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.

中文翻译：

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基于蛋白质组学的内皮细胞硫酸乙酰肝素相互作用组筛选显示C型凝集素14a（CLEC14A）是肝素结合蛋白。

动物细胞表达硫酸乙酰肝素蛋白聚糖，该蛋白通过硫酸乙酰肝素-蛋白质相互作用来执行许多重要的细胞功能。膜硫酸乙酰肝素结合蛋白的鉴定具有挑战性，因为它们的丰度低并且需要大量富集。在这里，我们报告蛋白质组学的工作流程，用于鉴定和表征结合硫酸乙酰肝素的膜锚定和细胞外蛋白。该技术基于在没有变性和固定，肝素亲和层析和高分辨率LC-MS / MS的情况下活细胞的有限蛋白水解作用，我们将其命名为LPHAMS。LPHAMS在U937单核和原代鼠及人内皮细胞中的应用鉴定出55种质膜，细胞外基质和可溶性分泌蛋白，包括许多以前未鉴定的肝素结合蛋白。该方法还促进了肝素结合结构域的作图，从而可以预测肝素结合位点的位置。为了验证LPHAMS的发现功能，我们表征了一种新发现的肝素结合蛋白C型凝集素14a（CLEC14A），它是C型凝集素家族中调节血管生成的成员。我们发现，CLEC14A的C型凝集素结构域以纳摩尔亲和力与肝素一对一结合，并使用分子建模和诱变作用，绘制了其肝素结合位点。CLEC14A与其他糖胺聚糖（包括内皮硫酸硫酸乙酰肝素和硫酸软骨素E）发生物理相互作用，但不与中性或唾液酸化寡糖发生相互作用。