Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry

Abstract

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.

Sulfated glycosaminoglycans inhibit transglutaminase 2 by stabilizing its closed conformation

“…Further physiological relevance of the GAG-TG2 interaction is given by the syndecan-4-dependent translocation into the extracellular space, which is initiated by the binding of TG2 to the HS epitopes of vesicle-associated intracellular syndecan-4 57 , 59 . Although the open conformation of TG2 (and therefore its transamidase activity) in the extracellular environment is probably not targeted by sulfated GAG derivatives, their interaction with TG2 in the closed conformation state could potentially already occur before the enzyme is released into the ECM since sulfated GAG derivatives have been shown to be internalized by various cell types 60 – 62 . In this light, the obtained results encourage further studies focusing on the applications of GAG derivatives towards the treatment of clinically relevant diseases in which TG2 is involved, such as cancer and fibrosis.…”

Sulfated glycosaminoglycans inhibit transglutaminase 2 by stabilizing its closed conformation

“…Further physiological relevance of the GAG-TG2 interaction is given by the syndecan-4-dependent translocation into the extracellular space, which is initiated by the binding of TG2 to the HS epitopes of vesicle-associated intracellular syndecan-4 57,59 . Although the open conformation of TG2 (and therefore its transamidase activity) in the extracellular environment is probably not targeted by sulfated GAG derivatives, their interaction with TG2 in the closed conformation state could potentially already occur before the enzyme is released into the ECM since sulfated GAG derivatives have been shown to be internalized by various cell types [60][61][62] . In this light, the obtained results encourage further studies focusing on the applications of GAG derivatives towards the treatment of clinically relevant diseases in which TG2 is involved, such as cancer and fibrosis.…”

Chemical Modification of Hyaluronan and Their Biomedical Applications

“…These selectively modified derivatives are intended, on the one hand, to mimic more complex GAG or GAG derivatives that can be synthesized only in time-consuming multi-step syntheses and high personal efforts. On the other hand, significant contributions to the detailed understanding of important interaction processes between GAG and their different reaction partners like ECM proteins, intracellular proteins or specific growth factors (Grosskopf et al, 2021) are expected.…”