Limits...
Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation.

Surya W, Chooduang S, Choong YK, Torres J, Boonserm P - PLoS ONE (2016)

Bottom Line: Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution.This oligomeric state did not change after incubation of these heterodimers with detergent.Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

No MeSH data available.


Related in: MedlinePlus

SDS-PAGE of the equimolar mixture act-A/act-B.(A) Equimolar mixture of act-A/act-B incubated in aqueous buffer (─), or in the presence of hydrophobic environments: liposomes (PC:PA) at the protein:lipid ratios shown, or detergents indicated. After SDS solubilization the samples were either heated (+Δ) or not heated (-Δ). The migration of monomeric act-A and act-B is indicated. All the lanes contain identical protein quantity (5 μg). The black arrows point to faint bands that correspond to a small proportion of homodimers or heterodimers.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4920411&req=5

pone.0158356.g001: SDS-PAGE of the equimolar mixture act-A/act-B.(A) Equimolar mixture of act-A/act-B incubated in aqueous buffer (─), or in the presence of hydrophobic environments: liposomes (PC:PA) at the protein:lipid ratios shown, or detergents indicated. After SDS solubilization the samples were either heated (+Δ) or not heated (-Δ). The migration of monomeric act-A and act-B is indicated. All the lanes contain identical protein quantity (5 μg). The black arrows point to faint bands that correspond to a small proportion of homodimers or heterodimers.

Mentions: The observed structural similarities between domains 3 and 4 of aerolysin and the C-terminal domain of BinB [25] suggest a somewhat similar mechanism of action for these toxins. Although both pro-aerolysin and aerolysin migrate as monomers in SDS, previous exposure to a hydrophobic environment, i.e., liposomes or non-ionic detergent polyethylene glycol monooctyl ether (octyl-POE), produced exclusively SDS-resistant heptameric oligomers [33]. To test if a similar behavior is observed in Bin toxins, an equimolar mixture of act-A and act-B was exposed to either PA/PC liposomes or detergents C14SB, DPC, and C8E5, and subsequently solubilized in SDS for electrophoresis. However, act-A and act-B only formed monomers in SDS (Fig 1). The results with and without heating were similar, although the bands appeared more compact in the former condition. The sample heated in C8E5 showed very faint bands (see arrow) that may correspond to a small percentage of homo and/or heterodimers. Therefore, in contrast with aerolysin, neither act-A or act-B, separately or as an equimolar mixture, form any SDS-resistant oligomers when exposed to hydrophobic environments, including lipid bilayers.


Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation.

Surya W, Chooduang S, Choong YK, Torres J, Boonserm P - PLoS ONE (2016)

SDS-PAGE of the equimolar mixture act-A/act-B.(A) Equimolar mixture of act-A/act-B incubated in aqueous buffer (─), or in the presence of hydrophobic environments: liposomes (PC:PA) at the protein:lipid ratios shown, or detergents indicated. After SDS solubilization the samples were either heated (+Δ) or not heated (-Δ). The migration of monomeric act-A and act-B is indicated. All the lanes contain identical protein quantity (5 μg). The black arrows point to faint bands that correspond to a small proportion of homodimers or heterodimers.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920411&req=5

pone.0158356.g001: SDS-PAGE of the equimolar mixture act-A/act-B.(A) Equimolar mixture of act-A/act-B incubated in aqueous buffer (─), or in the presence of hydrophobic environments: liposomes (PC:PA) at the protein:lipid ratios shown, or detergents indicated. After SDS solubilization the samples were either heated (+Δ) or not heated (-Δ). The migration of monomeric act-A and act-B is indicated. All the lanes contain identical protein quantity (5 μg). The black arrows point to faint bands that correspond to a small proportion of homodimers or heterodimers.
Mentions: The observed structural similarities between domains 3 and 4 of aerolysin and the C-terminal domain of BinB [25] suggest a somewhat similar mechanism of action for these toxins. Although both pro-aerolysin and aerolysin migrate as monomers in SDS, previous exposure to a hydrophobic environment, i.e., liposomes or non-ionic detergent polyethylene glycol monooctyl ether (octyl-POE), produced exclusively SDS-resistant heptameric oligomers [33]. To test if a similar behavior is observed in Bin toxins, an equimolar mixture of act-A and act-B was exposed to either PA/PC liposomes or detergents C14SB, DPC, and C8E5, and subsequently solubilized in SDS for electrophoresis. However, act-A and act-B only formed monomers in SDS (Fig 1). The results with and without heating were similar, although the bands appeared more compact in the former condition. The sample heated in C8E5 showed very faint bands (see arrow) that may correspond to a small percentage of homo and/or heterodimers. Therefore, in contrast with aerolysin, neither act-A or act-B, separately or as an equimolar mixture, form any SDS-resistant oligomers when exposed to hydrophobic environments, including lipid bilayers.

Bottom Line: Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution.This oligomeric state did not change after incubation of these heterodimers with detergent.Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

No MeSH data available.


Related in: MedlinePlus