Integrated glycoproteomics identifies a role of N-glycosylation and galectin-1 on myogenesis and muscle development

Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development but our molecular understanding of the precise glycans, catalytic enzymes and lectins involved remain only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N -linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown di-galactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labelling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins most notably the up-regulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N -glycosylation and the up-regulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration. ### Competing Interest Statement The authors have declared no competing interest. * ### Abbreviations AAV : adeno-associated virus ANOVA : analysis of variation CBDs : carbohydrate binding domains CDGs : congenital disorders of glycosylation CRISPR : clustered regularly interspaced short palindromic repeats CID : collisional induced dissociation DTT : dithiothreitol ECM : extracellular matrix EThcD : electron transfer dissociation with higher collisional dissociation supplemental activation FBS : fetal bovine serum FDR : false discovery rate FA : formic acid GBPs : glycan-binding proteins HCD : higher collisional dissociation HILIC : hydrophilic interaction liquid chromatography MCS : multiple cloning site MeCN : acetonitrile NMJ : neuromuscular junctions PCA : principal component analysis PGC : porous graphitized carbon PSM : peptide spectral match TEAB : triethylammonium bicarbonate TFA : trifluoroacetic acid TMT : tandem mass tags