Quantitative Phosphoproteomics Analysis Reveals Broad Regulatory Role of Heparan Sulfate on Endothelial Signaling

Abstract

Heparan sulfate (HS) is a linear, abundant, highly sulfated polysaccharide that expresses in the vasculature. Recent genetic studies documented that HS critically modulates various endothelial cell functions. However, elucidation of the underlying molecular mechanism has been challenging because of the presence of a large number of HS-binding ligands found in the examined experimental conditions. In this report, we used quantitative phosphoproteomics to examine the global HS-dependent signaling by comparing wild type and HS-deficient endothelial cells that were cultured in a serum-containing medium. A total of 7222 phosphopeptides, corresponding to 1179 proteins, were identified. Functional correlation analysis identified 25 HSdependent functional networks, and the top five are related to cell morphology, cellular assembly and organization, cellular function and maintenance, cell-to-cell communication, inflammatory response and disorder, cell growth and proliferation, cell movement, and cellular survival and death. This is consistent with cell function studies showing that HS deficiency altered endothelial cell growth and mobility. Mining for the underlying molecular mechanisms further revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, including ILK, integrin, actin cytoskeleton organization, tight junction and thrombin signaling. Intriguingly, this analysis unexpectedly determined that the top HS-dependent signaling is the IGF-1 signaling pathway, which has not been known to be modulated by HS. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/ growth hormone signaling pathways, including those both previously known, such as HGF and VEGF, and those unknown, such as IGF-1, erythropoietin, angiopoietin/Tie, IL-17A and growth hormones. Twelve of the identified 22 growth factor/cytokine/growth hormone signaling pathways, including IGF-1 and angiopoietin/Tie signaling, were alternatively confirmed in phospho-receptor tyrosine kinase array analysis. In summary, our SILAC-based quantitative phosphoproteomic analysis confirmed previous findings and also uncovered novel HS-dependent functional networks and signaling, revealing a much broader regulatory role of HS on endothelial signaling. Molecular & Cellular Proteomics 12: 10.1074/mcp.M112.026609, 2160-2173, 2013.Heparan sulfate (HS) is a linear, highly sulfated polysaccharide composed of glucosamine and hexauronic acid disaccharide repeating units (1). HS covalently attaches to core proteins to form HS proteoglycans (HSPG). Dictated by the location of the core proteins, HS chains present on cell surfaces, such as linking to syndecans and glypicans, and in the basement membrane by attaching to perlecan and agrin (1-3). HS biosynthesis is initiated by heterodimers formed by copolymerases Exostosin-1 (Ext1) and Exostosin-2 (Ext2) that elongate HS chains by alternatively adding glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) residues from their respective UDP-s...

Heparan sulfation is essential for the prevention of cellular senescence

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Heparan Sulfate Microarray Reveals That Heparan Sulfate–Protein Binding Exhibits Different Ligand Requirements

“…An immortalized mouse lung endothelial cell line 33 was used in the cell surface FGF-2 binding study. Briefly, endothelial cells (80% confluent) were harvested with trypsin (0.25%) and plated (15 000/100 μ L/well) onto a 96 well tissue culture plate (SIAL0596, Sigma).…”

Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells

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