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

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>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 <jats:italic>N</jats:italic>-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 <jats:italic>Lgals1</jats:italic> inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in <jats:italic>N</jats:italic>-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.</jats:p>

Multiattribute Glycan Identification and FDR Control for Glycoproteomics

“…However, we were able to generate a list of 248 entrapment glycans that have very similar masses (within 0.05 Da) of true mouse glycans using several substitutions of glycan residues (see Supplementary Data 1, "mouse entrapment" for the list of compositions). We generated NeuAc (27), NeuGc (17), Fuc (24), and phosphate (180)-containing entrapment glycans with similar masses to many of the most commonly observed mouse glycans (Table S5). Unlike in the yeast entrapment search, these had to be manually checked against the mouse glycan list to ensure that true mouse glycans were not included as potential entrapments, resulting in smaller numbers of these entrapment glycans overall and an imbalance between the phosphate-containing and other types.…”

“…Some search tools provide additional capabilities that can assist in controlling the false discovery rate (FDR) of modified peptides, such as the use of the extended mass model of PeptideProphet (21) to model distinct probabilities for modifications with different masses used with MSFragger (17), or distinguishing between rare and common modifications in Byonic (12). These tools and many others have increasingly been applied to large scale glycoproteomics analyses (14, 18,[22][23][24][25] utilizing peptide-focused FDR methods, often in conjunction with a second empirical filtering or manual validation step.…”

Strategies for Proteome-Wide Quantification of Glycosylation Macro- and Micro-Heterogeneity

“…Following the developments in proteomics, quantitative glycoproteomics approaches commonly use stable isotopes, such as 13 C, 15 N, 18 O and 2 H, to label glycopeptides. Such labeling creates mass shifts to glycopeptide precursors or fragmentation products so that glycopeptides from different samples can be differently labeled and pooled before measurement but remain distinguishable by mass spectrometry.…”

“…Its applications include the sitespecific quantification of N-and O-linked glycosylation on protein therapeutics [15], urinary N-glycoprotein profiling in prostate cancer patients [16] and the determination of Nglycoproteome dynamics associated with prostate cancer progression [17]. In addition, the Parker group integrated quantitative glycomics and glycoOf note, raw files generated by Glycoproteomics to reveal the functional roles of N-glycosylation in myogenesis and muscle development [18]. The Lu group also used iTRAQ and Byonic workflows to identify glycopeptides of serum mannose receptors as potential biomarkers to differentiate the subtypes of breast cancer [19].…”

Multi-attribute Glycan Identification and FDR Control for Glycoproteomics

“…Some search tools provide additional capabilities that can assist in controlling the false discovery rate (FDR) of modified peptides, such as the use of the extended mass model of PeptideProphet (21) to model distinct probabilities for modifications with different masses used with MSFragger (17), or distinguishing between rare and common modifications in Byonic (12). These tools and many others have increasingly been applied to large scale glycoproteomics analyses (14,18,(22)(23)(24)(25) utilizing peptide-focused FDR methods, often in conjunction with a second empirical filtering or manual validation step.…”