Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Hauk, Pricila; Guzzo, Cristiane R.; Ramos, Henrique Roman; Ho, Paulo Lee; Farah, Chuck S.

doi:10.1016/j.jmb.2009.05.034

Cited by 41 publications

(55 citation statements)

References 77 publications

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“…Wild-type LipL32 with or without an N-terminal His tag fusion and a recombinant C-terminal fragment corresponding to residues 185-272 plus an N-terminal His tag (LipL32 185-272_His tag ) were produced as described (12,14). Mutations were introduced into the lipL32 gene by a PCR-driven overlap extension protocol (17) using as template the previously described plasmid lipL32ϩpAE (14).…”

Section: Methodsmentioning

confidence: 99%

“…Intrinsic Tryptophan Fluorescence and Ca 2ϩ Binding StudiesFor calcium-binding studies, proteins were decalcified as previously described (14). Briefly, proteins were incubated for 60 min at 25°C in 3 mM EDTA followed by buffer exchange with chelating Sepharose-treated Tris-HCl buffer.…”

Section: Methodsmentioning

confidence: 99%

“…Fluorescence spectroscopy studies were carried out using a 1 cm path-length quartz cell and an Aviv ATF105 Fluorimeter or a Hitachi F4500 Fluorimeter with 1-nm excitation bandwidth and 10-nm emission bandwidth. Intrinsic tryptophan fluorescence was measured with an excitation wavelength set at 285 nm and emission spectra were recorded between 310 and 400 nm using 2 M LipL32 (or mutants) in 10 mM Tris-HCl (pH 8.0), 50 mM KCl in the presence or absence of 1 mM CaCl 2 (14). Calcium titration experiments were performed using 2 M LipL32 wild-type or mutant LipL32 in 10 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.5 mM EGTA.…”

Section: Methodsmentioning

confidence: 99%

“…Our group has recently demonstrated that LipL32 specifically binds Ca 2ϩ ions and that this binding increases LipL32 conformational stability (14). The crystal structure of apoLipL32 (14,15) and the calcium-bound structure (16) led to the characterization of the significant structural changes associated with metal binding that includes strand-coil transitions and a 10-Å translocation of an aspartate-rich loop to form part of the Ca 2ϩ -binding site.…”

mentioning

confidence: 99%

“…The crystal structure of apoLipL32 (14,15) and the calcium-bound structure (16) led to the characterization of the significant structural changes associated with metal binding that includes strand-coil transitions and a 10-Å translocation of an aspartate-rich loop to form part of the Ca 2ϩ -binding site. These observations raised the question regarding the role of Ca 2ϩ in the binding of LipL32 to its target proteins.…”

mentioning

confidence: 99%

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Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

Hauk

Barbosa

et al. 2012

Journal of Biological Chemistry

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LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca 2؉ . Recent crystal structures have been obtained for the protein in the apo-and Ca 2؉ -bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca 2؉ and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca 2؉ binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca 2؉ affinity as the wild-type protein. We then evaluated if Ca 2؉ binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca 2؉ ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

Hauk

Barbosa

et al. 2012

Journal of Biological Chemistry

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Multiepitope‐based vaccine design by exploring antigenic potential among leptospiral lipoproteins using comprehensive immunoinformatics and structure‐based approaches

Kumar

Shiraz

Akif

2022

Biotech and App Biochem

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Leptospirosis is a tropical and globally neglected zoonotic disease caused by pathogenic spirochetes, Leptospira. Although the disease has been studied for decades, a potent or effective vaccine is not available so far. Efforts are being made to design an efficient vaccine candidate using different approaches. Immunoinformatics approaches have been proven to be promising in terms of time and cost.Here, we used immunoinformatics and structure-based approaches to evaluate antigenic B-and T-cell epitopes present on the leptospiral lipoproteins (LipL).The promiscuous overlapping epitopes (B-cell, T-cell, interferon (IFN)-γ positive, and non-allergens), which can induce humoral, cell-mediated, and innate immunity, were selected to generate a multiepitope chimeric vaccine. To enhance the vaccine immunogenicity, a Toll-like receptor (TLR) agonist was fused to the vaccine with a suitable linker. The chimeric vaccine structure was predicted for molecular docking studies with immune receptors. Moreover, the stability of the vaccine-immune receptor complexes was analyzed by normal mode analysis (NMA). The potency of the vaccine construct was predicted by the immune simulation tool. The study provides additional information toward constructing peptide-based chimeric vaccines against Leptospira.

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Generation‐Dependent Host–Guest Interactions: Solution States of Polyglycerol Dendrimers of Generations 3 and 4 Modulate the Localization of a Guest Molecule

Lee

Ooya

2012

Chemistry A European J

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Host-guest interactions between polyglycerol dendrimers of generations 3 and 4 (PGD-G3 and G4) and 4-amino-3-hydroxynapthalene-2-sulphonic acid (AHSA) were investigated by fluorescence spectroscopy, isothermal titration calorimetry (ITC), and dynamic light scattering (DLS). PGD-G3 molecules were found to form an associated state with an average diameter of 82.7 nm in aqueous solution, in which PGD-G3 provided a much more polar microenvironment than glycerol. PGD-G3 and AHSA interacted attractively, showing a binding constant of 5.3×10(5) M(-1) with a 2:1 stoichiometry. On the other hand, AHSA interacted with the periphery of PGD-G4, the majority of which existed as a unimer, forming a less polar microenvironment. The driving force of the interactions for PGD-G3 and -G4 were mainly enthalpically and entropically driven, respectively. The generation-dependent host-guest interactions were described in conjunction with thermodynamic parameters.

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Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Cited by 41 publications

References 77 publications

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

Multiepitope‐based vaccine design by exploring antigenic potential among leptospiral lipoproteins using comprehensive immunoinformatics and structure‐based approaches

Generation‐Dependent Host–Guest Interactions: Solution States of Polyglycerol Dendrimers of Generations 3 and 4 Modulate the Localization of a Guest Molecule

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