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. 2013 Apr 26;288(17):12335-44.
doi: 10.1074/jbc.M112.418699. Epub 2013 Mar 13.

Essential calcium-binding cluster of Leptospira LipL32 protein for inflammatory responses through the Toll-like receptor 2 pathway

Yueh-Yu Lo  1 , Shen-Hsing Hsu, Yi-Ching Ko, Cheng-Chieh Hung, Ming-Yang Chang, Hsiang-Hao Hsu, Ming-Jeng Pan, Yen-Wei Chen, Ching-Hung Lee, Fan-Gang Tseng, Yuh-Ju Sun, Chih-Wei Yang, Rong-Long Pan
Affiliations

Essential calcium-binding cluster of Leptospira LipL32 protein for inflammatory responses through the Toll-like receptor 2 pathway

Yueh-Yu Lo et al. J Biol Chem. .

Abstract

Leptospirosis is the most widespread zoonosis caused by the pathogenic Leptospira worldwide. LipL32, a 32-kDa lipoprotein, is the most abundant protein on the outer membrane of Leptospira and has an atypical poly(Asp) motif ((161)DDDDDGDD(168)). The x-ray crystallographic structure of LipL32 revealed that the calcium-binding cluster of LipL32 includes several essential residues Asp(132), Thr(133), Asp(164), Asp(165), and Tyr(178). The goals of this study were to determine possible roles of the Ca(2+)-binding cluster for the interaction of LipL32 and Toll-like receptor 2 (TLR2) in induced inflammatory responses of human kidney cells. Site-directed mutagenesis was employed to individually mutate Ca(2+)-binding residues of LipL32 to Ala, and their effects subsequently were observed. These mutations abolished primarily the structural integrity of the calcium-binding cluster in LipL32. The binding assay and atomic force microscopy analysis further demonstrated the decreased binding capability of LipL32 mutants to TLR2. Inflammatory responses induced by LipL32 variants, as determined by TLR2 pathway intermediates hCXCL8/IL-8, hCCL2/MCP-1, hMMP7, and hTNF-α, were also lessened. In conclusion, the calcium-binding cluster of LipL32 plays essential roles in presumably sustaining LipL32 conformation for its proper association with TLR2 to elicit inflammatory responses in human renal cells.

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Figures

FIGURE 1.
FIGURE 1.
Characterization of LipL32. A, Ca2+-binding site of LipL32. The Ca2+-coordinated residues, including Asp132, Thr133, Asp164, Asp165, and Tyr178, are shown by bonds and sticks. Ca2+ is presented as a yellow-colored sphere. B, calcium-free LipL32. These figures were selected from the Protein Data Bank with code numbers of 2WFK and 3FRL for LipL32 and apo-LipL32, respectively. C, sequence alignment of LipL32 proteins (residues 129–183). Identical residues are shown in white with black background, and conserved residues with a high degree of similarity are shown in white with dark gray background. Residues mutated to Ala in this study are marked by dark asterisks.
FIGURE 2.
FIGURE 2.
Spectral analysis for LipL32 and variants. A, stains-all assay of LipL32 and its calcium-binding cluster variants. The ellipticity (θ) values are shown with a maximum peak at 660 nm. B, fluorescence spectrometry. C, CD spectra. D, thermal unfolding of LipL32 and its variants monitored by CD. Black, wild type LipL32; red, D132A variant; green, T133A variant; yellow, D164A variant; blue, D165A variant; pink, Y178A variant.
FIGURE 3.
FIGURE 3.
Interaction of LipL32 and TLR2 as determined by ELISA. A, schematic presentation of TLR2 protein. TLR2 (784 residues) is composed of leucine-rich repeats (LRR), transmembrane (TM), and Toll/IL-1 receptor (TIR) domains. Two TLR2 fragments, TLR2(49–538) and TLR2(589–784), were prepared according to methods described under “Experimental Procedures.” B, LipL32 binding to TLR2(49–538) and TLR2(589–784) with BSA as a negative control. C, binding of TLR2(49–538). The binding capability of TLR2(49–538) was examined by using synthetic lipopeptide Pam3CSK4 molecules and lipoteichoic acid (LTA), respectively; BSA was used as blank. The concentrations of LipL32, Pam3CSK4, lipoteichoic acid, and TLR2 fragments were all 2 μm.
FIGURE 4.
FIGURE 4.
Distribution of the interaction force between the LipL32-functionalized AFM tip and the TLR2(49–538) fragment on mica surface. A, AFM image of TLR2 adsorbed onto mica. The left panel is the height mode, and the right panel is the three-dimensional mode. The scale bar is 500 nm. B, control. Top panel, schematic presentation for the measurement; lower panels, interaction force between TLR2 on the mica surface and the tip modified with and without BSA, respectively. C, interaction force between LipL32 variants modified on the tip and the TLR2(49–538) fragment on the mica surface. Top panel, schematic presentation for the measurement; lower panels, interaction force between LipL32 variants modified on the tip and the TLR2(49–538) fragment on the mica surface. D, AbTLR2 neutralization of the interaction force between LipL32 and TLR2. Top panel, schematic presentation of AbTLR2 neutralization and force measurement; lower panels, interaction force between LipL32 variants modified on the tip and the TLR2(49–538) fragment on the mica surface in the presence of the AbTLR2. After treatment with AbTLR2, mica modified by TLR2(49–538) was washed to remove unbound antibody.
FIGURE 5.
FIGURE 5.
Inflammatory responses of HK2 cells induced by LipL32. A, stimulation of hCXCL8/IL8, hMMP7, hCCL2/MCP-1, and hTNF-α mRNA expression in HK2 cells. The expression was induced by wild type LipL32 treated with polymyxin, heat, and protease K, respectively. B, dose-dependent inflammatory responses in HK2 cells. The inflammatory responses were provoked by treatment with wild type LipL32 at different concentrations. C–F, inflammatory responses elicited by LipL32 variants. The expression was induced by incubation with 2.5 μg/ml of the respective LipL32 variants. Each point represents the mean absorbance of three independent experiments, and the error bars show the standard deviations.
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