Großkopf Henning, Vogel Sarah, Müller Claudia Damaris, Köhling Sebastian, Dürig Jan-Niklas, Möller Stephanie, Schnabelrauch Matthias, Rademann Jörg, Hempel Ute, von Bergen Martin, Schubert Kristin
Department of Molecular Systems Biology, Helmholtz Centre for Environmental Research GmbH - UFZ, Leipzig D-04318, Germany.
Institute of Physiological Chemistry, Medical Faculty, Technische Universität Dresden, Dresden D-01307, Germany.
Biol Chem. 2021 Sep 1;402(11):1427-1440. doi: 10.1515/hsz-2021-0167. Print 2021 Oct 26.
Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.
糖胺聚糖（GAGs）是细胞外基质（ECM）的重要功能成分。人工合成的GAGs，如硫酸化透明质酸（sHA），具有促骨生成特性并能促进愈合过程。因此，它们在支持骨再生和伤口愈合方面具有很高的研究价值。尽管硫酸化GAGs（sGAGs）出现在细胞内，但关于其细胞内作用和潜在相互作用伙伴的知识却很匮乏。在这里，我们使用基于亲和纯化质谱（AP-MS）的方法来鉴定人骨髓间充质干细胞（hBMSC）中新型的、特别是细胞内与sGAG相互作用的蛋白质。总体而言，发现有477种蛋白质与四种不同的sGAGs中的至少一种相互作用。蛋白质定位的富集分析表明，主要鉴定出了细胞内和细胞相关的相互作用蛋白。通过反向实验证实了sGAG与α2-巨球蛋白受体相关蛋白（LRPAP1）、输出蛋白-1（XPO1）和丝氨酸蛋白酶HTRA1（HTRA1）之间的相互作用。连续的通路和聚类分析导致鉴定出了生物学过程，即涉及核酸结合和加工、LRP1依赖性内吞作用和外泌体形成的过程。考虑到sGAG在囊泡样结构中优先定位于细胞内，相互作用数据也表明sGAG对基于囊泡的运输过程具有特异性调节作用。通过鉴定许多sGAG特异性相互作用蛋白，我们的数据为旨在研究sGAG细胞效应分子机制的后续研究提供了资源。
