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Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Published on: May 28, 2012
Purification and characterization of murine lipopolysaccharide-binding protein
P Gallay1, S Carrel, M P Glauser
1Department of Internal Medicine, CHUV-Lausanne, Switzerland.
Insights
Murine lipopolysaccharide-binding protein (LBP) was purified and characterized, showing high similarity to human and rabbit LBP. This protein enhances monocyte sensitivity to LPS, acting as an acute-phase protein in mice.
Area of Science:
- Immunology
- Biochemistry
Background:
- Lipopolysaccharide-binding protein (LBP) is a serum protein crucial for regulating host responses to lipopolysaccharide (LPS).
- LPS-LBP complexes stimulate immune cells like monocytes and macrophages via CD14, but murine LBP properties were largely unknown.
Purpose of the Study:
- To purify and characterize murine lipopolysaccharide-binding protein (LBP).
- To investigate the functional properties of mouse LBP in promoting LPS-monocyte interactions.
Main Methods:
- Ion-exchange chromatography and high-pressure liquid chromatography were used for murine LBP purification.
- NH2-terminal sequencing determined amino acid identity with other species' LBPs.
- Monocyte-LPS binding assays assessed LBP's functional role.
Main Results:
- Murine LBP exhibited high sequence similarity (80-90% amino acid identity) to human and rabbit LBP.
- Purified mouse LBP significantly promoted LPS binding to monocytes.
- LBP enhanced monocyte sensitivity to LPS by over 100-fold and was identified as an acute-phase protein.
Conclusions:
- Murine LBP shares structural and functional similarities with LBP from other species.
- Mouse LBP plays a key role in modulating innate immune responses to LPS.
- Further in vivo studies are warranted to elucidate LBP's precise role in endotoxemia models.
Abstract:
The serum protein lipopolysaccharide (LPS)-binding protein (LBP) seems to play an important role in regulating host responses to LPS. Complexes of LPS and LBP form in serum and stimulate monocytes, macrophages, or polymorphonuclear leukocytes after binding to CD14. Previous reports have described the structure and properties of LBP from human and rabbit sera. Since mice are used in some experimental models of endotoxemia or gram-negative bacterial infections, information is needed about the properties of murine LBP. Murine LBP was purified by ion-exchange chromatography and high-pressure liquid chromatography; its NH2-terminal sequence (TNPGLVTRIT) was very similar to those of human and rabbit LBPs (80 to 90% amino acid identity). Murine LBP resembled LBPs from other species in that it promoted the binding of LPS to monocytes and enhanced the sensitivity of monocytes to LPS at least 100-fold. Mouse LBP, like rabbit and human LBPs, was found to be an acute-phase protein. Further in vivo studies with mice and anti-CD14 or anti-LBP reagents should help determine the role of LBP in response to LPS challenges.
