Gesslbauer Bernd, Derler Rupert, Handwerker Claudia, Seles Elisabeth, Kungl Andreas J
Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria.
ProtAffin Biotechnologie AG, Graz, Austria.
Electrophoresis. 2016 Jun;37(11):1437-47. doi: 10.1002/elps.201600043. Epub 2016 Apr 9.
Glycosaminoglycans (GAGs) are linear, highly sulfated polysaccharides expressed by almost all animal cells. They occur as soluble molecules, or form proteoglycans by being O-linked to different core proteins on the cell surface and in the extracellular matrix. Due to their ability to interact with diverse proteins and to modulate their biologic functions, GAGs are main drivers of mammalian biology. However, to the present day, the human GAG binding proteome has only been insufficiently explored. The aim of this study was therefore to investigate the human GAG binding proteome of different sources by using the major GAG classes as ligands, and to explore the GAG-binding selectivity of the human plasma proteome. For this purpose, proteins were pulled down from immobilized low molecular weight heparin, heparan sulfate, and dermatan sulfate under different conditions and were identified by nano-LC/MS². Four hundred and fifty eight human GAG binding proteins have been identified, whereas plasma proteins showed clear differences in their GAG-binding specificity/selectivity and affinity. We were able to differentiate between proteins that bound to all three glycan ligands and proteins that showed selective binding to one or two glycan ligands. Moreover, step-gradient salt elution revealed different binding affinities toward different GAG ligands. On top of proteins with well-known GAG-binding properties we have identified formerly unknown GAG binders. Functional annotation of the identified GAG-binding proteins showed clusters of proteins that are involved in a variety of biological processes. The method described here is well suited for identifying GAG-binding proteins and for comparing human subproteomes with respect to binding to different GAG classes.
糖胺聚糖（GAGs）是几乎所有动物细胞表达的线性、高度硫酸化的多糖。它们以可溶性分子形式存在，或通过与细胞表面和细胞外基质中不同的核心蛋白O-连接形成蛋白聚糖。由于其能够与多种蛋白质相互作用并调节其生物学功能，GAGs是哺乳动物生物学的主要驱动因素。然而，时至今日，人类GAG结合蛋白质组的研究仍不充分。因此，本研究的目的是使用主要的GAG类别作为配体，研究不同来源的人类GAG结合蛋白质组，并探索人类血浆蛋白质组的GAG结合选择性。为此，在不同条件下从固定化的低分子量肝素、硫酸乙酰肝素和硫酸皮肤素中拉下蛋白质，并通过纳升液相色谱/串联质谱（nano-LC/MS²）进行鉴定。已鉴定出458种人类GAG结合蛋白，而血浆蛋白在其GAG结合特异性/选择性和亲和力方面表现出明显差异。我们能够区分与所有三种聚糖配体结合的蛋白质和对一种或两种聚糖配体表现出选择性结合的蛋白质。此外，分步梯度盐洗脱揭示了对不同GAG配体的不同结合亲和力。除了具有已知GAG结合特性的蛋白质外，我们还鉴定出了以前未知的GAG结合蛋白。对已鉴定的GAG结合蛋白的功能注释显示，参与各种生物学过程的蛋白质聚集在一起。这里描述的方法非常适合鉴定GAG结合蛋白，并比较人类亚蛋白质组在结合不同GAG类别方面的情况。
