Site mapping and characterization of O-glycan structures on alpha-dystroglycan isolated from rabbit skeletal muscle.
The main extracellular matrix binding component of the dystrophin-glycoprotein complex, alpha-dystroglycan (alpha-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown alpha-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 alpha-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on alpha-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 alpha-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.
Complex Carbohydrate Research Center, Department of Chemistry, University of Georgia, Athens, Georgia 30602-4712, USA.
Stephanie H
SH
Stalnaker
Sana
S
Hashmi
Jae-Min
JM
Lim
Kazuhiro
K
Aoki
Mindy
M
Porterfield
Gerardo
G
Gutierrez-Sanchez
James
J
Wheeler
James M
JM
Ervasti
Carl
C
Bergmann
Michael
M
Tiemeyer
Lance
L
Wells
D000818:Animals; D002241:Carbohydrates / Q000737:chemistry; D049030:Dystroglycans / Q000737:chemistry*; D006001:Glycoconjugates / Q000737:chemistry; D054794:Glycomics / Q000379:methods; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D007797:Laminin / Q000737:chemistry; D008358:Mannose / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry*; D011817:Rabbits; D020349:Surface Plasmon Resonance / Q000379:methods
D002241:Carbohydrates; D006001:Glycoconjugates; D006023:Glycoproteins; D007797:Laminin; D011134:Polysaccharides; C121346:laminin 1; D049030:Dystroglycans; D008358:Mannose