Isolation of a lipopolysaccharide-binding acute phase reactant from rabbit serum

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• Acute phase reactants are proteins whose plasma concentrations increase or decrease in response to inflammation.
• Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and a potent immune stimulator.

Purpose of the Study:

• To purify and characterize a novel acute phase reactant involved in lipopolysaccharide (LPS) binding.
• To investigate the interaction between the purified protein and LPS.

Main Methods:

• Purification of LPS-binding protein (LBP) using ion-exchange chromatography (Bio-Rex 70 and Mono-Q).
• Characterization of LBP by SDS-PAGE and N-terminal amino acid sequencing.
• Assessing LPS-LBP interaction via immunoprecipitation and photoaffinity labeling.

Main Results:

• Purified LBP approximately 2,000-fold, yielding two glycoproteins (60.5 kDa and 58 kDa) in a variable ratio.
• Identical N-terminal sequences for both glycoproteins suggest a common origin.
• Demonstrated direct binding of LBP to LPS through immunoprecipitation and cross-linking experiments.

Conclusions:

• LPS-binding protein (LBP) is a newly identified acute phase reactant.
• LBP directly interacts with LPS, suggesting a role in modulating LPS's biological effects.
• LBP may represent a novel component of the innate immune response to Gram-negative bacteria.