Identification and characterization of receptors for vacuolating activity of subtilase cytotoxin.
Some shiga toxin-producing Escherichia coli secrete a novel AB5 cytotoxin, named subtilase cytotoxin (SubAB), which induces vacuole formation in addition to cytotoxicity in susceptible cells. By immunoprecipitation with SubAB from Vero cells, we discovered proteins of 100 kDa, 135 kDa and 155 kDa as potential candidates for its receptor. These proteins were N-glycosylated in their extracellular domains, a modification that was necessary for interaction with SubAB. Biotinylated receptors were partially purified by Datura stramonium agglutinin affinity chromatography and avidin-agarose and analysed by TOF mass spectroscopy. The peptide sequences of p135 were identical to beta1 integrin, and its identification was confirmed with anti-integrin beta1 antibody. The p155 protein was identified as alpha2 integrin using anti-integrin alpha2 antibody. In addition, treatment of Vero cells with beta1 integrin RNAi before exposure to SubAB prevented vacuolating activity. These results suggested that SubAB recognizes alpha2beta1 integrin as a functional receptor; this first interaction may be an important key step leading to the SubAB-induced morphological changes in Vero cells.
Yahiro
Kinnosuke
K
Departments of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chiba, Japan.
Morinaga
Naoko
N
Satoh
Mamoru
M
Matsuura
Gen
G
Tomonaga
Takeshi
T
Nomura
Fumio
F
Moss
Joel
J
Noda
Masatoshi
M
D000818:Animals; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods; D006224:Cricetinae; D003603:Cytotoxins / Q000378:metabolism* / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D020868:Gene Silencing; D006031:Glycosylation; D045325:HCT116 Cells; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D039421:Integrin alpha2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D019012:Integrin beta1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D016023:Integrins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D011485:Protein Binding; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism*; D014709:Vero Cells
D003603:Cytotoxins; D039421:Integrin alpha2; D019012:Integrin beta1; D016023:Integrins