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 of glycan-binding proteins for further study.
Highlights
Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate–protein interactions
We describe a new simple proteomics strategy to identify plasma membrane and extracellular heparan sulfate-binding proteins (HSBPs) that permits the simultaneous determination of the binding domains that interact with heparin/heparan sulfate
Samples obtained after proteolytic digestion or mock digestions with PBS were bound to heparin-Sepharose, and weakly-bound proteins were washed out with low ionic strength buffer (0.3 M NaCl in 20 mM HEPES)
Summary
Limited proteolysis is a powerful tool to map conformational features of proteins. By using suboptimal conditions for proteolysis (limiting enzyme, reduced temperature, and omission of reducing agents and denaturants), limited cleavage occurs at exposed hinges or loops resulting in the liberation of intact protein domains [13, 14]. For THBS1, identified peptides from the input and flow-through aligned outside of the heparinbinding domain whereas peptides identified in the 0.3 M wash and high-salt eluate localized to the heparin-binding N-terminal thrombospondin domain (Fig. 1D) [17, 18] These findings show that the partial proteolysis released subdomains of accessible proteins and that fractionation of the released material by heparin-affinity chromatography enriched for subdomains that map to the heparin-binding site. We inspected peptides derived from previously unidentified HSBPs (Fig. 2B) and examined their position in available crystal structures or in generated molecular models based on related structures to search for patches of positively-charged amino acids fitting the constraints described for heparin-binding domains (Fig. 3, A–H) [1, 35, 36]. TSP1 and LamG are protein modules known to interact with heparin [10], whereas COLF1 has not been previously associated with heparin binding
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