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S. Lee, K. Lohr, S. Chakravarti; Kinetic Analysis to Determine the Interactions Between CD14 and Lumican. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6241.
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© ARVO (1962-2015); The Authors (2016-present)
Lumican is a leucine-rich repeat keratan sulfate proteoglycan of the corneal stromal extracellular matrix. We showed previously that lumican regulates the recognition of endotoxic lipopolysaccharide (LPS) of gram-negative bacteria through its interactions with CD14, the LPS-binding adaptor protein for the Toll-like receptor 4 (TLR4) signaling pathway (2007, JBC282:26409). To further characterize lumican-CD14 interactions, we aim to quantify the binding affinity between CD14 and recombinant lumican (rLum) or its mutants by means of Surface Plasmon Resonance (SPR) analysis.
A BIAcore 3000 instrument (Amersham Pharmacia Biotech, Uppsala, Sweden) was used to covalently link human recombinant CD14 to a CM5 (carboxymethylated) sensorchip. Stable and high immobilization of CD14 to the sensorchip was achieved with 50 µL of CD14 at 25 µg/mL in 10mM Sodium Acetate, pH 4.5, injected at 10 µL/min. The rate constant of association and dissociation were derived at eight different injection concentrations of rLum or Y30F mutant. Interactions of immobilized CD14 to non-specific rabbit IgG and an anti CD14 IgG were used as negative and positive controls respectively. Kinetic constants were analyzed using a model for 1:1 (Langmuir) fitting of the sensorgram data using BIAevaluation 4.1 Software (BIAcore AB, Sweden).
The shape of the sensorgrams from injecting rLum, compared to those obtained with nonspecific IgG as a negative control, indicated binding between rLum and CD14. The affinity constant (KA) of lumican for CD14 was 3.5 x 104 M-1 and the dissociation constant (KD) was 2.86 x 10-5 M. The KA of Y30F mutant lumican for CD14 was 6.1 x 104 M-1 and the KD was 1.65 x 10-5 M, showing a 50% decrease in interaction with CD14.
These results indicate that the N-terminal region of lumican may play a crucial role in the interaction between lumican and CD14. The findings reveal specific binding between lumican and CD14, underscoring an important role for lumican in innate immune response in the cornea. Further studies are underway to generate more mutant forms and fragments of lumican to better elucidate the specific interactions that occur between lumican and CD14.
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