Site Mapping and Characterization of O-Glycan Structures on α-Dystroglycan Isolated from Rabbit Skeletal Muscle

Abstract

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, ␣-dystroglycan (␣-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown ␣-DG to be modified by both O-GalNAc-and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle ␣-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on ␣-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from ␣-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.Defects in protein glycosylation related to human disease were first reported in the 1980s, and since then, about 40 various types of congenital disorders of glycosylation have been reported (1). The term congenital disorders of glycosylation was first used to describe alterations of the N-glycosylation pathway and was later expanded to include the O-glycosylation pathways (1-3). The importance and complexity of O-linked glycosylation have only recently begun to be appreciated (1, 3, 4). In particular, mutations in genes encoding (putative) glycosyltransferases, which catalyze the addition and extension of O-linked mannose-initiated glycans, have garnered increased attention in the last decade given that they are causative for several forms of congenital muscular dystrophy (5, 6).The most common forms of O-glycosylation on secretory proteins are the mucin-like O-GalNAc structures that are initiated by polypeptide N-␣-acetylgalactosaminyltransferases in the endoplasmic reticulum-Golgi intermediate compartment and/or early cis-Golgi (7). Additionally, other O-linked structures are initiated with alternative monosaccharides, such as O-mannose, O-glucose, O-fucose, O-xylose, and O-GlcNAc onSer/Thr residues and the O-galactose modification of hydroxylysine residues in collagen domains (4). The diversity of O-mannosylated proteins in mammals, although quite abundant in some tissues (ϳ30% of O-glycans released from mouse brains (8)), has not been well characterized. The only clearly identified mamma...

Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

“…S1). While only a few of these sites have been identified in man, the majority of the sites have previously been identified in mice and rabbits (20,25,(28)(29)(30). No glycopeptides were identified in the region previously shown to carry the phosphoryl-Man glycan (Fig.…”

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

“…In this analysis we readily detected glycopeptides from ␣-DG. We identified 14 glycosites in the C-terminal part of the mucin-like domain (Ala 417 -Ser 485 ), which is normally O-GalNAc-modified as reported previously (13,(31)(32)(33)(34). The relative quantification (Figs.…”

The O-Mannosylation Pathway: Glycosyltransferases and Proteins Implicated in Congenital Muscular Dystrophy

“…In the early 2000s, multiple groups established that deficiencies of enzymes in this pathway result in multiple forms of congenital muscular dystrophy (CMD) 2 that have now been termed dystroglycanopathies (2)(3)(4)(5)(6). Recent work has begun to unravel the structures of the functional glycans that are altered and to identify sites of modification on ␣-dystroglycan (7)(8)(9)(10)(11)(12), the most well characterized and clearly functionally relevant O-mannosylated protein (13,14). While briefly describing the dystrophin-dystroglycan complex and the diversity of O-mannosylated structures, this minireview will primarily highlight the enzymes and proteins that are known to be defective in dystroglycanopathies.…”

“…Although ␣-dystroglycan is both N-and O-linked glycosylated, it is the O-linked glycans that are essential for proper function (25). In terms of O-linked glycosylation, ␣-dystroglycan contains both classical mucin-like O-GalNAc-initiated glycans and the more unusual O-Man-initiated glycans (11). Multiple studies have clearly demonstrated that it is the O-mannosylated glycan structures that serve as binding sites for laminin and presumably other extracellular matrix proteins (18 -23).…”

“…Multiple sites of O-mannosylation (and O-GalNAcylation) on ␣-dystroglycan have been established (8,11,12). This O-mannose can then be extended to create a variety of glycan structures ( Fig.…”