MD-2-dependent human Toll-like receptor 4 monoclonal antibodies detect extracellular association of Toll-like receptor 4 with extrinsic soluble MD-2 on the cell surface
| Autor: | Ritsu Ito, Hiroki Tsukamoto, Yoshihisa Tomioka, Masao Kimoto, Yoshitaka Ikeda, Naoto Suzuki, Ippo Ukai, Hideyuki Ihara |
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| Rok vydání: | 2013 |
| Předmět: |
Lipopolysaccharides
Protein Conformation medicine.drug_class Antibody Affinity Lipopolysaccharide Receptors Lymphocyte Antigen 96 Biophysics CHO Cells Biology Monoclonal antibody Biochemistry Epitope Cell Line Cricetulus Cricetinae medicine Extracellular Animals Humans Receptor Molecular Biology Toll-like receptor Chinese hamster ovary cell Antibodies Monoclonal Cell Biology Molecular biology Toll-Like Receptor 4 Biotinylation TLR4 lipids (amino acids peptides and proteins) |
| Zdroj: | Biochemical and Biophysical Research Communications. 440:31-36 |
| ISSN: | 0006-291X |
| DOI: | 10.1016/j.bbrc.2013.09.004 |
| Popis: | MD-2 is essential for lipopolysaccharide (LPS) recognition of Toll-like receptor 4 (TLR4) but not for cell surface expression. The TLR4/MD-2 complex is formed intracellularly through co-expression. Extracellular complex formation remains a matter for debate because of the aggregative nature of secreted MD-2 in the absence of TLR4 co-expression. We demonstrated extracellular complex formation using three independent monoclonal antibodies (mAbs), all of which are specific for complexed TLR4 but unreactive with free TLR4 and MD-2. These mAbs bound to TLR4-expressing Ba/F3 cells only when co-cultured with MD-2-secreting Chinese hamster ovary cells or incubated with conditioned medium from these cells. All three mAbs bound the extracellularly formed complex indistinguishably from the intracellularly formed complex in titration studies. In addition, we demonstrated that two mAbs lost their affinity for TLR4/MD-2 on LPS stimulation, suggesting that these mAbs bound to conformation-sensitive epitopes. This was also found when the extracellularly formed complex was stimulated with LPS. Additionally, we showed that cell surface TLR4 and extrinsically secreted MD-2 are capable of forming the functional complex extracellularly, indicating an additional or alternative pathway for the complex formation. |
| Databáze: | OpenAIRE |
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