Abstract
Purpose: :
Lumican is a leucine-rich repeat keratan sulfate proteoglycan of the corneal stromal extracellular matrix. We showed previously that lumican regulates 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 the lumican-CD14 interactions, we aim to quantify the binding affinity between CD14 and recombinant lumican (rLum) by means of Surface Plasmon Resonance (SPR) analysis.
Methods: :
A Biacore 2000 instrument (GE Healthcare) was used to covalently link human recombinant CD14 to a CM5 (carboxymethylated) sensorchip. Stable and high immobilization of CD-14 to the sensorchip was achieved with 10uL of CD14 at 0.1mg/mL, diluted in 10mM Sodium Acetate, pH 4.0, injected at 5uL/min. The rate constant of association and dissociation were derived at three different injection concentrations of rLum. Interactions of immobilized CD-14 to non-specific rabbit IgG and an anti CD-14 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).
Results: :
The shape of the sensorgrams from injecting rLum compared to that obtained with nonspecific IgG as a negative control, indicated binding between rLum and CD14. The association rate constant (ka) of lumican for CD14 was 4.15 x 104M-1s-1, and the dissociation rate constant (kd) was 2.72 x 10-4s-1, revealing fast association and dissociation with an affinity constant KA of 1.53 x 108 M-1 at 25°C. As expected the positive control indicated a much higher KA of 2.36 x 1012 M-1 between CD-14 and anti CD-14 IgG.
Conclusions: :
The findings indicate strong and specific binding between lumican and CD14, underscoring an important role for lumican in innate immune response to bacterial LPS in the cornea. Further studies are underway to elucidate lumican, CD14 and LPS interactions.
Keywords: cornea: basic science • proteoglycans/glycosaminoglycans • inflammation