Heterodimeric integrin complexes containing beta1-integrin promote internalization and lethality of anthrax toxin.
To kill macrophages, the lethal factor component of Bacillus anthracis toxin binds to a carrier protein (PA), which then interacts with the CMG2 receptor protein on the cell surface and is endocytosed into the cytoplasm. CMG2, as well as TEM8, a second PA receptor not present on macrophages, contain a von Willebrand A domain that is crucial for toxin binding. Here we report that integrin beta1, another cell surface von Willebrand A domain protein, can mediate and potentiate anthrax toxin endocytosis. By using microarray-based analysis to globally correlate gene expression profiles with toxin sensitivity, we associated toxin effects with the integrin-activating proteins osteopontin and CD44. Further study showed that PA binds to alpha4beta1- and alpha5beta1-integrin complexes, leading to their conjoint endocytosis, and also interacts-weakly relative to CMG2 but comparably to TEM8--with purified alpha5beta1 complex in vitro. Monoclonal antibody directed against beta1-integrin or its alpha integrin partners reduced PA/integrin endocytosis and anthrax toxin lethality, and hyaluronic acid--which interferes with CD44-mediated integrin activation--had similar effects. Remarkably, whereas deficiency of CMG2 protected macrophages from rapid killing by large toxin doses (>50 ng/mL), by 24 h the toxin-treated cells were dead. Such late killing of CMG2-deficient cells by high dose toxin as well as the late death observed during exposure of CMG2-producing macrophages to low-dose toxin (<1 ng/mL), was dependent on integrin function. Effects of inactivating both CMG2 and integrin were synergistic. Collectively, our findings argue strongly that beta1-integrin can both potentiate CMG2-mediated endocytosis and serve independently as a low-affinity PA receptor.
Martchenko
Mikhail
M
Departments of Genetics and Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
Jeong
Sun-Young
SY
Cohen
Stanley N
SN
D000818:Animals; D000942:Antigens, Bacterial / Q000378:metabolism* / Q000494:pharmacology; D001427:Bacterial Toxins / Q000378:metabolism* / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004705:Endocytosis; D020869:Gene Expression Profiling; D018922:HL-60 Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D039041:Integrin alpha4beta1 / Q000378:metabolism; D039081:Integrin alpha5beta1 / Q000378:metabolism; D019012:Integrin beta1 / Q000737:chemistry / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D053495:Osteopontin / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D034622:RNA Interference; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D018000:Receptors, Peptide; D020133:Reverse Transcriptase Polymerase Chain Reaction
C473287:ANTXR1 protein, human; C447150:ANTXR2 protein, human; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D002352:Carrier Proteins; D018960:Hyaluronan Receptors; D039041:Integrin alpha4beta1; D039081:Integrin alpha5beta1; D019012:Integrin beta1; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C030325:anthrax toxin; D053495:Osteopontin