Characterization of S25-Nb10 and stx-Nb6

An important parameter of the nanobodies is their affinity as it influences the quality of staining in IF experiments. The conditions during SPR measurements caused both precipitation and degradation of the antigen due to long incubation times as mentioned in section 3.4.3.

I initially decided on this method as not only the affinity, indicated by the dissociation constant KD, but also the kinetic behavior of the nanobodies could be measured. The kinetic parameters such as association (kon) and dissociation (koff) provide additional information on the dynamic behavior.

Those values can be used to adjust the washing conditions accordingly to minimize the loss of nanobody during washing steps.

I found most nanobodies to exhibit similar values for kon causing the dissociation constant KD to directly determine the overall affinity of the probe [81,168]. Hence, a low dissociation constant was considered to indicate a strong affinity of my nanobody. To measure the KD value of the nanobody-antigen complex, MST was finally used as a more reliable and faster method.

The two nanobodies S25-Nb10 and stx-Nb6 binding SNAP-25 and syntaxin 1A, respectively, passed all validation criteria and bind their antigen with high affinity. Their dissociation constants were found to be in the low nanomolar range (15.5 ± 3.3 nM for S25-Nb10 and 5.0 ± 1.2 nM for stx-Nb6), which

112

indicates a high affinity of the nanobody comparable to other probes described in the literature [84,168]. Those nanobodies were therefore used as final probes for further experiments.

I decided to use the nanobodies in a concentration of 50 nM in immunostainings, which is well above the KD value and thus expected to saturate the detection of the epitopes. At the same time this concentration of nanobodies was still low enough to prevent unspecific staining in cells not expressing EGFP (see Figure 22). This is shown in Figure 28 where a GFP-nanobody also conjugated to atto647N was used at equivalent conditions to determine if the fluorophore contributes to background binding (negative control).

The sequences of rat syntaxin 1A and SNAP-25 were used for both phage display selection and validation experiments. Both proteins are virtually conserved among mammals including homo sapiens, mus musculus and rattus norwegicus with occasional deviations of single amino acids.

Therefore, the species type of the antigen is not expected to influence the result of nanobody binding. This is also supported by the fact that both nanobodies detected a single protein in immunoblots regardless of the species type as shown in Figure 20.

Besides these observations, it is a remarkable fact that the nanobodies are at all working in Western blots detecting the antigen after denaturing SDS-PAGE as demonstrated in section 3.4.4. Although nanobodies detecting linear epitopes have been reported before, the detection of unfolded proteins is generally rather untypical for those probes [51,79,84]. A possible explanation could be the incomplete denaturation or partial refolding of SNAP-25 and syntaxin 1A during SDS-PAGE.

Alternatively, the nanobodies might indeed bind to linear or unstructured epitopes, which do not require the antigen to acquire a specific conformation.

To narrow down the region of nanobody binding to the antigen, I generated truncated forms covering the antigen sequence piece by piece. As shown in Figure 23, individual domains of the antigen seem to be in fact sufficient to mediate nanobody binding. The epitope nanobody S25-Nb10 was found to localize in the N-terminal domain of SNAP-25 which constitutes one of the two SNARE domains of that protein. Generally, those domains are composed of an alpha-helical structure, which form intermolecular bundles resulting in a stable helical bundle [162]. As a single SNARE domain is not expected to acquire a tertiary structure, the epitope mapping shown in Figure 23 suggests that both nanobodies might indeed detect a linear epitope or a helical secondary structure.

If by future NMR analyses the epitope of the nanobody S25-Nb10 can be confirmed to reside within the SNARE domain, the nanobody could be an interesting tool to investigate the SNARE complexes.

By intracellular expression of the nanobodies in neurons, the SNARE complex formation could be

113

impaired due to sequestering of SNAP-25. Alternatively also the ratio of free SNAP-25 compared to per-assembled complexes or clusters could be determined if the nanobody is found to only bind free SNAP-25. This might be a valuable approach to investigate the dynamics of synaptic vesicle cycling and synaptic physiology in more details. A first indication that S25-Nb10 cannot detect SNAP-25 engaged in a SNARE complex can be inferred from Figure 30 where both nanobodies were imaged in dual channel STED microscopy.

Due to the interaction of SNAP-25 and syntaxin 1A on the plasma membrane to nucleate a SNARE complex for vesicle fusion, both proteins are expected in direct proximity [179,180]. The low colocalization of SNAP-25 and syntaxin 1A observed in Figure 30 might thus be caused by steric hindrance or conformational change in the SNARE bundle which masks the epitope. Similarly, antibodies often localize an epitope within the SNARE domain (see section 3.5) and therefore might likewise be impaired in binding their antigen in a complex. So far, SNAP-25 and syntaxin 1A have been only reported to localize in adjacent domains but not in the same SNARE complex as it would be expected [166].

Despite the apparent binding specificity for the SNARE domain of SNAP-25 and syntaxin 1A, the nanobodies merely detect other closely related SNAP or syntaxin proteins (see section 3.4.4 and Figure 21). The high specificity of S25-Nb10 was expected as SNAP-25 shares only little homology with other SNAP proteins (see Figure 21 and Figure 31). On the contrary, currently 16 different syntaxin proteins are known in human, most of which show high degree of similarity [181]. I tested three syntaxin variants that show the highest level of sequence conservation to syntaxin 1A, indicated by the green box in Figure 31. Still the stx-Nb6 shows specificity for syntaxin 1A with little cross-reactivity to syntaxin 3 but no reactivity for the phylogenetically close protein syntaxin 1B.

Taking into account that the nanobody might detect a linear part of the protein this narrows down the potential position for the epitope drastically. In practice, an amino acid sequence found in both syntaxin 1A and syntaxin 3 but not in syntaxin 1B could be a likely position for the nanobody to bind.

An exemplary region is located in between the amino acid residues 53-59, where syntaxin 1A possesses higher sequence similarity to syntaxin 3 than to syntaxin 1B. Interestingly, this region is also part of the truncation construct showing nanobody binding in Figure 23. Hence, recombinant expression of this peptide to use for dot blot or blocking assays could pinpoint the epitope to a short peptide. If that peptide shows binding to the nanobody, it may also be used as a ligand for NMR to gain structural information on the interaction between stx-Nb6 and syntaxin 1A.

114

In further experiments, the specificity of stx-Nb6 for syntaxin 1A can be used to quantify the ratio between cellular levels of endogenous syntaxin 1A and 1B. Despite the high degree of sequence conservation in between those molecules, syntaxin 1A and 1B have a different impact on synaptic physiology and neuronal survival [182,183]. The nanobody could thus be used as a tool to selectively detect endogenous levels of syntaxin 1A as many commercially available antibodies cannot distinguish between those two syntaxin variants (see Figure 21 and Figure 31).

Yet my primary focus lies on the application of nanobodies as a versatile tool for super-resolution microscopy in fixed samples. High affinity and high specificity were therefore considered to be the main criteria to define my nanobodies, regardless of the epitope position. After identification of S25-Nb10 and stx-Nb6 as the final candidates, I therefore focused on their implementation in IF experiments.

Figure 31: Phylogenetic alignment of the SNAP and syntaxin protein family. During this study, I focused on SNAP-25 and syntaxin 1A as two major SNARE proteins present in neuronal synapses. The highlighted syntaxin variants were used to analyze the specificity of nanobodies directed against syntaxin 1A. Alignment was created with CLC sequence viewer 7.

115