Association of Botulinum Neurotoxin Serotype A Light Chain with Plasma Membrane-bound SNAP-25

Sheng Chen,Joseph T Barbieri

doi:10.1074/jbc.m111.224493

Abstract

The Clostridium botulinum neurotoxins (BoNTs) cleave SNARE proteins, which inhibit binding and thus fusion of neurotransmitter vesicles to the plasma membrane of peripheral neurons. BoNTs comprise an N-terminal light chain (LC) and C-terminal heavy chain, which are linked by a disulfide bond. There are seven serotypes (A-G) of BoNTs based upon immunological neutralization. Although the binding and entry of BoNT/A into neurons has been subjected to considerable investigation, the intracellular events that allow BoNT/A to efficiently cleave SNAP-25 within neurons is less well understood. Earlier studies showed that intracellular LC/A bound to the plasma membrane of neurons. In this study, intracellular LC/A is shown to directly bind SNAP-25 on the plasma membrane. Solid phase binding showed that the N-terminal residues of LC/A bound residues 80-110 of SNAP-25, which was also observed in cultured neurons. Association of the N-terminal 8 amino acids of LC/A and residues 80-110 of SNAP-25 also enhanced substrate cleavage. These findings explain how LC/A associates with SNAP-25 on the plasma membrane and provide a basis for LC/A cleavage of SNAP-25 within the SNARE complex.

Highlights

botulinum neurotoxins (BoNTs) are zinc proteases that cleave soluble NSF attachment protein receptor (SNARE) proteins [9]
The current study shows that light chain (LC)/A binds directly to SNAP-25 on the plasma membrane of neurons
The N terminus of LC/A contributes to high affinity binding of LC/A to SNAP-25 on the plasma membrane and to the efficient cleavage of SNAP-25

Summary

EXPERIMENTAL PROCEDURES

Materials—Neuro-2A cells were purchased from the ATCC (CCL131). pEGFP, pERFP, and pEYFP were purchased from Invitrogen. ␣-SNAP-25 IgG and ␣-myelin IgG were purchased from Santa Cruz Biotechnology. Expression plasmids encoding LC/A, SNAP-25, and the respective truncated derivatives were constructed by PCR-am-. BoNT/A Binds SNAP-25 on the Plasma Membrane plifying DNA encoding LC/A, SNAP-25, and their different truncation mutations into pET-15b. Plasmids encoding these constructs were transformed into Escherichia coli BL21(DE3) RIL or BL21(DE3) (Stratagene). Linear Velocity Assay—Linear velocity of SNAP-25 cleavage by LC/A and LC/A(8 – 438) was performed in a 20-␮l reaction containing: 2 ␮M SNAP-25 derivatives and the indicated amounts of LC in 10 mM Tris-HCl (pH 7.6) and 20 mM NaCl. Reactions were incubated at 37 °C for 15 min and subjected to SDS-PAGE. The percentage of substrate cleavage and the concentration of LCs were plotted, and the LCs required to cleave 50% of substrate were derived

RESULTS

Peptides matched

DISCUSSION