Article

Heat shock protein 60: specific binding of lipopolysaccharide.

German Diabetes Clinic, German Diabetes Center, Leibniz Institute, Heinrich Heine University, Dusseldorf, Germany.
The Journal of Immunology (impact factor: 5.79). 03/2005; 174(3):1298-305. pp.1298-305
Source: PubMed

ABSTRACT Human heat shock protein 60 (HSP60) has been shown to bind to the surface of innate immune cells and to elicit a proinflammatory response. In this study we demonstrate that the macrophage stimulatory property of recombinant human HSP60 is tightly linked to the HSP60 molecule and is lost after protease treatment. However, inhibition of macrophage stimulation was reached by the LPS-binding peptide magainin II amide. Indeed, HSP60 specifically bound [(3)H]LPS. [(3)H]LPS binding to HSP60 was saturable and competable by the unlabeled ligand. To identify the epitope region of the HSP60 molecule responsible for specific LPS binding, we analyzed the effect of several anti-HSP60 mAbs on HSP60-induced production of inflammatory mediators from macrophages. We identified only one mAb, clone 4B9/89, which blocked the macrophage stimulatory activity of the chaperone. The epitope specificity of this mAb points to the region aa 335-366 of HSP60. Clone 4B9/89 also strongly inhibited [(3)H]LPS binding to HSP60. A more detailed analysis was performed by screening with selected overlapping 20-mer peptides of the HSP60 sequence, covering the region aa 331-380. Only one peptide blocked LPS binding to HSP60, thereby restricting the potential LPS-binding region to aa 351-370 of HSP60. Finally, analysis of selected 15-mer peptides and a 13-mer peptide of the HSP60 sequence revealed that most of the LPS-binding region was accounted for by aa 354-365 of HSP60, with the motif LKGK being critical for binding. Our studies identified a defined region of HSP60 involved in LPS binding, thereby implicating a physiological role of human HSP60 as LPS-binding protein.

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Keywords

15-mer peptides
 
defined region
 
detailed analysis
 
epitope region
 
HSP60 sequence
 
HSP60-induced production
 
Human heat shock protein 60
 
human HSP60
 
innate immune cells
 
LPS binding
 
LPS-binding peptide magainin II amide
 
LPS-binding protein
 
LPS-binding region
 
macrophage stimulation
 
macrophage stimulatory property
 
motif LKGK
 
potential LPS-binding region
 
proinflammatory response
 
recombinant human HSP60
 
specific LPS binding
 

Christiane Habich