title: Essential calcium-binding cluster of Leptospira LipL32 protein for inflammatory responses through the Toll-like receptor 2 pathway.
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.
authors.affiliation: Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, National Tsing Hua University, Hsin Chu 30013, Taiwan.
authors.forename: Yueh-Yu
authors.initials: YY
authors.lastname: Lo
authors.forename: Shen-Hsing
authors.initials: SH
authors.lastname: Hsu
authors.forename: Yi-Ching
authors.initials: YC
authors.lastname: Ko
authors.forename: Cheng-Chieh
authors.initials: CC
authors.lastname: Hung
authors.forename: Ming-Yang
authors.initials: MY
authors.lastname: Chang
authors.forename: Hsiang-Hao
authors.initials: HH
authors.lastname: Hsu
authors.forename: Ming-Jeng
authors.initials: MJ
authors.lastname: Pan
authors.forename: Yen-Wei
authors.initials: YW
authors.lastname: Chen
authors.forename: Ching-Hung
authors.initials: CH
authors.lastname: Lee
authors.forename: Fan-Gang
authors.initials: FG
authors.lastname: Tseng
authors.forename: Yuh-Ju
authors.initials: YJ
authors.lastname: Sun
authors.forename: Chih-Wei
authors.initials: CW
authors.lastname: Yang
authors.forename: Rong-Long
authors.initials: RL
authors.lastname: Pan
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018932:Chemokine CCL2 / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics; D007668:Kidney / Q000378:metabolism* / Q000473:pathology; D007919:Leptospira / Q000235:genetics / Q000378:metabolism*; D007922:Leptospirosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D020783:Matrix Metalloproteinase 7 / Q000096:biosynthesis / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D015398:Signal Transduction; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; C111835:CCL2 protein, human; C508606:CXCL8 protein, human; D018932:Chemokine CCL2; D016209:Interleukin-8; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins; C495342:TLR2 protein, human; D051195:Toll-Like Receptor 2; D014409:Tumor Necrosis Factor-alpha; C060932:MMP7 protein, human; D020783:Matrix Metalloproteinase 7