Dynamic glycoproteomics identifies a role of N-glycosylation and Galectin-1 on myogenesis and muscle development
Blazev R, Ashwood C, Abrahams JL, Chung LH, Francis D, Yang P, Watt KI, Qian H, Quaife-Ryan GA, Hudson JE, Gregorevic P, Thaysen-Andersen M, Parker BL. Integrated glycoproteomics identifies a role of N-glycosylation and galectin-1 on myogenesis and muscle development. Mol Cell Proteomics. 2020 Sep 16:mcp.RA120.002166. doi: 10.1074/mcp.RA120.002166. Epub ahead of print. PMID: 32938751.
- Organism: Rattus norvegicus
- Instrument: 6330 Ion Trap LC/MS
N-glycan, rat, muscle, myoblast
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. Defects in glycan conjugation have been associated with the development of several muscular diseases suggesting an essential function 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 myogenesis in cell culture. We observed wide-spread changes in the abundance of several receptors and important enzymes regulating glycosylation. Quantification of released N-linked glycans via glycomic analysis confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases including a switch in sialic acid linkages, and changes in di-galactosylation and paucimannosylation. Quantitative glycoproteomic analysis with multiplexed stable isotope labelling and analysis of enriched glycopeptides with multiple fragmentation approaches identified precise glycoproteins containing these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins including the up-regulation of Galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early defect in the myogenic program. We also observed similar changes in N-glycosylation and the up-regulation of LGALS1 during postnatal skeletal muscle development in mice. Treatment of new-born mice with recombinant adeno-associated viruses to express LGALS1 in the musculature resulted in enhanced muscle mass. Our data will be a valuable resource to further understand the role of glycosylation and lectins on myogenesis and may aid in the development of intervention strategies aimed at promoting healthy muscle development or regeneration.
This panorama page only contains Skyline assays for released N-glycan analysis.
These are LFQ analyses of N-glycans released by PNGase-F from rat myoblasts and tissue.
Cells were washed twice with ice-cold PBS and lysis in ice-cold 100 mM sodium carbonate containing protease inhibitor cocktail (Roche) by tip-probe sonication. Lysates were rotated at 4℃ for 1 h then centrifuged at 150,000 x g for 60 min at 4℃ to pellet microsomal protein fraction. The pellet was resuspended in 6 M urea, 2M thiourea, 1% SDS containing 25 mM Triethylammonium bicarbonate (TEAB), pH 8.0 and protein precipitated with chloroform:methanol:water (1:3:4). Precipitated protein was washed with methanol and resuspended in 6 M urea, 2M thiourea containing 25 mM TEAB, pH 8.0. Protein concentration was determined via Qubit (Invitrogen), normalized and stored at -80℃.
Created on 8/17/20, 3:30 PM