Abstract
α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.
Highlights
Α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein
The biosynthesis of O-mannosyl glycans is initiated by protein O-mannosyltransferases (POMT1 and POMT2) at Ser/Thr residues in the ER6
Our structural and biochemical analyses revealed the acceptor substrate recognition mechanism by dimer fukutin-related protein (FKRP): the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit
Summary
Α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. Fukutin and fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Protein O-linked mannose β-1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) forms core M1 using UDP-GlcNAc as a donor substrate[7]. Protein O-linked mannose β-1,4-N-acetylglucosaminyltransferase 2 (POMGNT2, formerly GTDC2 or AGO61), β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2), and protein-Omannose kinase (POMK) sequentially modify the O-Man residue, forming a phospho-core M3 structure [GalNAcβ1-3GlcNAcβ[1,2,3,4] (phospho-6)Man]5. Fukutin (FKTN) and fukutin-related protein (FKRP) add a tandem ribitol-phosphate (RboP), and ribitol xylosyltransferase 1 (RXYLT1, formerly TMEM5) and β-1,4-glucuronyltransferase 1 (B4GAT1) add a single GlcAβ1-4Xylβ[1,2,3,4] unit to the phosphocore M310–13. A GlcA-Xyl (GlcAβ1-3Xylα1-3) repeat is added by α-1,3-xylosyl- and β-1,3-glucuronyltransferase
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