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

Abstract

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.

Towards structure-focused glycoproteomics

“…Robust probability-based false discovery rate-driven search engines and algorithms for intact glycopeptides are still required to propel the field forward. Adding to the informatics advances needed to achieve such important improvements, ‘ glycomics-assisted glycoproteomics ' represents a complementary strategy that can guide the glycoproteomics data analysis to lower the glycopeptide false discovery rates while simultaneously addressing the complexity of the glycome [ 35 , 43 , 67 , 246 , 247 ].…”

“…In the surveyed studies, data were most frequently collected using data-dependent acquisition (DDA) by employing higher-energy collision dissociation (HCD-), stepped collision energy (SCE)-HCD-, electron-transfer dissociation (ETD)- and/or electron-transfer/higher-energy collision dissociation (EThcD-) MS/MS acquisition strategies (discussed below). Our survey also illustrated that targeted re-isolation and orthogonal fragmentation of glycopeptide ions are increasingly performed using diagnostic oxonium ions arising from HCD-MS/MS commonly referred to as product-dependent (pd) acquisition [ 60 , 66 , 67 , 71 ]. Data-independent acquisition (DIA), albeit less common, has also been used to profile mixtures of glycopeptides [ 57 , 72–76 ].…”

Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of SerumN- andO-Glycopeptide Data

“…Facilitated by recent advances in separation science, mass spectrometry (MS) and informatics, glycoproteomics has matured over the past decade and is now ready to tackle biological questions and generate new insights into the complex glycoproteome in biological systems from a new angle [4][5][6][7] . While liquid chromatography tandem mass spectrometry (LC-MS/MS)based glycoproteomics studies routinely report thousands of N-and O-linked glycopeptides [8][9][10][11][12][13] , accurate and confident identification of glycopeptides from large volumes of mass spectral data remain a significant bottleneck in the field.…”

Oligomannosylation and MAN1A1 expression associate strongly with a subset of human cancer types

“…Cell surface glycoepitopes are known to play important roles in cellcell and cell-extracellular matrix communication [1,24,73]. To this end, we explored the cell surface expression of oligomannosidic epitopes of the promyelocytic HL- 60…”