Presentation Title

Multicleavable Protein Substrates for the Mass Spectrometric Detection of Botulinum Neurotoxins

Faculty Mentor

Markus Kalkum

Start Date

17-11-2018 12:30 PM

End Date

17-11-2018 2:30 PM

Location

HARBESON 54

Session

POSTER 2

Type of Presentation

Poster

Subject Area

health_nutrition_clinical_science

Abstract

Botulinum neurotoxins (BoNTs) are proteins produced by anaerobic bacteria belonging to the genus Clostridium. Their categorization as the most potent biological toxin on Earth has generated much interest in their biological function and therapeutic potential in medicine. BoNTs exist in 8 different serotypes and contain two amino acid chains, the light (L) chain (~50 kDa) and the heavy (H) chain (~100 kDa). BoNT’s toxicity stems from its ability to initiate peripheral neuroparalysis, affecting the skeletal and autonomic nervous systems. BoNT interrupts neurotransmitter release through a five-step process in which it targets intracellular substrates:

1. BoNT uniquely binds to a nerve terminal through independent receptor(s) embedded in the presynaptic membrane.

2. BoNT is then internalized within the nerve terminal.

3. The BoNT L chain migrates across the vesicle membrane.

4. The L chain is released into cytosol.

5. The L chain cleaves SNARE proteins.

My project involves measuring BoNT enzymatic activity by measuring the cleavage of SNARE protein-like substrates. These substrates do not exist in nature but were designed computationally. In contrast to naturally occurring SNARE proteins, the substrates contain multiple cleavage sites. They were also designed such that they generate isobaric products upon BoNT cleavage. These novel substrates are referred to as Isobaric Signal Amplifying Protein Substrates (ISAS). The goal of this project is to develop protein substrates that can interact with traces of the toxin to detect its presence. The toxin will cleave the protein substrate’s amino acid chain at designated positions. The masses of cleaved peptides from the substrate proteins can be analyzed by mass spectrometry to identify whether a particular serotype of BoNT was responsible for the cleavage.

Keywords: proteomics, mass spectrometry, botulinum neurotoxins, enzymes, cleavage, isobaric products, detection

Summary of research results to be presented

- The BoNT/A, BoNT/E and BoNT/HA/F5 protein substrates were expressed and successfully purified.

- These substrates were subjected to BoNT cleavage.

- The cleavage products were analyzed and confirmed using SDS PAGE and MALDI methodologies.

- The single-mass, Isobaric Signal Amplification Substrates, were able to produce amplified signals as translated by sharper, more specific spectra peaks because their single-mass fragments share the same amino acid sequence.

- More intense fragment signaling occupying a narrow spectrum range improves the sensitivity of the cleaved ISAS peptide in responding to low concentrations of botulinum toxin wherever it is suspected to be present.

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Nov 17th, 12:30 PM Nov 17th, 2:30 PM

Multicleavable Protein Substrates for the Mass Spectrometric Detection of Botulinum Neurotoxins

HARBESON 54

Botulinum neurotoxins (BoNTs) are proteins produced by anaerobic bacteria belonging to the genus Clostridium. Their categorization as the most potent biological toxin on Earth has generated much interest in their biological function and therapeutic potential in medicine. BoNTs exist in 8 different serotypes and contain two amino acid chains, the light (L) chain (~50 kDa) and the heavy (H) chain (~100 kDa). BoNT’s toxicity stems from its ability to initiate peripheral neuroparalysis, affecting the skeletal and autonomic nervous systems. BoNT interrupts neurotransmitter release through a five-step process in which it targets intracellular substrates:

1. BoNT uniquely binds to a nerve terminal through independent receptor(s) embedded in the presynaptic membrane.

2. BoNT is then internalized within the nerve terminal.

3. The BoNT L chain migrates across the vesicle membrane.

4. The L chain is released into cytosol.

5. The L chain cleaves SNARE proteins.

My project involves measuring BoNT enzymatic activity by measuring the cleavage of SNARE protein-like substrates. These substrates do not exist in nature but were designed computationally. In contrast to naturally occurring SNARE proteins, the substrates contain multiple cleavage sites. They were also designed such that they generate isobaric products upon BoNT cleavage. These novel substrates are referred to as Isobaric Signal Amplifying Protein Substrates (ISAS). The goal of this project is to develop protein substrates that can interact with traces of the toxin to detect its presence. The toxin will cleave the protein substrate’s amino acid chain at designated positions. The masses of cleaved peptides from the substrate proteins can be analyzed by mass spectrometry to identify whether a particular serotype of BoNT was responsible for the cleavage.

Keywords: proteomics, mass spectrometry, botulinum neurotoxins, enzymes, cleavage, isobaric products, detection