The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome

Abstract

Endogenous regeneration and repair mechanisms are re-sponsible for replacing dead and damaged cells to main-tain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms in-volves skeletal muscle. Although the molecular mecha-nisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environ-ment 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 glycosylphosphatidyl-inositol-anchored, five extracellular matrix, and two mem-brane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the oc-cupancy for 235 N-linked glycosylation sites. The identifi-cation 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 annota-tions, whereas ambiguous annotations for 14 other pro-teins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and ␤-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myo-tubes. 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 im-portant characterization of cell intermediates during myo-blast differentiation into myotubes. Molecular & Cellular Proteomics 8:2555–2569, 2009.