The facts mentioned above indicate that glycine function

The facts mentioned above indicate that glycine function in brain is tightly regulated or pathologically altered mostly by glycine transporters. Modification of cytosolic regions of transporters, which interconnect them with intracellular regulatory pathways, could be one of the ways to provoke brain glycine concentration changes. We recently described N-terminal truncation of GlyT1 with calpain protease (Baliova and Jursky, 2005). Because the major epitopes of previously used antibodies were located outside the short C-terminal fragment of GlyT1 removed by calpain, we were unable to detect the modification of GlyT1 on distal C-terminal region reported here. We hypothesize that such truncation/modification during the pathological calcium overload could either additionally contribute to abnormal GlyT1 function or it represents certain compensation regulatory feedback.
Materials and methods
Results Despite the presence of several calpain cleavage sites in the recombinant GlyT1C fusion protein sequence, our previous screening of spinal cord synaptosomes did not show marked changes of GlyT1C-terminal immunoreactivity following calcium increase. Polyclonal antigenic determinants are often distributed heterogeneously and successful detection of proteolytic truncation in vivo could depend on position of major antibody epitopes (Baliova et al., 2009). To verify this possibility we decided to determine the exact position of calpain cleavage sites in recombinant GlyT1C fusion protein and to separate possible groups of antibody epitopes directed against peptide fragments released by calpain cleavage. Because protein sequencing by Edman degradation proceeds through the protein N-terminal end, it was possible to use only one set of the proteolytic fragments to determine the calpain cleavage sites. Some of the released fragments were too small to be separated by PAGE. To overcome this limitation, we took advantage of the fact that calpain specificity is mostly determined by 11 Gliotoxin (Tompa et al., 2004). In an alternative approach, which we used previously (Baliova et al., 2009), we introduced methionine at the beginning of the GlyT1C-terminal fragment and removed the stop codon from its end. This allowed us to fuse the GlyT1C-terminus with a bigger protein tag in the opposite orientation. Following calpain cleavage, the fragments bearing the N-terminal part of GlyT1C remain attached to the larger protein tag and allowed their isolation using the standard 12% SDS gel. When we made this construct, we found that GlyT1C-protein sequence in Meth-GlyT1C-GST fusion protein was cleaved by unknown endogenous E. coli proteolytic system (not shown). This activity interfered with subsequent calpain cleavage and isolation of proteolytic fragments for purpose of protein sequencing. In order to recover full-length protein we inserted initiating methionine and 6Xhistidine tag coding DNA linker upstream of GlyT1C-GST DNA coding sequence. Two steps isolation procedure using both 6Xhistidine and GST tag allowed as recovering the full-length protein. Calpain cleavage of Meth-6Xhis-GlyT1C-GST fusion protein released three major proteolytic fragments indicating the existence of minimally three calpain cleavage sites (Fig. 1). Predicted size deduced from the gel mobility of the fragments suggested that all cleaved sites are located in GlyT1C moiety of the fusion protein. The Edman protein sequencing of these fragments showed that cleavage sites are located in positions equivalent to R567/T68, T603/T604 and G625/S626 of whole mouse GlyT1B protein sequence. Location of the calpain cleavage site in position G625/S626 revealed that calpain removes 12 amino acids from GlyT1C-terminus (Fig. 1). To test if antibody epitopes against this short amino acid region are present in our polyclonal serum, we constructed fusion of cellulose binding protein (CBD) with the last 12 amino acids of GlyT1C and probed it with total anti-GlyT1C antibody. Following the detection of specific antibody staining (not shown), epitopes anti-GlyT1C626–638 were purified on immobilized CBD-GlyT1C626–638 fusion protein. Using the same fresh immobilized fusion protein, the epitopes anti-GlyT1C626–638 were removed from polyclonal anti-GlyT1C554–638 antibodies, resulting in anti-GlyT1C554–625 antibodies. In subsequent experiments we verified the position of immunoreactivity on individual bands of calpain cleaved GST-GlyT1C fusion protein for these two sets of antibodies (Fig. 2). Appearance of second band in close vicinity of intact GST-GlyT1C fusion protein in calpain cleaved sample, indicated that calpain removes short stretch of amino acids on its distal end, which corroborated the results obtained by protein sequencing of cleavage products of Meth-6Xhis-GlyT1C-GST fusion protein. As expected, removal of these amino acids correlated with disappearance of anti-GlyT1C626–638 epitopes in one of the closely located bands. Interestingly 12 amino acid truncated GST-GlyT1CD12 runs higher than intact protein, which indicates that distal 12 amino acid peptide has significant influence on fusion protein secondary structure. Fig. 2 also shows that following removal 12 amino acids and elimination of anti-GlyT1C626–638 epitopes, there are still comparably strong anti-GlyT1C554–625 epitopes suitable for detection of upstream located calpain cleavage sites. Epitopes were however completely eliminated by the next calpain cleavage site in position T603/T604 indicating that these epitopes reside between amino acids 604 and 626. Fig. 2 also indicates that in GST-GlyT1C additional calpain cleavage site exists, previously not observed in Meth-6Xhis-GlyT1C-GST which is located between position R567/T568 and T603/T604.