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On the development of the BoNTA specific PL50 and PL51 substrates
156AcIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANNopRAPyaKNleLNH2203 (PL50)
187AcSNKTRIDEANNopRAPyaKNleLNH2203 (PL63)
Botulinum toxin A exclusively cleaves SNAP25, a membrane protein involved in vesicle docking and neurotransmitter release. Moreover, it cleaves this 206 amino-acid protein at a single amide bond, Q197- R198 .
Based on the sequence of SNAP 25, we have designed specific BoNT/A substrates1,2.
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Starting from the « minimal » active site binding region surrounding the cleavage site, we synthesized a 17 mer quenched peptide termed PL63. Study of the kinetic parameters of this substrate towards BoNT/A LC reveals that it binds the toxin with an affinity of about 15µM whereas its catalytic constant is of 1.95 105 M1.s1.
In order to see whether extending the substrate to the α-exosite identified by Breidenbach and Brunger3 could impact the kinetic parameters of the substrate, we synthesized the PL50 quenched peptide which covers amino acids 156 to 203 of the SNAP-25 peptide sequence.
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The PL50 substrate bound the BoNT/A toxin with an affinity of 0,8 µM, i.e. with almost a 20 fold increase compared to PL63. Although the catalytic constant was not significantly modified, the resulting specificity constant of PL50 was increased by more than 40 fold.
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In order to increase the stability of the PL50 substrate in solution, the 3 methionine residues within its sequence were replaced by their isosteric analogue norleucine. Although these latter changes should not induce structural changes, the resulting PL51 substrate was shown to possess a significantly improved affinity for the toxin, and a more than 7 fold increase in its specificity constant (Km/kcat).
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These results show that extending the PL63 peptide substrate to the α-exosite sequence significantly increases substrate binding, as suggested by Breidenbach and Brunger3, making both PL50 and PL51 the best available substrates on the market for BoNTA quantification.
1Fournie-Zaluski MC et al. (2005) PCT WO 2005121354 3Breidenbach & Brunger (2004) Nature, 432:925
2Poras H., Ouimet T., et al. (2009) AEM ,75 : 4382
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