MassIVE MSV000084510

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Proteomics-based screening of the endothelial heparan sulfate interactome reveals CLEC14A as a heparin binding protein

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Description

We report a novel proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind to heparin. The technique is based on Limited Proteolysis of live cells in the absence of denaturation and fixation, Heparin-Affinity chromatography, and high-resolution LC-MS/MS, which we designate as LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells led to the identification of 64 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitates the mapping of the heparin-binding domains, making it possible to make predictions on the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly discovered heparin-binding proteins, CLEC14A, a member of the C-type lectin family that modulates angiogenesis. The C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity and the heparin-binding site was mapped by molecular modeling and mutagenesis. CLEC14A can physically interact with other glycosaminoglycans including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The combination of limited proteolysis and mass spectrometry led to the identification of previously undocumented glycosaminoglycan-binding proteins and mapping of their ligand binding sites. The technique should be applicable to other cells and glycans and provides a way to expand the repertoire glycan-binding proteins for further study. [doi:10.25345/C5M66F] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Proteomics, heparin, heparin-binding proteins, heparan sulfate, glycosaminoglycans, endothelium, vascular biology, C-type lectin 14a (CLEC14A)

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Principal Investigators:
(in alphabetical order) 		Jeffrey D. Esko, Cellular and Molecular Medicine, UC San Diego, USA
Submitting User: alejandro
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