Mapping and separation of the

Mapping and separation of the anti-GlyT1C epitopes allowed us to verify the existence of calpain cleavage sites in isolated mouse synaptosomes. In order to allow calcium to enter the intrasynaptosomal compartment and activate endogenous calpain, we incubated synaptosomes in hypoosmotic high (200μM) calcium buffer. As shown on upper part of the Fig. 3, high calcium showed only small effect on distribution of anti-GlyT1C554–625 epitopes, indicating that calpain cleavage sites detected in recombinant fusion protein upstream of anti-GlyT1C626–638 epitopes are not extensively cleaved in synaptosomal preparations. On the contrary there was a significant decrease of anti-GlyT1C626–638 antibodies following calcium increase (lower part of Fig. 3). Decrease of immunoreactivity was strongly suppressed by calcium chelation and inhibited by calpain inhibitor. As it is shown on lower part of the Fig. 3 inhibition of calpain did not fully recover decrease of distal GlyT1C immunoreactivity, which suggested that some additional calcium dependent modifications could be involved. The presence of asparagine (d) in position 635 indicated possibility that distal end of GlyT1C could be a substrate of other calcium-induced enzymes, caspases. Removal of last three Ellipticine (-SRI), which mimics potential caspase cleavage, resulted in complete loss of anti-GlyT1C626–638 epitopes, similarly as we observed in calpain mediated removal of last 12 amino acids of GST-GlyT1C (Fig. 4). The presence of general caspase inhibitor Z-VAD-FMK in synaptosomal samples indeed inhibited the decrease of distal GlyT1C immunoreactivity, with slightly lesser extend as observed with calpain inhibitor (Fig. 3). Some of the previous reports however indicated that general caspase inhibitor Z-VAD-FMK acts as a calpain inhibitor (Bizat et al., 2005), therefore we cannot exclude the sole effect of Z-VAD-FMK on calpain. Another calcium-induced modification, which can significantly alter the immunoreactivity is phosphorylation (Susarla et al., 2004). Phosphorylation prediction analysis of distal 12 amino acids peptide removed by calpain by software NetPhos 2.0 Server (www.cbs.dtu.dk/services/NetPhos/; Blom et al., 1999) showed that serines in positions 627, 630, 631 and 636 in GlyT1C could be potentially phosphorylated (score 0.124; 0.290; 0.118; 0.461, on scale 0–1). Replacement of most probably phosphorylated serine 636 with asparagine, which mimics phosphoserine, resulted in significant loss of anti-GlyT1C626–638 immunoreactivity in GST-GlyT1C fusion protein (Fig. 4). The presence of magnesium in synaptosomal samples stimulated effect of calcium mediated decrease of anti-GlyT1C626–638 immunoreactivity, which indicated that phosphorylation in this region indeed can occur (Fig. 5). Presence of chelerythrine suppressed magnesium stimulated decrease of anti-GlyT1C626–638 immunoreactivity, indicating that in addition to proteolysis, PKC mediated phosphorylation can take place in region of last 12 amino acids of GlyT1 during the calcium overload (Fig. 5). Activity of cysteine proteases such as calpain and caspases is influenced by various metal ions. To investigate their influence, we substituted calcium with Zn2+, Mn2+ and Co2+ (Fig. 6). Neither of these metals induced decrease of GlyT1C626–638 immunoreactivity in synaptosomes. When ions were supplemented together with calcium, manganese did not interfere with calcium-induced decrease of immunoreactivity. In contrast zinc and cobalt totally inhibited decrease of GlyT1C626–638 immunoreactivity induced by calcium overload.
Discussion We previously showed that cytosolic domains of several neurotransmitter transporters are substrates of calpain protease (Baliova et al., 2004, Baliova et al., 2009, Baliova and Jursky, 2005, Franekova et al., 2008). In glycine transporter GlyT1C recombinant fusion protein we observed several calpain cleavage sites, which we were unable to sufficiently confirm in vivo. Absence of cleavage in synaptosomes could be explained via protein interactions, protective phosphorylation, but detection of calpain cleavage in vivo also depends on position of antibody epitopes used for detection of cleaved fragments in complex mixture of proteins. Here we investigated whether separation of different anti-GlyT1C polyclonal antibody epitopes will allow us to detect in vivo some of the calpain cleavage sites, found in recombinant GlyT1C-terminus.