Dataset Information

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 and hence are of high interest for supporting wound healing. Although sulfated GAGs (sGAGs) are internalized into cells, knowledge of intracellular effects and 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 from human bone marrow stromal cells (hBMSC). Overall, 478 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. Only a limited fraction of about 10 % was annotated for extracellular regions. The interactions of the 2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and Serine protease HTRA1 (HTRA1) were confirmed in reverse assays.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Bone Marrow

SUBMITTER: Henning Großkopf

LAB HEAD: Martin von Bergen

PROVIDER: PXD023362 | Pride | 2021-11-03

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 31

Publications

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

Großkopf Henning H Vogel Sarah S Müller Claudia Damaris CD Köhling Sebastian S Dürig Jan-Niklas JN Möller Stephanie S Schnabelrauch Matthias M Rademann Jörg J Hempel Ute U von Bergen Martin M Schubert Kristin K

Biological chemistry 20210901 11

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- ...[more]

PMID: 34472763

Similar Datasets

Project description:The inter-alpha-trypsin inhibitor (IαI) complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The IαI complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcA3Gal3Gal4Xyl-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the non-sulfated non-reducing end, (GalNAc4GlcA3)n ,to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation (HCD). The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal two GalNAc residues. The fragmentation behavior of CS glycopeptides under variable HCD conditions displays an energy dependency that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.

			Action	DRS
	6psHA_363.raw	Raw
	6psHA_528.raw	Raw
	6psHA_530.raw	Raw
	6psHA_532.raw	Raw
	6psHA_633.raw	Raw