Identification and characterization of bovine Lipopolysaccharide-binding protein

Author

Lajwanti S. Khemlani

Date of Award

5-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Philip N. Bochsler

Committee Members

David Slauson, Albert Ichiki, Roher Carroll

Abstract

Lipopolysaccharides (LPSs, endotoxin) are a major component of the outer membrane of gram-negative bacteria. LPS possesses little intrinsic biological activity. However, within the host, it is capable of playing a major role in the production of several potent proinflammatory mediators. These mediators or cytokines in moderate amounts are crucial for host defense. However, excessive production of these mediators can lead to septicemia, which can prove to be fatal. Several recent studies have indicated that the host has developed endogenous regulatory mechanisms in order to deal with bacterial infections. It has also been shown that some of these immunoregulatory mechanisms include proteins which, upon binding to LPS, can either potentiate or attenuate its effects. A 60 kDa LPS-binding protein (LBP) has recently been identified and characterized from rabbit, murine and human serum. LBP binds to LPS with high affinity and the LPS-LBP complex interacts with a membrane bound receptor called CD 14. This interaction has been shown to shift the threshold for LPS induced activation of cells such as monocytes and macrophages. Previous studies done with other systems has led to the speculation of the existence of such an analog in cattle. This research project shows the presence of an analogous LPS-binding protein in the bovine serum. Using photoaffinity labeling studies, a 60 kDa LPS-binding protein has been identified in the adult normal bovine serum. Fractionation of this serum using ion-exchange chromatography (Bio-Rex 70 column and HPLC, Mono Q column) and photoaffinity labeling studies utilizing ¹²⁵I-ASD-LPS has enabled the identification of a pool or fraction of serum that contains the 60 kDa LPS-binding protein. These studies show that the 60 kDa protein elutes at 220 mM NaCl. NH₂-terminal sequencing of the first 20 amino acids has shown that this 60 kDa protein has significant sequence homology with previously characterized LBPs. Furthermore, tissue factor assays done with bovine alveolar macrophages stimulated with 1 ng/ml LPS in combination with normal bovine serum and bovine serum fractions containing the 60 kDa LPS-binding protein, obtained at two different stages of purification, has shown that the action of LPS is augmented from 100-1600 fold in the presence of bovine serum and the above protein fractions. The stimulatory effect of LPS-LBP complex has been shown to be mediated through the CD 14 receptor. This has been shown by including two concentrations of anti-human CD14 monoclonal antibodies (10 and 20 <μg/ml) in the biological assays. Heating protein fractions at 56°C, 60°C and 75°C prior to addition to macrophages has shown that this is a heat labile protein. Incubation of polymyxin B (10 μg/ml) with LPS before the tissue factor assay has shown a reduction in the tissue factor expression. This study suggests that the protein fractions used in the assays bind to the lipid A region of LPS.

Recommended Citation

Khemlani, Lajwanti S., "Identification and characterization of bovine Lipopolysaccharide-binding protein. " PhD diss., University of Tennessee, 1994.
https://trace.tennessee.edu/utk_graddiss/10388