Wei-Jen Lin

Complete DNA sequences of the botulinum neurotoxin complex of Clostridium botulinum type A-Hall (Allergan) strain ☆

BOTOX is manufactured with the purified native 900-kDa type A neurotoxin complex from Clostridium botulinum type A-Hall (Allergan) strain. This complex is composed of the botulinum neurotoxin (BoNT) and several toxin associated proteins known as the hemagglutinins (HAs) and the non-toxic non-hemagglutinin protein (NTNH). We describe here the complete gene sequences of the BoNT complex of type A-Hall (Allergan) strain. Using a polymerase chain reaction-based approach, we sequenced six open reading frames (ORFs) encoding BoNT (1296 amino acids), the toxin-associated proteins: HA70, 625 aa; HA17, 147 aa; HA34, 291 aa; NTNH, 1193 aa; and the regulatory component botR/OrfX, 178 aa. Comparative alignments of the amino acid sequence of BoNT/A shows a 98-100% sequence identity among different strains of the type A, except for the Kyoto-F strain (90%), whereas the sequence identity between BoNT/A and other toxin serotypes is only 30.4-39.1%. Similar to the neurotoxin, the toxin-associated proteins and botR from type A-Hall strain also share more than 95% identity to the homologous proteins found in type A-NCTC2916 strain. Among all the toxin associated proteins, NTNHs and HA70s are the most conserved with 65-87% identity across different serotypes. On the other hand, HA34s, present only in serotypes A-D, show greater diversity than all other toxin-associated proteins; HA34/A has 90% identity to HA34/B and only approximately 35% identity to HA34/C and HA34/D. Relatively higher sequence identity ( approximately 60%) is seen in HA17 and botR of Hall A when compared to their counterparts in serotypes C or D. Of all proteins within the toxin complex, NTNH and HA70 have the highest degree of conservation across serotypes and this may underscore a critical role for these proteins in the formation of the complexes. Physiologically, different duration of action in different serotypes may be due to different modifications of toxins by neuronal enzymes, which lead to different compartmentalization of different toxins. Computer-assisted motif analysis reveals that toxins contain several potential sites for phosphorylation by casein kinase II, protein kinase C, tyrosine kinases, glycogen synthase kinase 3, cGMP dependent protein kinase (PKG) that are well conserved. The reported sequence information for type A-Hall strain will potentially facilitate elucidation of the toxin interactions with the nontoxin proteins in the complex.