Apolipoproteins are abundant serum proteins and well known for their role in lipid transport processes. Their importance in health and disease, in particular cardiovascular disease has been well established. It has become increasingly clear that apolipoproteins are an important component of the innate immune system. In that role, apolipoproteins have the ability to bind and neutralize lipopolysaccharides (LPS), which are abundantly present in the outer membrane of Gram-negative bacteria. When released in the circulation, LPS cause septic shock, a major cause of death in intensive care units. In the current proposal we aim to provide a molecular basis for the apolipoprotein-LPS interaction. To accomplish this, we will use invertebrate apolipophorin III (apoLp-III) as a model, since a wealth of structural information is available for this protein. Human apoA-I will be employed to complement our studies. It is hypothesized that apoLp-III is a pattern recognition protein, binding and neutralizing a variety of cell wall components of microbial invaders, most noticeably LPS. The flexible a-helical structure of the protein accommodates for large conformational changes, and is a key feature that facilitates the LPS binding interaction. Using recombinant apolipoprotein and various LPS variants, the binding interaction will be studied in solution using a combination of molecular biology, biochemical and biophysical analysis. The research plan includes the following specific aims. (i) A thorough biophysical characterization of the LPS/apoLp-III complex to gain insight in the apolipoprotein-LPS binding interaction. (ii) Elucidate the role of LPS-carbohydrate in the binding interaction. The importance of LPS carbohydrate and the need for a large protein conformational change will be investigated using molecular spectroscopy with single tryptophan and double cysteine mutant proteins. (iii) Since charge plays an important role in the LPS binding interaction with human apoA-I, key lysine residues in apoA-I necessary for LPS binding will be identified. Using a site-directed mutagenesis approach, lysine residues which are part of the apolipoprotein-LPS binding interaction will be identified. In conclusion, by employing a well established model protein for the structure-function relationship of exchangeable apolipoproteins in conjunction with human apoAI, important insights in the molecular mechanism of apolipoprotein-LPS interaction will be obtained. This knowledge can be used to improve treatment and develop new strategies to treat Gram-negative sepsis.

Public Health Relevance

Bacterial sepsis is a common threat causing more than 200,000 fatalities each year in the US. Apolipoproteins, well known for their role in lipid and cholesterol transport, are likely to play a vital role in innate immunity, by neutralizing lipopolysaccharides released from invading bacteria which are responsible for sepsis which often results in shock and death. The proposed research aims to understand the molecular basis of the protective role of apolipoproteins, thereby providing a foundation for improving the treatment of bacterial sepsis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Continuance Award (SC3)
Project #
5SC3GM089564-03
Application #
8208976
Study Section
Special Emphasis Panel (ZGM1-MBRS-X (CH))
Program Officer
Rogers, Michael E
Project Start
2010-01-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
3
Fiscal Year
2012
Total Cost
$106,549
Indirect Cost
$32,299
Name
California State University Long Beach
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
006199129
City
Long Beach
State
CA
Country
United States
Zip Code
90840
Beck, Wendy H J; Adams, Christopher P; Biglang-Awa, Ivan M et al. (2013) Apolipoprotein A-I binding to anionic vesicles and lipopolysaccharides: role for lysine residues in antimicrobial properties. Biochim Biophys Acta 1828:1503-10
Moreno-Habel, Daniela A; Biglang-awa, Ivan M; Dulce, Angelica et al. (2012) Inactivation of the budded virus of Autographa californica M nucleopolyhedrovirus by gloverin. J Invertebr Pathol 110:92-101
Wan, Chung-Ping Leon; Chiu, Michael H; Wu, Xinping et al. (2011) Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks. Biochim Biophys Acta 1808:606-13