Neural cells secrete a unique repertoire of proteins

Abstract

Proteins that are released from cells consist of those in the extracellular matrix, as well as extracellular signaling and adhesion molecules. The majority of these extracellular proteins are, however, unknown. To determine their identity, we have used a proteomics approach to define proteins released from neurons, astrocytes and neural precursor cells. Using two-dimensional gels and liquid chromatography/mass spectrometry technology, it is shown that while astrocytes release a relatively small number of proteins, neurons and neuronal precursor cells release a larger number of proteins with more functional diversity. Although there is overlap between the different cell types, the exact composition of the extracellular protein pool is unique for each cell population. The various subsets of extracellular neural proteins include those involved in cellular Redox regulation and chaperones. In addition, many proteolytic enzymes are found outside of the cell. These data show that the extracellular space within the nervous system has a more diverse protein composition than previously thought. Keywords: astrocytes, neuronal cells, neurons, proteomics, secreted proteins. proteins and their diversity was much greater with the neural cells than with mesodermal cells such as muscle and fibroblasts. In addition, unlike nerve cells, the secreted proteins were very similar between the different mesodermal cell types. It was concluded that much of the protein diversity in the nervous system is in proteins that are found in the extracellular space. However, at the time of that publication, the technology was not available to readily determine the identity of this extracellular protein population. Indeed, no studies have previously identified and compared secreted proteins from CNS cell populations. We have therefore used 2-D gel and liquid chromatography/mass spectrometry (LC/ MS) technology to identify a subset of extracellular proteins released by cortical nerve and astrocytes as well as by several clonal nerve precursor cell populations isolated from the embryonic and adult rat cortex. Of the 200 proteins identified, it is shown that a large number of proteins with molecular chaperone and antioxidant properties are secreted, and that distinct subsets of proteins are secreted by nerve, astrocytes, and neural precursor cells. Materials and methods Cell LinesCell lines and preparation of secreted proteins The nerve-like precursor cells B35, B50, B103, and B104 were cloned from nitrosoethylurea-induced brain tumors (Schubert 1974). The major criterion on which the classification of the nerve cells was made was electrical excitability and veratridine-stimulated sodium flux. A large number of other markers, such as cell-surface antigens, are in agreement with the physiology (Schubert et al. 1986). L6 myoblasts were derived from neonatal skeletal muscle (Yaffe 1968).The clonal adult rat hippocampal cell line, adult hippocampal precursor (AHP), was obtained from J. Ray (Salk Institute) (Ray et al. 1993;Taupin et al. 2000). Cel...

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“…Samples were then subject to LDS-PAGE and silver-staining (Invitrogen). Individual lanes of the gel were cut into 10-15 slices and digested with trypsin prior to mass spectrometry analysis using ESI-MS/ MS as described Schubert et al 2009). Data were analyzed using the Mascot algorithm (Matrix Science).…”

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“…Conditioned media were obtained as previously described (Schubert et al, 2009) and desalted by means of ion-exchange sepharose columns (GE Healthcare, Piscataway, NJ, USA), frozen in dry ice, and lyophilized. Lyophilized proteins were resuspended in 0.5 ml of sample buffer [8 M urea, 4% (w/v) 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, 40 mM Tris, 0.2% Bio-Lyte 3–10 ampholytes (Bio-Rad, Hercules, CA, USA), 50 mM dithiothreitol].…”

“…Gels were fixed in 50% methanol overnight and stained with silver. Gel spots from 2-D gels were excised and in-gel digested with trypsin and analyzed by LC electrospray ionization MS, as described previously (Schubert et al, 2009). …”

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“…The characterization of proteins released from neurons, astrocytes and neural precursor cells by 2DE and LC-MS has been recently reported [47,48]. These studies revealed that the secreted extracellular pool is specific for a given cell population, including proteins involved in cell-cell interactions, energy metabolism, redox regulations and several molecular chaperones.…”

“…Curiously, several 'housekeeping' proteins (e.g. lactate dehydrogenase, creatine kinase, malate dehydrogenase, proteasome subunits, vimentin) were identified among the secreted proteins [48,49]. It has been hypothesized that this observation may reflect the selective release of a subset of internal cellular contents via exosomes, which contain a variety of cytoplasmic proteins that are deposited outside the cell [50].…”