The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome: IDENTIFICATION, GLYCOSITE OCCUPANCY, AND MEMBRANE ORIENTATION*

Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function. One of the best examples of endogenous repair mechanisms involves skeletal muscle, which has innate regenerative capacity (for reviews, see Refs. 1–4). Skeletal muscle repair begins with satellite cells, a heterogeneous population of mitotically quiescent cells located in the basal lamina that surrounds adult skeletal myofibers (5, 6), that, when activated, rapidly proliferate (7). The progeny of activated satellite cells, known as myogenic precursor cells or myoblasts, undergo several rounds of division prior to withdrawal from the cell cycle. This is followed by fusion to form terminally differentiated multinucleated myotubes and skeletal myofibers (7, 8). These cells effectively repair or replace damaged cells or contribute to an increase in skeletal muscle mass.The molecular mechanisms that regulate differentiation of satellite cells and myoblasts toward myofibers are not fully understood, although it is known that the cell surface proteome plays an important biological role in skeletal muscle differentiation. Examples include how cell surface proteins modulate myoblast elongation, orientation, and fusion (for a review, see Ref. 8). The organization and fusion of myoblasts is mediated, in part, by cadherins (for reviews, see Refs. 9 and 10), which enhance skeletal muscle differentiation and are implicated in myoblast fusion (11). Neogenin, another cell surface protein, is also a likely regulator of myotube formation via the netrin ligand signal transduction pathway (12, 13), and the family of sphingosine 1-phosphate receptors (Edg receptors) are known key signal transduction molecules involved in regulating myogenic differentiation (14–17). Given the important role of these proteins, identifying and characterizing the cell surface proteins present on myoblasts in a more comprehensive approach could provide insights into the molecular mechanisms involved in skeletal muscle development and repair. The identification of naturally occurring cell surface proteins (i.e. markers) could also foster the enrichment and/or characterization of cell intermediates during differentiation that could be useful therapeutically.Although it is possible to use techniques such as flow cytometry, antibody arrays, and microscopy to probe for known proteins on the cell surface in discrete populations, these methods rely on a priori knowledge of the proteins present on the cell surface and the availability/specificity of an antibody. Proteomics approaches coupled with mass spectrometry offer an alternative approach that is antibody-independent and allows for the de novo discovery of proteins on the surface. One approach, which was used in the current study, exploits the fact that a majority of the cell surface proteins are glycosylated (18). The method uses hydrazide chemistry (19) to immobilize and enrich for glycoproteins/glycopeptides, and previous studies using this chemistry have successfully identified soluble glycoproteins (20–24) as well as cell surface glycoproteins (25–28). A recently optimized hydrazide chemistry strategy by Wollscheid et al. (29) termed cell surface capturing (CSC)¹ technology, reports the ability to identify cell surface (plasma membrane) proteins specifically with little (<15%) contamination from non-cell surface proteins. The specificity stems from the fact that the oligosaccharide structure is labeled using membrane-impermeable reagents while the cells are intact rather than after cell lysis. Consequently, only extracellular oligosaccharides are labeled and subsequently captured. Utilizing information regarding the glycosylation site then allows for a rapid elimination of nonspecifically captured proteins (i.e. non-cell surface proteins) during the data analysis process, a feature that makes this approach unique to methods where no label or tag is used. Additionally, the CSC technology provides information about glycosylation site occupancy (i.e. whether a potential N-linked glycosylation site is actually glycosylated), which is important for determining the protein orientation within the membrane and, therefore, antigen selection and antibody design.To uncover information about the cell surface of myoblasts and to identify potential markers of myoblast differentiation, we used the CSC technology on the mouse myoblast C2C12 cell line model system (30, 31). This adherent cell line derived from satellite cells has routinely been used as a model for skeletal muscle development (e.g. Refs. 1, 32, and 33), skeletal muscle differentiation (e.g. Refs. 34–36), and studying muscular dystrophy (e.g. Refs. 37–39). Additionally, these cells have been used in cell-based therapies (e.g. Refs. 40–42). Using the CSC technology, 128 cell surface N-linked glycoproteins were identified, including several that were found to change in overall abundance as the myoblasts differentiate toward myotubes. The current data also confirmed the occupancy of 235 N-linked glycosites of which 226 were previously unconfirmed. The new information provided by the current study is expected to facilitate the development of useful tools for studying the differentiation of myoblasts toward myotubes.