Expression and properties of an aerolysin--Clostridium septicum alpha toxin hybrid protein.
Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosyl-phosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.
Diep
D B
DB
Department of Biochemistry and Microbiology, University of Victoria, BC, Canada.
Nelson
K L
KL
Lawrence
T S
TS
Sellman
B R
BR
Tweten
R K
RK
Buckley
J T
JT
D000333:Aeromonas / Q000737:chemistry; D000818:Animals; D001427:Bacterial Toxins / Q000032:analysis / Q000737:chemistry*; D001798:Blood Proteins / Q000494:pharmacology; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002352:Carrier Proteins / Q000502:physiology; D015816:Cell Adhesion Molecules, Neuronal / Q000494:pharmacology; D002470:Cell Survival; D003013:Clostridium / Q000737:chemistry* / Q000502:physiology*; D058966:Contactins; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D047091:Eosinophil Granule Proteins; D004912:Erythrocytes / Q000378:metabolism; D058973:Folate Receptors, GPI-Anchored; D006460:Hemolysin Proteins; D006801:Humans; D051379:Mice; D052899:Pore Forming Cytotoxic Proteins; D051381:Rats; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012260:Ribonucleases; D018800:Thy-1 Antigens / Q000494:pharmacology; D013997:Time Factors; D014018:Tissue Distribution; D010738:Type C Phospholipases / Q000032:analysis / Q000737:chemistry* / Q000502:physiology; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000494:pharmacology
D001427:Bacterial Toxins; D001798:Blood Proteins; D002352:Carrier Proteins; D015816:Cell Adhesion Molecules, Neuronal; D058966:Contactins; D047091:Eosinophil Granule Proteins; D058973:Folate Receptors, GPI-Anchored; D006460:Hemolysin Proteins; D052899:Pore Forming Cytotoxic Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D018800:Thy-1 Antigens; D014643:Variant Surface Glycoproteins, Trypanosoma; C107096:proaerolysin; C032676:aerolysin; D012260:Ribonucleases; D010738:Type C Phospholipases