Lipopolysaccharide binding of an exchangeable apolipoprotein, apolipophorin III, from Galleria mellonella

A new role of apolipophorin III (apoLp-III) as an immune activator has emerged recently. To gain insight into this novel function, the interaction of apoLp-III with lipopolysaccharide (LPS) was investigated. Apol-p-III from Galleria mellonella was incubated with LPS from Escherichia coli 055:135, and analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE). Protein staining showed that apoLp-III mobility was significantly reduced. In addition, silver and LPS fluorescent staining demonstrated that LPS mobility was increased upon incubation with apoLp-III. This result suggests association of apoLp-III with LPS. Sodium dodecyl sulfate (SDS) PAGE analysis showed decreased apol-p-III mobility upon LPS addition, indicative of LPS apol-p-III interaction in the presence of SDS. The unique tyrosine residue that resides in apoLp-III was used to provide additional evidence for LPS binding interaction. In the absence of LPS, apol-p-III tyrosine fluorescence was relatively low. However, LPS addition resulted in a progressive increase in the fluorescence intensity, indicating tertiary rearrangement in the environment of tyrosine 142 upon LPS interaction. Other well-characterized apoLp-IIIs were also examined for LPS binding. Manduca sexta, Bombyx mori and Locusta migratoria apol-p-III were all able to interact with LPS. The ability of apoLp-III to form complexes with LPS supports the proposed role of apol-p-III in innate immunity.