Department of Laboratory Medicine, Sahlgrenska Academy, University of Gothenburg, Bruna Stråket 16, SE 413 45 Gothenburg, Sweden.
Proteomics Core Facility, Sahlgrenska Academy, University of Gothenburg, Medicinaregatan 9E, SE 405 30 Gothenburg, Sweden.
Glycobiology. 2021 Sep 9;31(8):916-930. doi: 10.1093/glycob/cwab035.
Proteoglycans (PGs) are proteins with glycosaminoglycan (GAG) chains, such as chondroitin sulfate (CS) or heparan sulfate (HS), attached to serine residues. We have earlier shown that prohormones can carry CS, constituting a novel class of PGs. The mapping of GAG modifications of proteins in endocrine cells may thus assist us in delineating possible roles of PGs in endocrine cellular physiology. With this aim, we applied a glycoproteomic approach to identify PGs, their GAG chains and their attachment sites in insulin-secreting cells. Glycopeptides carrying GAG chains were enriched from human pancreatic islets, rat (INS-1 832/13) and mouse (MIN6, NIT-1) insulinoma cell lines by exchange chromatography, depolymerized with GAG lyases, and analyzed by nanoflow liquid chromatography tandem mass spectrometry. We identified CS modifications of chromogranin-A (CgA), islet amyloid polypeptide, secretogranin-1 and secretogranin-2, immunoglobulin superfamily member 10, and protein AMBP. Additionally, we identified two HS-modified prohormones (CgA and secretogranin-1), which was surprising, as prohormones are not typically regarded as HSPGs. For CgA, the glycosylation site carried either CS or HS, making it a so-called hybrid site. Additional HS sites were found on syndecan-1, syndecan-4, nerurexin-2, protein NDNF and testican-1. These results demonstrate that several prohormones, and other constituents of the insulin-secreting cells are PGs. Cell-targeted mapping of the GAG glycoproteome forms an important basis for better understanding of endocrine cellular physiology, and the novel CS and HS sites presented here provide important knowledge for future studies.
蛋白聚糖（PGs）是一类带有糖胺聚糖（GAG）链的蛋白质，如硫酸软骨素（CS）或硫酸乙酰肝素（HS），这些 GAG 链连接在丝氨酸残基上。我们之前已经证明，前激素可以携带 CS，构成了一类新的 PGs。因此，对内分沁细胞中蛋白质 GAG 修饰的研究可以帮助我们描绘 PGs 在内分泌细胞生理学中的可能作用。基于此目的，我们应用糖蛋白质组学方法鉴定了胰岛素分泌细胞中的 PGs、它们的 GAG 链及其连接位点。通过交换层析从人胰岛、大鼠（INS-1 832/13）和小鼠（MIN6、NIT-1）胰岛素瘤细胞系中富集携带 GAG 链的糖肽，用 GAG 裂解酶解聚，并用纳流液相色谱串联质谱分析。我们鉴定了 CgA、胰岛淀粉样多肽、分泌颗粒蛋白 1、分泌颗粒蛋白 2、免疫球蛋白超家族成员 10 和蛋白 AMBP 的 CS 修饰。此外，我们还鉴定了两种 HS 修饰的前激素（CgA 和分泌颗粒蛋白 1），这令人惊讶，因为前激素通常不被认为是 HSPGs。对于 CgA，糖基化位点携带 CS 或 HS，使其成为所谓的混合位点。还在 syndecan-1、syndecan-4、神经连接蛋白-2、蛋白 NDNF 和 testican-1 上发现了额外的 HS 位点。这些结果表明，几种前激素和其他胰岛素分泌细胞的成分也是 PGs。GAG 糖蛋白质组的细胞靶向作图为更好地理解内分泌细胞生理学奠定了重要基础，而本文中呈现的新型 CS 和 HS 位点为未来的研究提供了重要的知识。
