Widespread Sequence Variations in VAMP1 across Vertebrates Suggest a Potential Selective Pressure from Botulinum Neurotoxins

Abstract

Botulinum neurotoxins (BoNT/A-G), the most potent toxins known, act by cleaving three SNARE proteins required for synaptic vesicle exocytosis. Previous studies on BoNTs have generally utilized the major SNARE homologues expressed in brain (VAMP2, syntaxin 1, and SNAP-25). However, BoNTs target peripheral motor neurons and cause death by paralyzing respiratory muscles such as the diaphragm. Here we report that VAMP1, but not VAMP2, is the SNARE homologue predominantly expressed in adult rodent diaphragm motor nerve terminals and in differentiated human motor neurons. In contrast to the highly conserved VAMP2, BoNT-resistant variations in VAMP1 are widespread across vertebrates. In particular, we identified a polymorphism at position 48 of VAMP1 in rats, which renders VAMP1 either resistant (I48) or sensitive (M48) to BoNT/D. Taking advantage of this finding, we showed that rat diaphragms with I48 in VAMP1 are insensitive to BoNT/D compared to rat diaphragms with M48 in VAMP1. This unique intra-species comparison establishes VAMP1 as a physiological toxin target in diaphragm motor nerve terminals, and demonstrates that the resistance of VAMP1 to BoNTs can underlie the insensitivity of a species to members of BoNTs. Consistently, human VAMP1 contains I48, which may explain why humans are insensitive to BoNT/D. Finally, we report that residue 48 of VAMP1 varies frequently between M and I across seventeen closely related primate species, suggesting a potential selective pressure from members of BoNTs for resistance in vertebrates.

Activity of botulinum neurotoxin type D (strain 1873) in human neurons

“…Several reasons might account for that, including preferential growth of BoNT/D producing Clostridia in animal feed, ingestions of larger amounts of contaminated foods (and BoNT/D) by animals, potential colonization of animal's digestive tracts with toxin producing Clostridia, potential poor absorption of BoNT/D from the human gastrointestinal tract, and certainly VAMP1 polymorphism (29), and as our data indicate, potentially variable cell entry.…”

“…Compared to BoNT/A1, this is more than 150 times lower sensitivity in iCell Neurons and about 25 times reduced sensitivity in HIP Neurons (28). Cleavage of VAMP-1 could not be evaluated due to low expression in these cells, but has been examined in detail in another recent study (29). …”

“…These data indicate potential distinct pharmacological properties of BoNT/D compared to BoNT/A1 in the ICR mouse strain and greater similarity with BoNT/B1, which also cleaves VAMP1 and 2 and causes milder botulism than BoNT/A1. A recent study has identified a VAMP1 polymorphism (M/148) in the BoNT/D cleavage site in the commonly used Sprague Dawley rat strain, which affects BoNT/D sensitivity (29). Even though no such polymorphism is known for ICR mice or suggested by the data, such or similar variation cannot be entirely excluded in the outbred ICR mouse strain at this point.…”

“…BoNT/D is not considered to be a cause of human botulism, and this was supported by a trial study in humans which indicated a poor electrophysiological response to up to 10 U of BoNT/D injected in the EDB muscle (19) and studies that indicate that human VAMP1 is less sensitive to cleavage by BoNT/D than VAMP2 (16-18, 29). Another study has demonstrated that at high concentrations of BoNT/D, human intercostal muscle preparations are sensitive to BoNT/D (21).…”

“…All of these data together suggest that humans might be susceptible to BoNT/D intoxication if exposed to large enough quantities, but that potency of BoNT/D in humans is significantly lower than for BoNT/A, B, E, or F. The single human botulism outbreak described for BoNT/D supports this hypothesis (4). The absence of other reports of human botulism caused by BoNT/D may be due to several factors including non-reporting or misdiagnosis of mild cases, low occurrence of intoxication, differential sensitivity of human motor-neurons to BoNT/D due to a VAMP-1 polymorphism (29), poor intestinal absorption, and possibly low occurrence of toxigenic strains producing BoNT/D in the environment, as well as low levels of BoNT/D production by toxigenic strains.…”