indicated in the schematic representations of the antigens.

Both S25-Nb10 and stx-Nb6 show a single band in the blot indicating specific detection of their respective target antigen while showing no cross-reactivity to other proteins in lysates or tissue samples.

Figure 22 shows that both nanobodies specifically detect cells transfected with their target antigen but virtually show no background signal in non-transfected cells or expressing the complementary construct. This also confirms that the nanobodies bind to the overexpressed antigen and not the bind to EGFP also overexpressed in the cells. This is an important control as EGFP was used as a common reporter also in further experiments conducted in this project. In addition to the specific binding of the nanobodies, Figure 22 shows a different cellular localization of SNAP-25 and syntaxin 1A.

Whereas SNAP-25 is distributed all over the cell, the overexpressed syntaxin 1A accumulates in a perinuclear region. Previous studies showed that syntaxin 1A accumulates in the endoplasmatic reticulum (ER) region if cofactors such as Munc-18 are not present [160,161]. This would explain the phenotype observed in cells overexpressing only syntaxin 1A fused to EGFP as in Figure 22.

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Figure 21: Analysis of cross-reactivity to alternative SNAP and syntaxin variants and sequence alignment.

Equimolar amount of purified protein were spotted on a nitrocellulose membrane and detected with fluorescently labeled nanobodies. A: S25-Nb10 binds specifically to SNAP-25 and only shows minimal cross-reactivity to SNAP-47 and no binding to SNAP-23 and SNAP-29. B: stx-Nb6 specifically detects syntaxin 1A and additionally shows a mild cross-reactivity for syntaxin3. However, as the signal for syntaxin3 made only around 7 % of the specific signal, this background binding can presumably be neglected in IF experiments. The stx-Nb6 also shows only minimal binding to syntaxin 1B and syntaxin 2, which can be neglected in IF microscopy.

C: Sequence alignment of the tested SNAP- (top) and syntaxin-variants (bottom). The level of conservation is color-coded while green indicates the highest level of sequence identity. The alignment shows that the different SNAP variant share only little sequence identity whereas the tested syntaxin variant are conserved to a high level. Still, the stx-Nb6 almost exclusively detects syntaxin 1A indicating binding to a specific epitope.

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Figure 22: Specificity analysis of S25-Nb10 and stx-Nb6 by IF microscopy. COS-7 cells were transfected with SNAP-25 of syntaxin 1A fused to EGFP. Nanobodies conjugated to atto647N were applied at a concentration of 50 nM to detect the antigens. A: The fluorescence signal from S25-Nb10 overlaps with the fluorescence of the SNAP-25-EGFP construct but does not show any binding to syntaxin 1A EGFP. B: Complementarily, stx-Nb6 only detects syntaxin 1A-EGFP but not the SNAP-25 fusion protein. These results confirm the specificity of both nanobodies for their target antigen, also confirmed by no background signal on non-transfected cells (Hoechst-staining). Images were recorded with a Nikon Ti-E epifluorescence microscope.Scale bar represents 20 µm.

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