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Membrane interaction of Pasteurella multocida toxin involves sphingomyelin
Author(s) -
Brothers Michael C.,
Ho Mengfei,
Maharjan Ram,
Clemons Nathan C.,
Bannai Yuka,
Waites Mark A.,
Faulkner Melinda J.,
Kuhlenschmidt Theresa B.,
Kuhlenschmidt Mark S.,
Blanke Steven R.,
Rienstra Chad M.,
Wilson Brenda A.
Publication year - 2011
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2011.08365.x
Subject(s) - sphingomyelin , surface plasmon resonance , membrane , cell membrane , biology , biochemistry , biophysics , chemistry , microbiology and biotechnology , materials science , nanoparticle , nanotechnology
Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N‐terminus of PMT (PMT‐N) is considered to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C‐terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: (a) TLC‐overlay binding experiments with 125 I‐labeled PMT, PMT‐N or the C‐terminus of PMT; (b) pull‐down experiments using reconstituted membrane liposomes with full‐length PMT; (c) surface plasmon resonance analysis of PMT‐N binding to reconstituted membrane liposomes; (d) and surface plasmon resonance analysis of PMT‐N binding to HEK‐293T cell membranes without or with sphingomyelinase, phospholipase D or trypsin treatment. The results obtained revealed that, in our experimental system, full‐length PMT and PMT‐N did not bind to gangliosides, including monoasialogangliosides GM 1 , GM 2 or GM 3 , but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin or phosphatidylcholine with other lipid components. Collectively, these studies demonstrate the importance of sphingomyelin for PMT binding to membranes and suggest the involvement of a protein co‐receptor.