BoNT/A-LC (pBN4)
no toxin
Glu 224 (wt)
Gln 224
Lys 224
TeTx-LC (pLC2)
BoNT/A-LC
BoNT/A-HC
BoNT/A, red.
30 ā
SNAP-25
14.2 ā
FIG. 4 The L chain of BoNT/A cleaves SNAP-25 and requires Glu 224 for activity. In the experiments shown in the left part of the figure, SNAP-25 was translated in vitro in the presence of [35S]methionine and combined at the end of the incubation with BoNT/A-L chain and TeTx-L chain that were translated in vitro in parallel incubations using unlabelled amino acids. In the experiments shown in the right three lanes, BoNT/A components were used that were purified from bacteria. Note that neither the H chain nor the two mutant L chains or TeTx L chains caused cleavage of SNAP-25.
METHODS. For the construction of the Gln 224 and Lys 224 mutants, two new singular restriction sites, BamHI and Nrul were generated by PCR in positions 1,002 and 1,063ā¹. The sites flank the sequence coding for a conserved HExxHxxH motif that is essential for ZnĀ²āŗ binding, thus allowing replacement of the internal sequence by appropriate synthetic oligonucleotides. In vitro translation was as given in Fig. 3 but linearized DNA was used as template for the generation of mRNA. SNAP-25 mRNA was translated in the presence of [35S]methionine. BoNT/A-LC mRNA was translated in an unlabelled form, both under control of SP6 promoter in pSP72 (Promega, Madison, WI). At the end of the translation period (60 min, 30 Ā°C), cycloheximide was added to both translation assays (250 Ī¼M final concentration) before mixing of the samples. Incubation was for 2 h at 37 Ā°C. Electrophoresis and fluorography were as described in Fig. 3.