Phospholipid binding

The phospholipid-binding behavior of Otof-C2A was tested using floatation assays.

For this, liposomes are produced from defined lipid mixtures via gel filtration and added to protein solution. After a long, fast centrifugation in a Nycodenz-gradient, the liposomes float to the upper phase of the experimental tube and take potentially bound protein with them.

To test phospholipid-binding for Otof-C2A, several lipid mixes were used (Table 4).

Three of them were artificially produced and contain

1. 0% PS and 0% PIP2

2. 16% PS and 0% PIP2

3. 16% PS and 1.6% PIP2.

Experiments with liposomes formed from the first three mixtures were done to clarify the question whether Otof-C2A binds to liposomes (only) in presence of the anionic phospholipid PS, like for other C2-domains like Syt1-C2A or PKCγ-C2. Also, it needed to be clarified whether binding is enhanced in presence of PIP2, which was found for example for Syt1-C2B (for review see Cho and Stahelin, 2006).

As a fourth lipid mixture, a natural total brain lipid extract was used. It was used to study Otof-C2A’s phospholipid-binding behavior also under more natural conditions, though the brain total lipid extract does not necessarily represent the lipid composition of the inner hair cell plasma membrane. All experiments were done in presence (+ 1 mM CaCl2) and absence (+ 1 mM EGTA) of Ca²⁺-ions.

In contrast to the previously described ITC- and CD-spectroscopy-experiments, this time also the C2B-domain of Syt1 was part of the construct of the positive/negative control besides Syt1-C2A. This was done because Syt1-C2B is known for Ca²⁺

-independent PIP2-binding with its so called “β-groove” (Schiavo et al., 1996), a positively charged region at the concave face of the β-sandwich (Fukuda et al., 1994) that has been found in many C2-domains (for review see Cho and Stahelin, 2006). As Syt1-C2B is known to be hard to purify on its own, it was not used separately but in combination with Syt1-C2A as Syt1-C2AB tandem domain.

Figure 28: Floatation assays of Otof-C2A with Syt1-C2AB as positive control with three different lipid mixtures. Helfmann et al., 2011. Gel pictures: lanes from left to right are the collected fractions from top to bottom of the tube. Graphs show band staining intensities from top to bottom of the tube with mean +/- standard error of the mean (SEM; n=3).

A: SDS-Gel of experiment with liposomes containing PS.

B: SDS-Gel of experiment with liposomes containing no PS.

C: SDS-Gel of experiment with liposomes containing PS and PIP2.

D+G: Experiment with liposomes containing PS. Syt1-C2AB (D) and Otof-C2A (G).

E+H: Experiment with liposomes containing no PS for Syt1-C2AB (E) and Otof-C2A (H).

F+I: Graph of experiment with liposomes containing PS and PIP2 for Syt1-C2AB (F) and Otof-C2A (I).

A B C

D E F

G H I

• Results •

For each floatation assay, one 12%-SDS-gel was prepared with the lanes 1-9 containing 10 µl of the 20 µl-fractions 1-8 and 10 µl of the 40 µl-fraction 9 from top to bottom of the reaction tube.

In presence of PS and Ca²⁺, Syt1-C2AB was mainly found in the middle of the gradient (Fig. 28A, first line of gel; Fig. 28D, black trace), while in absence of Ca²⁺ it accumulated in the bottom of the tube (Fig. 28A, second line of gel; Fig. 28D, grey trace). Thus, the Ca²⁺-dependency of phospholipid-binding of Syt1-C2AB is confirmed.

In contrast, Otof-C2A is mainly found in the lower part of the gradient independently of the presence or absence of Ca²⁺, and was hardly detected in the highest fractions (Fig. 28A, third and fourth line of gel; Fig. 28G).

When liposomes without PS were used, the amount of protein in the upper fractions in Syt1-C2AB in presence of Ca²⁺ was reduced compared to experiments with PS (Fig. 28B, first and second line of gel; Fig. 28E), so that no difference between the Ca²⁺- and EGTA-trace was detectable anymore. This means, in absence of PS Syt1-C2AB does not bind to the liposomes anymore. For Otof-C2A, the experiment without PS showed no binding of liposomes (Fig. 28B, third and fourth line of gel;

Fig. 28H).

Figure 28C displays the results for the experiments with PS and PIP2: Syt1-C2AB accumulates in the middle part of the tube in presence of Ca²⁺ (Fig. 28C, first line of gel; Fig. 28F, black trace), but also in absence of Ca²⁺ some protein is detected in the low density fractions (Fig. 28C, second line of gel; Fig. 28F, grey trace). This was expected as Syt1-C2B was shown to bind to PIP2 independently of Ca²⁺ with its β-groove. In contrast, Otof-C2A is found in the bottom of the tube in both experiments (Fig. 28C, third and fourth line of gel; Fig. 28I).

When brain total lipid extract was used in the floatation, both proteins accumulated in the lower fractions (Fig. 29). In presence of Ca²⁺, around 5% of Syt1-C2AB appeared in fraction 1, while in presence of EGTA there was no protein at all (Fig.

29A). Brain total lipid extract contains only about two thirds of the amount of PS (10.5%) compared to the 16%-PS-mixtures. As Syt1-C2AB preferentially binds to the anionic phospholipid PS, this could be a reason for the little binding of the protein. The distribution of Otof-C2A in the gradient shows no difference in presence and absence of Ca²⁺ (Fig. 29B).

A B

Ca²⁺ Ca²⁺

EGTA EGTA

Figure 29: Floatation assays of Otof-C2A and Syt1-C2AB with brain total lipid extract. Helfmann et al., 2011. Graph shows band staining intensities from top to bottom of the tube with mean +/- SEM (three experiments). Gel shows distribution of protein in the tube from left to right = top to bottom.

A: Syt1-C2AB.

B: Otof-C2A.

Summarizing, Otof-C2A did not bind to liposomes with four different lipid compositions, independently from the presence or absence of Ca²⁺-ions. Presence of PS or PIP2 in the liposomes, which enhanced the lipid-binding affinity of Syt1-C2AB, could not render Otof-C2A phospholipid-binding.