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Transglutaminase participates in the blockade of neurotransmitter release by tetanus toxin: evidence for a novel biological function

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Abstract

Inhibition of neuroexocytosis by tetanus neurotoxin (TeNT) involves VAMP-2/synaptobrevin-2 cleavage. However, deletion of the TeNT activity does not completely abolish its inhibitory action. TeNT is a potent activator of the cross-linking enzyme transglutaminase 2 (TGase 2) in vitro. The role of the latter mechanism in TeNT poisoning was investigated in isolated nerve terminals and intact neurons. TeNT-induced inhibition of glutamate release from rat cortical synaptosomes was associated with a simultaneous activation of neuronal transglutaminase (TGase) activity. The TeNT-induced blockade of neuroexocytosis was strongly attenuated by pretreatment of either live Aplysia neurons or isolated nerve terminals with specific TGase inhibitors or neutralizing antibodies. The same treatments completely abolished the residual blockade of neuroexocytosis of a non-proteolytic mutant of TeNT light chain. Electrophysiological studies indicated that TGase activation occurs at an early step of TeNT poisoning and contributes to the inhibition of transmitter release. Bioinformatics and biochemical analyses identified synapsin I and SNAP-25 as potential presynaptic TGase substrates in isolated nerve terminals, which are potentially involved in the inhibitory action of TeNT. The results suggest that neuronal TGase activity plays an important role in the regulation of neuroexocytosis and is one of the intracellular targets of TeNT in neurons.

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Abbreviations

BPA:

5-Biotinamidopentylamine

DTT:

Dithiotreitol

G h :

GTP-binding protein type h

HBS:

HEPES buffered saline

MDC:

Monodansylcadaverine

PAGE:

Polyacrylamide gel electrophoresis

PMSF:

Phenylmethylsulfonyl fluoride

SDS:

Sodium dodecylsulfate

SV:

Synaptic vesicle

SNAP-25:

Synaptosomal-associated protein Mr 25

SNARE:

SNAP (soluble NSF attachment protein) receptors

SRS:

Sequence retrieval system

SynI:

Synapsin I

Syn-peptide:

Rat synapsin Ia658–668

TeNT:

Tetanus neurotoxin

TeNT-LC:

TeNT light chain

TGase:

Transglutaminase

TRANSIT:

TRANSglutaminase SITes

VAMP:

Vesicle-associated membrane protein/synaptobrevin

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Acknowledgments

We are grateful to Dr. S. Peluso for the gift of Clostridium tetani culture broth and to Dr. Daniela D’Arcangelo for the important support to the experimental design and the critical reading of manuscript. This work was supported by grants from Italy–USA Oncoproteomic Program (to F.F. and A.F.), the Italian National Research Council (Convenzione CNR-Consorzio M.Negri and P. Finalizzato) and Italian Association for Cancer Research (to A.L.), Ministero dell’Università e Ricerca (PRIN 2006 to F.B.). The support of Telethon–Italy (Grant GCP05134 to F.B. and GTF08002 to F.F.), Compagnia di San Paolo-Torino and Fondazione Pierfranco e Luisa Mariani (to F.B.) is also acknowledged.

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Correspondence to Francesco Facchiano.

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Supplementary Fig. 1. TeNT markedly inhibited [3H]-glutamate release during the early phase of K+-evoked SV exocytosis.

A. Purified intact synaptosomes were loaded with [3H]-glutamate and incubated with either vehicle (closed symbols) or bichainal TeNT (10 nM; open symbols) as described in Materials and Methods. [3H]-glutamate release was assessed in three consecutive 2-min periods during the initial resting phase (wash), followed by three 2-min periods in depolarizing medium (high KCl) and three further 2-min periods in HBS for repolarization (wash). B. Purified synaptosomes, permeabilized and intoxicated with pre-activated TeNT were subjected to the same experimental protocol described for panel A. The rate of [3H]-glutamate released per min is expressed in percent of the total [3H]-glutamate content at the beginning of each 2 min superfusion period and plotted as means ± SD from at least 3 independent experiments. Statistical analysis was performed using the Tukey’s multiple comparison test. TeNT-intoxicated synaptosomes displayed a significantly (p<0.001) decreased KCl-evoked [3H]-glutamate release. (TIFF 2333 kb)

Supplementary Fig 2. Correlation between activation of synaptosomal TGase and inhibition of [3H]-glutamate release by TeNT.

TGase activation, evaluated as either spermidine derivatives (closed symbols) or ε-γ-glutamyl)lysine isodipeptides (open symbols) is plotted against the corresponding extent of inhibition of [3H]-glutamate release by TeNT. Data refer to the experiments reported in Fig.1. Pearson’s correlation coefficients were 0.983 (n=12; p<0.01) and 0.987 (n=12; p<0.01) for spermidine derivatives and (glutamyl)lysine dipeptides, respectively. Basal levels of mono-[3H[-, bis-[3H[-Spd-derivatives and (glutamyl)lysine dipeptides were 8.3, 300.5 and 10.2 picomoles/mg of total proteins, respectively. (TIFF 2203 kb)

Supplementary Fig 3. The Syn-peptide does not affect the TeNT-induced VAMP-2 cleavage in synaptosomal lysates.

Increasing concentrations of reduced TeNT were preincubated for 10 min at 37°C in the absence (open symbols) or presence (closed symbols) of Syn-peptide (300 µM) before incubation for 3 h at 37°C with synaptosomal lysate (4 mg protein/ml). Samples were then subjected to SDS-PAGE and immunoblotting to determine the uncleaved VAMP content. After quantitative analysis of the immunoblots by densitometric scanning, data were normalized to the control VAMP immunoreactivity and expressed as means ± SD from at least 3 independent experiments. The residual immunoreactivity detected at the highest TeNT concentration corresponds to the toxin-resistant VAMP-1. Methods. The synaptosomal lysate was prepared by resuspending rat cerebrocortical synaptosomes in 10 mM HEPES/0.1 mM PMSF, pH 7.4, followed by homogenization and three freeze-thaw cycles. The suspension was diluted 4-fold in 100 mM HEPES/0.1 mM PMSF, pH 7.0 and re-homogenized. Increasing concentrations of TeNT, which had been previously reduced by a 30 min preincubation at 37°C in 100 mM HEPES/20 mM DTT (pH 7.0), were incubated for 10 min at 37°C and finally added to the synaptosomal lysate. Samples were incubated at 37°C for various times, added with Laemmli sample buffer, resolved on 16% SDS-polyacrylamide gels, blotted onto PVDF membranes, and probed as described with a purified rabbit antibody to VAMP-1/2 (Ashton et al., 1995). (TIFF 1893 kb)

Supplementary Fig 4. Toxicity assay of Syn-peptide.

Effects on melanoma cell growth of 12 or 24 hours exposure to Syn or Scrambled peptide. Cells (Sk mel 110) were grown as published and plated at density of 40.000/ well, in 6 well plates. After 12 or 24 hours of growth in the presence of 10% FCS, cell viability and proliferation was evaluated by trypan blue exclusion assay. Data are calculated as percent of cells plated at time zero and are shown as means ± standard deviation. (TIFF 117 kb)

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Facchiano, F., Deloye, F., Doussau, F. et al. Transglutaminase participates in the blockade of neurotransmitter release by tetanus toxin: evidence for a novel biological function. Amino Acids 39, 257–269 (2010). https://doi.org/10.1007/s00726-009-0436-3

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