The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome

Abstract

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.

Gene expression profiling of the hyperplastic growth zones of the late trout embryo myotome using laser capture microdissection and microarray analysis

“…The Ig superfamily members CD 166 and CD 276, which were up-regulated in our microarray, have also been detected at the surface of mouse C2C12 myoblasts [33], suggesting a role for these two proteins in early myoblast-myoblast interactions. Other membrane proteins that were both up-regulated in superficial hyperplastic zones in our study and that have been detected at the surface of C2C12 cells included cleft lip and palate transmembrane protein1, trophoblast glycoprotein, tissue factor/CD142, Ephrin type A receptor 2, tetraspanin 3 and 4 and fibroblast growth factor 4 (FGFR4) [33]. FGFR4 is highly expressed during chick embryo muscle differentiation [34] (Marics et al, 2002) and muscle regeneration [35].…”

“…Interestingly, we found that Junctional adhesion molecule Jam2b which is closely related to jam2a (Jam-B) and Jam3b (JAM-C), both critical for myocyte fusion [32], was also up-regulated along with protogenin which is closely related to the promyogenic transmembrane protein Neogenin [30]. The Ig superfamily members CD 166 and CD 276, which were up-regulated in our microarray, have also been detected at the surface of mouse C2C12 myoblasts [33], suggesting a role for these two proteins in early myoblast-myoblast interactions. Other membrane proteins that were both up-regulated in superficial hyperplastic zones in our study and that have been detected at the surface of C2C12 cells included cleft lip and palate transmembrane protein1, trophoblast glycoprotein, tissue factor/CD142, Ephrin type A receptor 2, tetraspanin 3 and 4 and fibroblast growth factor 4 (FGFR4) [33].…”

Cystathionine γ-lyase Accelerates Osteoclast Differentiation

“…23, 24 Itfg3 is a single pass transmembrane protein with confirmation as a glycoprotein. 25, 26 Serpinb6b is a member of the widely studied serine protease inhibitor family, the serpins, that inhibit their enzyme targets by covalent modification, an activity that is consequential to numerous and widespread biological processes. 27 Adseverin belongs to the villin superfamily of proteins, which function in actin remodeling in response to calcium signaling.…”

Chemical Glycoproteomics

“…CSC technology has also been applied to mouse myoblast cells, mouse β -cells, hepatoma cells, leukemia cells, pluripotent stem cells, Drosophila Kc167 cells, and neuronal progenitor cells. 145,156–163 Recently, Wollscheid and co-workers used CSC technology to identify cell-surface proteins in 41 human and 31 mouse cell lines. 164 This work is summarized in a publically available resource: the Cell Surface Protein Atlas (CSPA).…”