Integrated Glycoproteomics Identifies a Role of N-Glycosylation and Galectin-1 on Myogenesis and Muscle Development

Abstract

•Proteomic, glycomic and glycoproteomic analysis of myogenesis.•Mechanistic insights into site-specific glycoproteome regulation via discrete glycosidases and glycosyltransferases.•Quantification and validation of glycan-binding proteins.•Functional analysis of LGALS1 reveals a role in 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 remains 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 digalactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labeling 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 upregulation 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 upregulation 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. 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 remains 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 digalactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labeling 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 upregulation 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 upregulation 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. The bulk of skeletal muscle is composed of postmitotic multinucleated myofibers that form via the fusion of mononucleated progenitor myoblasts. Myofiber formation is achieved via myogenesis, a highly ordered process including differentiation, elongation, migration, cell adhesion, membrane alignment, and ultimately cell fusion of myoblasts (1Dittmar T. Zanker K.S. Cell fusion in health and disease. Volume II: cell fusion in disease. Introduction.Adv. Exp. Med. Biol. 2011; 714: 1-3PubMed Google Scholar). The initial differentiation of myoblasts is regulated by external growth factors, cytokines, steroid hormones, and signal transduction pathways that activate a series of muscle-specific and pleiotropic transcription factors (2Braun T. Gautel M. Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasis.Nat. Rev. Mol. Cell Biol. 2011; 12: 349-361Crossref PubMed Scopus (366) Google Scholar). Elongation of myoblasts is achieved by extension of filopodia and lamellipodia to contact surrounding muscle cells. Myoblasts subsequently migrate to each other, which requires extracellular matrix (ECM) remodeling to facilitate cell motility before cell recognition and adherence. Here, interactions between multiple adherence molecules trigger integrin signaling and a rearrangement of the actin-cytoskeleton. This is coupled to the regulation of several GTPases and guanine nucleotide exchange factors that contribute to membrane remodeling and cell fusion via the ARP2/3, WASP, and WAVE protein complexes (3Kim J.H. Jin P. Duan R. Chen E.H. Mechanisms of myoblast fusion during muscle development.Curr. Opin. Genet. Dev. 2015; 32: 162-170Crossref PubMed Scopus (129) Google Scholar). In mammals, distinct phases of myogenesis contribute to the formation of mature skeletal muscle (4Kablar B. Rudnicki M.A. Skeletal muscle development in the mouse embryo.Histol. Histopathol. 2000; 15: 649-656PubMed Google Scholar, 5Schiaffino S. Dyar K.A. Ciciliot S. Blaauw B. Sandri M. Mechanisms regulating skeletal muscle growth and atrophy.FEBS J. 2013; 280: 4294-4314Crossref PubMed Scopus (682) Google Scholar). Muscle patterning is established by the fusion of embryonic myoblasts. The second phase involves fusion of fetal myoblasts followed by the formation of the basal lamina and the expansion of adult precursor satellite cells (muscle stem cells). Accompanying this second phase is innervation of the myofibers leading to the formation of the neuromuscular junctions. Finally, postnatal myogenesis is achieved via myoblasts derived from satellite cells that are responsible for growth and regeneration of mature skeletal muscle. Myogenesis and muscle development involve the interaction of cell surfaces and ECM with hundreds of glycosylated proteins. It is therefore not surprising that defects in glycosylation have been associated with several developmental disorders. More than 50 congenital disorders of glycosylation (CDGs) have been identified in humans, and these typically present as abnormalities in development of the nervous system and/or skeletal muscle during infancy (6Scott K. Gadomski T. Kozicz T. Morava E. Congenital disorders of glycosylation: new defects and still counting.J. Inherit. Metab. Dis. 2014; 37: 609-617Crossref PubMed Scopus (89) Google Scholar). The majority of CDGs are inherited defects in one or more of the multiple enzymes responsible for glycosylation of asparagine residues (N-linked glycosylation) that occur on membrane-associated, cell surface, and secreted proteins. For example, several loss-of-function mutations have been identified in the PMM2 gene involved in the synthesis of GDP-mannose, a nucleotide-sugar donor responsible for the transfer of mannose residues to and for their with have and and S. The of PubMed Scopus Google Scholar). in have CDGs in with N-glycosylation and defects in muscle and/or nervous system development. This the of mutations in the and the catalytic of the S. R. in and congenital disorders of Mol. Genet. 2013; PubMed Scopus Google and which are involved in the regulation of N-linked glycosylation R. P. P. E. M. an Genet. 2013; PubMed Scopus Google Scholar). the mutations in regulating glycosylation and their with muscle we the regulation of N-glycosylation during myogenesis and muscle development. Furthermore, the of glycan-binding proteins as lectins on myogenesis and muscle development only partially understood. For example, of Lgals1 in defects in muscle development in mice and R. J. K. of galectin-1 in defects in myoblast fusion and muscle PubMed Scopus Google Scholar, of a protein in defects in skeletal muscle J. PubMed Scopus Google Scholar). The molecular mechanisms but in galectin-1 to a of ECM proteins including and integrins the to signaling M. of the interaction of integrin with and by during skeletal muscle Cell PubMed Google Scholar). are by muscle and of muscle of healthy and muscle cells with has several changes in suggesting discrete changes in glycosylation or abundance the healthy skeletal muscle and changes in PubMed Scopus Google Scholar). of recombinant galectin-1 in a mouse of muscular muscle and suggesting of galectin-1 in myogenesis P. P. P. galectin-1 and muscle in the mouse of muscular Dev. PubMed Scopus Google Scholar). 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