Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase

Hung

et al. 2017

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Proteins belonging to the toll-like receptor (TLR) family, particularly TLR2, are the major components of innate immunity against Leptospira infection. The ligands for TLR2 harbor several conserved patterns such as lipidation molecules, leucine-rich repeat (LRR) domains, TLR2 binding motifs, and TLR2 binding structure. In Leptospira, LipL32 interacts with TLR2 on human kidney cells concomitantly stimulating inflammatory responses. However, the binding mechanism of LipL32 to TLR2 is unknown. The computational prediction suggests that β1β2, loop-α3-loop, and α4 domains of LipL32 play vital roles in LipL32-TLR2 complex formation. To test these predictions, protein truncation experiments revealed that LipL32ΔNβ1β2 significantly decreased the affinity to TLR2 while LipL32ΔCα4 slightly reduced it. Interestingly, LipL32ΔCenα3 retained affinity to TLR2 in the absence of Ca2+ ions, indicating that Cenα3 play a role preventing the interaction between LipL32 and TLR2. Furthermore, the critical residues of LipL32 involved in interacting with TLR2 suggested that V35S, L36S and L263S variants significantly decreased the affinity to TLR2. The results further confirm that LipL32 interacts with TLR2 through Nβ1β2 and Cα4 domains of LipL32 as well as LipL32-TLR2 complex formation results from hydrophobic interactions. This study provides a detailed mechanism of the interaction between LipL32 and TLR2 and the residues involved in complex formation.

Section: Methodsmentioning

confidence: 99%

Active Components of Leptospira Outer Membrane Protein LipL32 to Toll-Like Receptor 2

Hung

et al. 2017

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“…The AFM cantilever made of silicon nitride, PNP-TR (NanoWorld, Neuchâtel, Switzerland), was cleaned by sonication in a series of solvents: 2-propanol, methanol, and deionized water (5 min each), successively. The tips were functionalized according to a previous method with minor modifications (29,30,42). At first, the probes were transferred into a 0.1% (v/v) solution of 3-aminopropyltrimethoxysilane (APTES) (Sigma, St. Louis, MO) in toluene for 2 h. Following silanization, the tips were sonicated in methanol and deionized water (5 min each), consecutively.…”

Section: Afm Tip and Mica Surface Modificationmentioning

confidence: 99%

“…The force applied to the protein modified mica surface was kept below 400 pN. The distance-force curves and force parameters were obtained according to the methods previously described (29,42). For antibody neutralization analysis, the protein modified mica was treated with anti-TLR2 antibodies (diluted 1:1,000 in PBS) for 1 h followed by five washes with PBS to remove unbound antibodies.…”

Section: Afm Force-distance Curves Measurement and Analysismentioning

confidence: 99%

Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

et al. 2019

Preprint

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Leptospirosis is an overlooked zoonotic disease caused by pathogenic Leptospira. The kidney is the major organ infected by Leptospira which causes tubulointerstitial nephritis. Leptospira outer membrane components contain several virulence factors that play important roles in the pathogenesis of leptospirosis. Among them, OmpA-like protein Loa22 is essential for leptospiral virulence.However, the pathogenic mechanisms of tubulointerstitial nephritis involving this virulence factor are still unclear and need further investigation. In this study, pull-down assays suggested that Toll-like receptor 2 (TLR2) proteins interacted with Loa22 from Leptospira outer membrane extractions. Combination of Atomic force microscopy (AFM) and side-directed mutagenesis suggested that Loa22 exhibited high affinity for Leptospira peptidoglycan (LPGN) and the residues of Loa22 were involved in LPGN interaction. Mutation of two key residues within the OmpA-like domain of Loa22, Asp 122 and Arg 143 , significantly attenuated their relative affinities for LPGN indicating that these two residues were responsible for LPGN binding. Thus Loa22 OmpA domain was responsible for interacting with LPGN and the two indicated residues may participate in binding to LPGN. Recombinant Loa22 (rLoa22) protein was further complexed with LPGN and incubated with HEK293-TLR2 cells to monitor inflammatory responses. Inflammatory responses were provoked by rLoa22-LPGN complexes, but not rLoa22 alone, involved CXCL8/IL8, hCCL2/MCP-1, and hTNF-α activation. Confocal microscopy further identified the co-localization of Loa22-LPGN complexes and TLR2 receptors on HEK293-TLR2 cell surface. The affinity between Loa22-LPGN complexes and TLR2 were further confirmed and measured by AFM and ELISA. Downstream signals from TLR2 including p38, ERK, and JNK were observed by western blotting induced by Loa22-LPGN complexes. In summary, this study identified LPGN in leptospira mediates interactions between Loa22 and TLR2 and induces downstream signals to trigger inflammatory responses.Interactions between Loa22-LPGN-TLR2 reveal a novel binding mechanism for the innate immune system and infection induced by leptospira. (295 words) 3 Author summaryLeptospirosis is one of the most overlooked zoonotic diseases caused by pathogenic Leptospira in warm and humid regions worldwide. With the infection by Leptospira, many organs are invaded and can result in multiple-organ failure (Weil's syndrome). Kidney is the major organ infected by pathogenic Leptospira, which would manifest as tubulointerstitial nephritis. In this study, we focused on the outer membrane lipoprotein Loa22 (Leptospiral OmpA-like domain 22) from pathogenicLeptospira which triggers inflammatory responses on renal tubular cell. Protein domain prediction indicated that Loa22 contains an important domain termed OmpA-like domain and the function of this domain is peptidoglycan (PGN) binding. From sequence alignments of Loa22 with other OmpA proteins, two important amino acids, Asp 122 and Arg 143 , were found to be highl...

“…AFM Measurement and Analysis. The AFM cantilever tips were functionalized according to previous methods to modify the rLoa22 protein and its variants 25,26,41 . The mica surface was modified for deposition of rTLR2 protein according to previous report 42,43 .…”

Section: Expression and Purification Of Loa22 And Antibody Preparatiomentioning

confidence: 99%

“…A commercial atomic force microscope (Nanoscope III, Digital Instruments, Santa Barbara, CA) with a J type scanner was employed throughout this study. The distance-force curves and force parameters were obtained according to the methods previously described 25,41 . All the measurements described above were performed with modified tips and showed repeatedly similar results.…”

Section: Expression and Purification Of Loa22 And Antibody Preparatiomentioning

confidence: 99%

Peptidoglycan mediates Leptospira outer membrane protein Loa22 to toll-like receptor 2 for inflammatory interaction: a novel innate immune recognition

Lin

et al. 2021

Sci Rep

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Leptospirosis is an overlooked zoonotic disease caused by pathogenic Leptospira depended on virulence of Leptospira and the host–pathogen interaction. Kidney is the major organ infected by Leptospira which causes tubulointerstitial nephritis. Leptospira outer membrane contains several virulence factors and an outer membrane protein A (OmpA) like protein (Loa22) is essential for virulence. Pull-down assays suggested that Loa22 was a potential Toll-Like Receptor 2 (TLR2) binding candidates from pathogenic Leptospira. Confocal microscopy was employed to observe the co-localization of TLR2 and Loa22-LPGN (Leptospira peptidoglycan) complexes. Atomic force microscopy (AFM), side-directed mutagenesis, and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the affinity between rLoa22, LPGN, and TLR2. Real time PCR was applied to measure the cytokines expression. Downstream signal transduction components were verified by western blot to evaluate the gene regulations. Mutation of two Loa22 key residues (Asp122 and Arg143) attenuated the affinities for LPGN. rLoa22-LPGN complexes were observed to co-localize with TLR2 and provoked inflammatory responses including CXCL8/IL8, hCCL2/MCP-1, and hTNF-α. Affinity studies suggested that Loa22-LPGN complexes elevated the affinity to TLR2 as compared to Loa22 protein. Downstream signals from TLR2 including p38, ERK, and JNK were regulated under rLoa22-LPGN complexes treatments. This study identified LPGN mediates interactions between Loa22 and TLR2 and induces downstream signals to trigger inflammatory responses. rLoa22-LPGN-TLR2 complexes reveal a novel binding mechanism for the innate immune system.