Proteomics-based screening of the endothelial heparan sulfate interactome reveals that C-type lectin 14a (CLEC14A) is a heparin binding protein

ABSTRACT: Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan-sulfate binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. 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, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and, using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire glycan-binding proteins for further study.

INSTRUMENT(S): Orbitrap Fusion Lumos, TripleTOF 5600, Q Exactive Plus

ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Mus Musculus (ncbitaxon:10090)

SUBMITTER: Jeffrey D. Esko  

PROVIDER: MSV000084782 | MassIVE | Thu Jan 09 14:00:00 GMT 2020

REPOSITORIES: MassIVE