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FN3 protein fragment containing two type III fibronectin domains from B. longum GT15 binds to human tumor necrosis factor alpha in vitro.

ABSTRACT: Most species of the genus Bifidobacterium contain the gene cluster PFNA, which is presumably involved in the species-specific communication between bacteria and their hosts. The gene cluster PFNA consists of five genes including fn3, which codes for a protein containing two fibronectin type III domains. Each fibronectin domain contains sites similar to cytokine-binding sites of human receptors. Based on this finding we assumed that this protein would bind specifically to human cytokines in vitro. We cloned a fragment of the fn3 gene (1503 bp; 501 aa) containing two fibronectin domains, from the strain B. longum subsp. longum GT15. After cloning the fragment into the expression vector pET16b and expressing it in E. coli, the protein product was purified to a homogenous state for further analysis. Using the immunoferment method, we tested the purified fragment's ability to bind the following human cytokines: IL-1?, IL-6, IL-10, TNF?. We developed a sandwich ELISA system to detect any specific interactions between the purified protein and any of the studied cytokines. We found that the purified protein fragment only binds to TNF?.

SUBMITTER: Dyakov IN

PROVIDER: S-EPMC7409735 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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FN3 protein fragment containing two type III fibronectin domains from B. longum GT15 binds to human tumor necrosis factor alpha in vitro.

Dyakov Ilya N IN Mavletova Dilara A DA Chernyshova Irina N IN Snegireva Nadezda A NA Gavrilova Marina V MV Bushkova Kristina K KK Dyachkova Marina S MS Alekseeva Maria G MG Danilenko Valery N VN

Anaerobe 20200806

Most species of the genus Bifidobacterium contain the gene cluster PFNA, which is presumably involved in the species-specific communication between bacteria and their hosts. The gene cluster PFNA consists of five genes including fn3, which codes for a protein containing two fibronectin type III domains. Each fibronectin domain contains sites similar to cytokine-binding sites of human receptors. Based on this finding we assumed that this protein would bind specifically to human cytokines in vitro ...[more]

PMID: 32771620

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Project description:BackgroundThe blood-spinal cord barrier (BSCB) is composed of a monolayer of endothelium linked with tight junctions and extracellular matrix (ECM)-rich basement membranes and is surrounded by astrocyte foot processes. Endothelial permeability is regulated by interaction between endothelial cells and ECM proteins. Fibronectin (FN) is a principal ECM component of microvessels. Excessive FN deposition disrupts cell-cell adhesion in fibroblasts through β1 integrin ligation. To determine whether excessive FN deposition contributes to the disruption of endothelial integrity, we used an in vitro model of the endothelial monolayer to investigate whether the FN inhibitor pUR4 prevents FN deposition into the subendothelial matrix and attenuates endothelial leakage.MethodsTo correlate the effects of excessive FN accumulation in microvessels on BSCB disruption, spinal nerve ligation-which induces BSCB leakage-was applied, and FN expression in the spinal cord was evaluated through immunohistochemistry and immunoblotting. To elucidate the effects by which pUR4 modulates endothelial permeability, brain-derived endothelial (bEND.3) cells treated with tumor necrosis factor (TNF)-α were used to mimic a leaky BSCB. A bEND.3 monolayer was preincubated with pUR4 before TNF-α treatment. The transendothelial electrical resistance (TEER) measurement and transendothelial permeability assay were applied to assess the endothelial integrity of the bEND.3 monolayer. Immunofluorescence analysis and immunoblotting were performed to evaluate the inhibitory effects of pUR4 on TNF-α-induced FN deposition. To determine the mechanisms underlying pUR4-mediated endothelial permeability, cell morphology, stress fiber formation, myosin light chain (MLC) phosphorylation, and β1 integrin-mediated signaling were evaluated through immunofluorescence analysis and immunoblotting.ResultsExcessive FN was accumulated in the microvessels of the spinal cord after spinal nerve ligation; moreover, pUR4 inhibited TNF-α-induced FN deposition in the bEND.3 monolayer and maintained intact TEER and endothelial permeability. Furthermore, pUR4 reduced cell morphology alteration, actin stress fiber formation, and MLC phosphorylation, thereby attenuating paracellular gap formation. Moreover, pUR4 reduced β1 integrin activation and downstream signaling.ConclusionspUR4 reduces TNF-α-induced β1 integrin activation by depleting ECM FN, leading to a decrease in endothelial hyperpermeability and maintenance of monolayer integrity. These findings suggest therapeutic benefits of pUR4 in pathological vascular leakage treatment.

Dataset Information

FN3 protein fragment containing two type III fibronectin domains from B. longum GT15 binds to human tumor necrosis factor alpha in vitro.

Publications

FN3 protein fragment containing two type III fibronectin domains from B. longum GT15 binds to human tumor necrosis factor alpha in vitro.

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