RESUMEN
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.
Asunto(s)
Glicosaminoglicanos/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas/metabolismo , Carioferinas/metabolismo , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Células Cultivadas , Cromatografía Liquida , Glicosaminoglicanos/química , Serina Peptidasa A1 que Requiere Temperaturas Altas/química , Serina Peptidasa A1 que Requiere Temperaturas Altas/aislamiento & purificación , Humanos , Carioferinas/química , Carioferinas/aislamiento & purificación , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL/química , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL/aislamiento & purificación , Células Madre Mesenquimatosas/química , Células Madre Mesenquimatosas/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/aislamiento & purificación , Espectrometría de Masas en Tándem , Proteína Exportina 1RESUMEN
Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-beta (TGF-beta) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of approximately 620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology.