1, a heterodimer of CD11a/CD18, and to perform preliminary studies of. Ltx/LFA-1 interaction in aqueous solution. A leucine zipper sequence was engineered ...
# 1096
Interaction of Actinobacillus actinomycetemcomitans leukotoxin (Ltx) with soluble LFA-1 (sLFA-1) Somesh Baranwal, Irene R. Kieba, Meryl Sava, Karen P. Fong Sharon Willis, Ann Rux and Edward T. Lally University of Pennsylvania Philadelphia PA, 19104
Introduction and Background
1b Analysis of purified soluble LFA-1
Actinobacillus actinomycetemcomitans is the primary cause of localized juvenile periodontitis in children. It produces several virulence factors to evade the host immune system. One of these virulence factors is leukotoxin which belongs to the RTX group of toxins. A. actinomycetemcomitans leukotoxin induces cell death in immune cells such as PMNs, lymphocytes and macrophages of humans, Great Apes and Monkeys. Although we have identified LFA-1 as a receptor for the leukotoxin and established that lipid rafts (membrane microdomains rich in cholesterol and sphingolipids) play a critical role in toxin activity, little is known about the kinetics of toxin/receptor interaction, since native LFA-1 is a transmembrane protein and insoluble in the aqueous environment.
Purpose and Hypothesis The primary aim of this project is to express and purify the soluble form of LFA1, a heterodimer of CD11a/CD18, and to perform preliminary studies of Ltx/LFA-1 interaction in aqueous solution. A leucine zipper sequence was engineered into the carboxy-terminal sequences of CD11a and CD18, and functions as a molecular clamp holding the LFA-1 αβ heterodimer together.
Results 1a. Expression and purification of soluble LFA-1
Figure 5a. Principle of Surface Plasmon Resonance
Figure 2. Native, SDS-PAGE and Western blot analysis of purified sLFA-1. Gels were stained with Coomassie Blue. sLFA-1 was detected in Western blots using mouse anti-human CD11a (Pharmingen) and mouse anti- human CD18 (CBR LFA1/2) primary antibodies and goat anti-mouse HRP secondary antibodies.
2. Soluble LFA-1 inhibits Ltx-mediated cytotoxicity A cytotoxicity assay was performed to analyze the interaction of sLFA-1 with leukotoxin. Varying concentrations of purified sLFA-1 were incubated with 10-9M leukotoxin for 30 min at 370C before the addition of Jurkat (Jn.9) cells and further incubated at 37oC for 2 hours. The viability of Jn.9 cells was determined after staining with trypan blue. 2x10-7 M sLFA-1 completely inhibited cell death mediated by leukotoxin.
3. Soluble LFA-1 binds to Ltx
The genes encoding CD11a and CD18 were truncated at their transmembrane region and fused in frame with 47 amino acid acidic and basic leucine zippers respectively. The leucine zipper was synthesized from GENEART and is optimized to express in mammalian cells by codon usage software. The genes encoding CD11a-ALZ in pCDNA6-V5 and CD18-BLZ in pCDNA3.1 were conucleofacted in CHO lec3.2.8.1 and positive clones were selected in presence of geneticin (G418) and blasticidin S. Clones secreting high amounts of sLFA-1 were screen by monoclonal antibody to leucine zipper (α-velcro). Soluble LFA-1 (sLFA-1) was purified from the cell culture supernatant by immunoaffinity chromatography (coupling TS2/4 to Sepharose 4B) and then by gel filtration chromatography over a superdex 200 column. The average yield of sLFA-1 was 800 µg/l of supernatant.
printed by
www.postersession.com
Enzyme linked immunosorbent assay (ELISA) was performed to determine if sLFA-1 binds leukotoxin. Either sLFA-1 or leukotoxin was dissolved in PBS and coated onto wells of a 96-well microtiter plate and kept overnight at 4oC. Wells were blocked with 1%BSA+PBST and varying concentrations of Ltx and sLFA-1 were added respectively. The plate was washed and either monoclonal antibody to leucine zipper or rabbit polyclonal antibody to leukotoxin was added. Peroxidase activity was detected at OD 450nm using HRP conjugated secondary antibody with TMB as a substrate.
The Biacore 2000 instrument was used to determine the real time interaction of leukotoxin with sLFA-1. All experiments were performed in HBS-P buffer. 800 RU of leukotoxin (dissolved in sodium acetate pH 5.5) were immobilized on a CM5 chip by the amine coupling method. Varying concentrations of sLFA-1 (in HBS-P buffer) were passed over the immobilized Ltx at the flow rate of 10 µl/min. Data was recorded for 2 min for association and 4 min for dissociation.. Ka, Kd and KD was determined using BIAEVALUATION® software version 4.1 following the 1:1 langmuir binding model. The data represent an average of 3 independent injections.
Figure 5b. Response vs time plot (sensogram).
Figure 6. Biacore Analysis of LTX/sLFA-1 interaction
Kinetic constant of LTX/sLFA-1 interaction.
Figure 3. Cytotoxicity analysis of sLFA-1. Values represent mean + SEM of triplicate readings of a representative experiment. *p
