Dataset Information

Proteome analysis of the MO3.13 cell line from human oligodendrocytes

ABSTRACT: MO3.13 is an immortal human-human hybrid cell line that express phenotypic characteristics of primary oligodendrocytes. It was created by fusing a 6-thioguanine-resistant mutant of the human rhabdomyosarcoma RD (cancer of skeletal muscle) with adult human oligodendrocytes by a lectin-enhanced polyethylene glycol procedure. In contrast to the tumor parent, MO3.13 expressed surface immunoreactivity for galactosyl cerebroside(GS) and intracellular immunoreactivity for myelin basic protein (MBP), proteolipid protein (PLP), and glial fibrillary acidic protein (GFAP). MO3.13 also exhibit the markers of immature oligodendrocytes GalC (galactosylceramidase) and CNPase. Upon differentiation, the MO3.13 cells have been also shown to express the MBP and MOG markers. Data analysis: Each MS raw file was processed using ProteoWizard for the generation of a MGF file. These were processed in SearchGUI, which runs the search engines Open Mass Spectrometry Search Algorithm (OMSSA) and X!Tandem against the UniProt human protein database (release 2013_08, 20,266 sequences). Search parameters were: peptide and fragment ion mass accuracy 10 ppm and 0.5 Da, respectively; protein and peptide FDRs 1%; two miss cleavages; trypsin as enzyme; fixed modifications: cysteine carbamidomethylation; variable modifications: methionine oxidation. In order to generate one single proteome dataset of MO3.13, resulting data was processed in PeptideShaker.

INSTRUMENT(S): LTQ Orbitrap, instrument model

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

SUBMITTER: Daniel Martins-de-Souza

PROVIDER: PXD000263 | Pride | 2013-10-09

REPOSITORIES: Pride

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Publications

The human oligodendrocyte proteome.

Iwata Keiko K Café-Mendes Cecilia C CC Schmitt Andrea A Steiner Johann J Manabe Takayuki T Matsuzaki Hideo H Falkai Peter P Turck Christoph W CW Martins-de-Souza Daniel D

Proteomics 20131120 23-24

Myelination of the CNS is performed by oligodendrocytes (OLs), which have been implicated in brain disorders, such as multiple sclerosis and schizophrenia. We have used the human oligodendroglial cell line MO3.13 to establish an OL reference proteome database. Proteins were prefractionationated by SDS-PAGE and after in-gel digestion subjected to nanoflow LC-MS analysis. Approximately 11 600 unique peptides were identified and, after stringent filtering, resulted in 2290 proteins representing nin ...[more]

PMID: 24167090

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Project description:Multiple sclerosis is a chronic neuroinflammatory disorder characterized by demyelination, oligodendrocyte damage/loss and neuroaxonal injury in the context of immune cell infiltration in the central nervous system. No neuroprotective therapy is available to promote the survival of oligodendrocytes and protect their myelin processes in immune-mediated demyelinating diseases. Pro-inflammatory CD4 T helper (Th)17 cells can directly interact with oligodendrocytes in multiple sclerosis and its animal model, causing injury to myelinating processes and cell death through direct contact. However, the molecular mechanisms underlying the close contact and subsequent detrimental interaction of Th17 cells with oligodendrocytes remain unclear. In this study we conducted single cell RNA sequencing of human primary oligodendrocytes cultured alone, in contact or separated by an insert with primary Th17 polarized T cells and identified several cell adhesion molecules that may modulate T cell contact mediated damage of oligodendrocytes. Furthermore, we used flow cytometry and immunofluorescence studies on central nervous system tissue from multiple sclerosis subjects, its animal model and controls to characterize the expression of cell adhesion molecules by mature oligodendrocytes. We found that a significant proportion of human and murine mature oligodendrocytes express melanoma cell adhesion molecule and activated leukocyte cell adhesion molecule in multiple sclerosis, EAE and controls although their regulation differ between human and mouse. We observed that exposure to pro-inflammatory cytokines or to human activated T cells are associated with a marked downregulation of the expression of melanoma cell adhesion molecule but not activated leukocyte cell adhesion molecule at the surface of human primary oligodendrocytes. Furthermore, we used in vitro live-imaging, immunofluorescence, and flow cytometry to determine the contribution of these molecules to Th17-polarized cell adhesion and cytotoxicity towards human oligodendrocytes. Silencing and blocking activated leukocyte cell adhesion molecule but not melanoma cell adhesion molecule limited prolonged interactions between oligodendrocytes and Th17-polarized cells, resulting in decreased adhesion of Th17-polarized cells to oligodendrocytes and conferred significant protection of oligodendrocytic processes.

Dataset Information

Proteome analysis of the MO3.13 cell line from human oligodendrocytes

Publications

The human oligodendrocyte proteome.

Similar Datasets

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