Dataset Information

Chemoenzymatic bio-orthogonal chemistry for site-specific double modification of recombinant thrombomodulin.

ABSTRACT: Best of both worlds: A one-pot strategy for site-specific PEGylation through strain-promoted alkyne-azide cycloaddition (SPAAC) and fluorescent labeling through sortase A-mediated ligation (SML) of recombinant thrombomodulin without prior chemical modification and without diminishing the protein activity has been developed.

SUBMITTER: Jiang R

PROVIDER: S-EPMC3926800 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Chemoenzymatic bio-orthogonal chemistry for site-specific double modification of recombinant thrombomodulin.

Jiang Rui R Wang Lin L Weingart Jacob J Sun Xue-Long XL

Chembiochem : a European journal of chemical biology 20131107 1

Best of both worlds: A one-pot strategy for site-specific PEGylation through strain-promoted alkyne-azide cycloaddition (SPAAC) and fluorescent labeling through sortase A-mediated ligation (SML) of recombinant thrombomodulin without prior chemical modification and without diminishing the protein activity has been developed. ...[more]

PMID: 24357004

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Project description:During traumatic joint injuries, impact overloading can cause mechanical damage to the cartilage. In the following inflammation phase, excessive inflammatory cytokines (e.g., interleukin-1β (IL-1β)) can act on chondrocytes, causing over-proliferation, apoptosis, and extracellular matrix (ECM) degradation that can lead to osteoarthritis. This study investigated the combined effects of traumatic overloading and IL-1β challenge on the metabolic activities of chondrocytes. Bovine cartilage explants underwent impact overloading followed by IL-1β exposure at a physiologically relevant dosage (1 ng/mL). New click chemistry-based methods were developed to visualize and quantify the proliferation of in situ chondrocytes in a nondestructive manner without the involvement of histological sectioning or antibodies. Click chemistry-based methods were also employed to measure the ECM synthesis and degradation in cartilage explants. As the click reactions are copper-free and bio-orthogonal, i.e., with negligible cellular toxicity, cartilage ECM was cultured and studied for 6 weeks. Traumatic overloading induced significant cell death, mainly in the superficial zone. The high number of dead cells reduced the overall proliferation of chondrocytes as well as the synthesis of glycosaminoglycan (GAG) and collagen contents, but overloading alone had no effects on ECM degradation. IL-1β challenge had little effect on cell viability, proliferation, or protein synthesis but induced over 40% GAG loss in 10 days and 61% collagen loss in 6 weeks. For the overloaded samples, IL-1β induced greater degrees of degradation, with 68% GAG loss in 10 days and 80% collagen loss in 6 weeks. The results imply a necessary immediate ease of inflammation after joint injuries when trauma damage on cartilage is present. The new click chemistry methods could benefit many cellular and tissue engineering studies, providing convenient and sensitive assays of metabolic activities of cells in native three-dimensional (3D) environments.

Dataset Information

Chemoenzymatic bio-orthogonal chemistry for site-specific double modification of recombinant thrombomodulin.

Publications

Chemoenzymatic bio-orthogonal chemistry for site-specific double modification of recombinant thrombomodulin.

Similar Datasets

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