ABSTRACT
Botulinum neurotoxins (BoNTs) are the most potent natural toxins known. The effects of BoNT serotype A (BoNT/A) can last several months, whereas the effects of BoNT serotype E (BoNT/E), which shares the same synaptic target, synaptosomal-associated protein 25 (SNAP25), last only several weeks. The long-lasting effects or persistence of BoNT/A, although desirable for therapeutic applications, presents a challenge for medical treatment of BoNT intoxication. Although the mechanisms for BoNT toxicity are well known, little is known about the mechanisms that govern the persistence of the toxins. We show that the recombinant catalytic light chain (LC) of BoNT/E is ubiquitylated and rapidly degraded in cells. In contrast, BoNT/A LC is considerably more stable. Differential susceptibility of the catalytic LCs to ubiquitin-dependent proteolysis therefore might explain the differential persistence of BoNT serotypes. In this regard we show that TRAF2, a RING finger protein implicated in ubiquitylation, selectively associates with BoNT/E LC and promotes its proteasomal degradation. Given these data, we asked whether BoNT/A LC could be targeted for rapid proteasomal degradation by redirecting it to characterized ubiquitin ligase domains. We describe chimeric SNAP25-based ubiquitin ligases that target BoNT/A LC for degradation, reducing its duration in a cellular model for toxin persistence.
Subject(s)
Botulinum Toxins, Type A/metabolism , Botulinum Toxins, Type A/toxicity , Botulinum Toxins/metabolism , Botulinum Toxins/toxicity , Clostridium botulinum type A/physiology , Clostridium botulinum type A/pathogenicity , Clostridium botulinum type E/physiology , Clostridium botulinum type E/pathogenicity , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Amino Acid Sequence , Animals , Botulinum Toxins/genetics , Botulinum Toxins, Type A/genetics , Cell Line , Clostridium botulinum type A/genetics , Clostridium botulinum type E/genetics , Genes, Bacterial , Humans , Mice , Molecular Sequence Data , Neurons/drug effects , Neurons/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Synaptosomal-Associated Protein 25/genetics , Synaptosomal-Associated Protein 25/metabolismABSTRACT
Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A-G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay.
