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 of glycan-binding proteins for further study.

A Systems View of the Heparan Sulfate Interactome

“…22,23 Although many HSBPs have been reported over the years, membrane-bound and membrane-associated HSBPs are often difficult to characterize due to low abundance, solubility issues, protease resistance, and contamination by intracellular proteins (e.g., histones) that strongly bind to heparin-affinity matrices and outcompete less abundant membrane proteins. To circumvent these problems, cell-surface biotinylation, 24 membrane fractionation, 25 and limited proteolysis 26 strategies have been applied before heparin-affinity chromatography. These approaches have revealed a large number of cell-surface HSBPs, including novel cellular receptors and adhesion molecules.…”

NMR Characterization of the Interactions Between Glycosaminoglycans and Proteins

“…The interactions between GAGs and proteins are closely related to many factors, including saccharide unit composition, degree of sulfation, sulfation pattern, chain length, monosaccharide ring conformation and glycosidic linkage. The research methods used to characterize the interaction between GAGs and proteins mainly include gel electrophoresis (GE) ( Nogueira et al, 2019 ), affinity chromatography (AC) ( Sandoval et al, 2020 ), surface plasmon resonance (SPR) ( Przybylski et al, 2020 ), biological layer interferometry (BLI) ( Xiao et al, 2016 ), isothermal titration (ITC) ( Zsila et al, 2018 ), microarray methods ( Pomin and Wang, 2018b ), crystal diffraction methods (X-ray) ( Dahms et al, 2015 ), mass spectrometry (MS) ( Yang and Chi, 2017 ), and nuclear magnetic resonance spectroscopy (NMR) ( Kato and Peters, 2017 ). NMR is an insensitive technique compared with other analytical method for the study of interactions between biomolecules.…”

MS-proteomics provides insight into the host responses towards alginate microspheres

“…pI was calculated along the primary sequence of the protein using the Bjellqvist scale [ 61 ]. Heparin- or heparan sulfate-binding proteins [ [64] , [65] , [66] , [67] ] are encircled in orange. …”