3.2.1 p 15 in NGF-differentiated PC 12 cells
Polysialogangliosides bind CNTs through the H^ domain with a in the nanomolar range
(Halpem and Neale, 1995; Helting et al., 1977; Schengmnd et al., 1991; van Heyningen, 1974). The relatively low binding affinity to polysialogangliosides suggests however that these glycolipids are not the only CNT receptors. Additional protein receptor(s) with a higher affinity may participate in CNTs binding as suggested by the observation of protease-sensitive TeNT binding sites in different neuronal preparations (Bakry et al., 1991b; Parton et al., 1988; Pierce et al., 1986; Yavin and Nathan, 1986). Alternatively, a binary complex containing both protein and polysialogangliosides could represent the high
Control CNT“ CNT+ Hc“ (+%)"
BoNT/A 37 ± 6 65 ± 8 (+75)
BoNT/B 23 ±5 60 ± 9 (+160)
BoNTE 24 ±3 61 ± 7 (+154)
TeNT 111 ± 11 195 ±20 (+75)
“The values given are the 50% paralysis time values (in min) observed for preparations (n = 3) exposed
to CNTs alone or in the presence of the correspondent recombinant fragment. Each value represents
the mean ± S.D.
‘’Increase in the 50 % of paralysis time following pretreatment with He expressed as percentage of the control.
Chapter 3_______________________________________________________________Results
affinity receptor (Montecucco, 1986). Crosslinking experiments performed on NGF- differentiated PCI2 cells suggested the presence of a putative 20-kDa receptor protein for TeNT (Schiavo et al., 1991). We sought to characterise this protein using the non-toxic
recombinant TeNT fragment in NGF-differentiated PC12 cells, a widely used
neuronal-like model system. After differentiation, these cells show extended neurites and an increased number of synaptic-like microvesicles (Tao-Cheng et al., 1995). In our investigation, we used a PC12 subclone that is sensitive to TeNT (Sandberg et al., 1989; Schiavo et al., 1991) and displays high-affinity receptors with pharmacological features similar to those found in mammalian brain (Sandberg et al., 1989). This subclone expresses complex polysialogangliosides before NGF-differentiation (Walton et al., 1988). However, TeNT-induced blockade of ACh release is strictly dependent upon NGF (Figliomeni and Grasso, 1985; Sandberg et al., 1989), suggesting the appearance of one or more TeNT-specific receptors upon NGF differentiation. In agreement with this hypothesis, our recombinant TeNT H^did not bind to undifferentiated PC12 cells (Figure 3.6 E), but gave a punctate, discontinuous staining of the plasma membrane in NGF- differentiated cells (Figure 3.6 A), similar to the surface distribution of TeNT or native
(Figure 3.6 C and D). Binding of the recombinant TeNT is specific as it was
completely abolished by pretreatment with an excess of native (Figure 3.6 B) and is
functional, since is leads to internalisation of the toxin fragment into an endocytic compartment when the temperature is raised to 37°C (Figure 3.6 G).
To assess TeNT binding to putative protein receptors, binding and chemical
crosslinking of [^^P]-labelled recombinant TeNT (200 pM) to intact NGF-differentiated
PC12 cells were performed (experiments carried out by Dr. J. Herreros). In the presence of the homobifunctional crosslinker bis[2(succinimidyloxycarbonyloxy)ethyl]sulphone
native Hq no toxin
com petition no toxin
Figure 3.6 Binding and internalisation of TeNT and its fragments in NGF-differentiated PC 12 ceils.
Recombinant TeNT He binding at 4°C was detected with an antibody against the VSV-G tag (A, B and E-H), whereas binding of TeNT (C)
and of its native He (D) was detected with polyclonal antibodies recognising TeNT holotoxin. Note the similarity of the staining patterns
using all forms of TeNT. In B, the interaction of the recombinant He is competed by an excess (300X) of native He. Binding of He to
undifferentiated cells (E) is indistinguishable from the control without He (F). (G) NGF-differentiated PC 12 cells internalise recombinant
TeNT He incubated for 1 h at 37°C in vesicular structures absent in control cells where the toxin fragment was omitted (//). The amount
Chapter 3_______________________________________________________________Results
(BSOCOES), these experiments yielded a = 65-kDa radioactive band in addition to [^^P]- Hc (Figure 3.7, lane 3, black arrow), indicating the interaction of with a protein of ~ 15-20 kDa (pl5). The 65-kDa crosslinking product was almost absent in undifferentiated PC I2 cells (Figure 3.7, lane 1), indicating that the expression of pl5 is strongly induced
by NGF-differentiation. In similar experiments radiolabelled native generated a
crosslinking product identical to that observed with the recombinant (Figure 3.7, lane
5). This result confirms that the recombinant behaves as the native fragment and that it
is fully competent for binding to NGF-differentiated PCI2 cells.
Additional crosslinking products running at ~ 83 kDa (Figure 3.7, asterisk) and 90 kDa (not shown) were also obtained. Gel filtration experiments had shown that isolated TeNT He has the ability to self-associate and form homodimers (Herreros et al., 2000b). The appearance after crosslinking of radioactive bands with molecular mass multiple of that of TeNT He is therefore expected. Both the 83- and 90-kDa radioactive products were also observed in crosslinking experiments in the absence of cells (J. Herreros, unpublished observations). Based on these observations, the 65-kDa band appears to be the unique product formed after interaction of H^ with a putative receptor protein.
Preincubation of cells with an excess of unlabelled recombinant, native H^ or TeNT resulted in a strong inhibition of the [^^P]-Hc binding and a total disappearance of the 65- kDa band (Figure 3.8, lanes 3-5). In contrast, equal amounts of recombinant BoNT/A or /B HcS did not affect He binding nor the appearance of the crosslinking products (Figure 3.8, lanes 6-7). BoNT/E He was the only BoNT fragment that weakly competed both the binding of TeNT He and the appearance of the 65-kDa band (Figure 3.8, lane 8) (see Discussion). These experiments indicate that the 65-kDa crosslinking product results from the interaction of TeNT He with a putative protein receptor highly specific for TeNT.