Abstract

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.

中文翻译：

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人、大鼠和小鼠胰岛素分泌细胞的蛋白多糖分析

摘要

蛋白聚糖 (PG) 是具有与丝氨酸残基相连的糖胺聚糖 (GAG) 链的蛋白质，例如硫酸软骨素 (CS) 或硫酸乙酰肝素 (HS)。我们之前已经证明激素原可以携带 CS，构成一类新的 PG。因此，对内分泌细胞中蛋白质的 GAG 修饰作图可能有助于我们描述 PG 在内分泌细胞生理学中的可能作用。为此，我们应用糖蛋白组学方法来鉴定 PG、它们的 GAG 链及其在胰岛素分泌细胞中的附着位点。通过交换色谱法从人胰岛、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，并通过纳流液相色谱串联质谱法进行分析. 我们鉴定了嗜铬粒蛋白-A (CgA)、胰岛淀粉样蛋白多肽、分泌粒蛋白-1 和分泌粒蛋白-2、免疫球蛋白超家族成员 10 和蛋白 AMBP 的 CS 修饰。此外，我们发现了两种 HS 修饰的激素原（CgA 和分泌粒蛋白-1），这令人惊讶，因为激素原通常不被视为 HSPG。对于 CgA，糖基化位点携带 CS 或 HS，使其成为所谓的混合位点。在 syndecan-1、syndecan-4、nerurexin-2、蛋白质 NDNF 和 testican-1 上发现了额外的 HS 位点。这些结果表明，胰岛素分泌细胞的几种激素原和其他成分是 PG。GAG 糖蛋白组的细胞靶向定位为更好地了解内分泌细胞生理学奠定了重要基础，