Abstract
α-Dystroglycan (α-DG) is a highly glycosylated cell-surface laminin receptor. Defects in the O-mannosyl glycan of an α-DG with laminin-binding activity can cause α-dystroglycanopathy, a group of congenital muscular dystrophies. In the biosynthetic pathway of functional O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP), whose mutated genes underlie α-dystroglycanopathy, sequentially transfer ribitol phosphate (RboP) from CDP-Rbo to form a tandem RboP unit (RboP-RboP) required for the synthesis of the laminin-binding epitope on O-mannosyl glycan. Both RboP- and glycerol phosphate (GroP)-substituted glycoforms have recently been detected in recombinant α-DG. However, it is unclear how GroP is transferred to the O-mannosyl glycan or whether GroP substitution affects the synthesis of the O-mannosyl glycan. Here, we report that, in addition to having RboP transfer activity, FKTN and FKRP can transfer GroP to O-mannosyl glycans by using CDP-glycerol (CDP-Gro) as a donor substrate. Kinetic experiments indicated that CDP-Gro is a less efficient donor substrate for FKTN than is CDP-Rbo. We also show that the GroP-substituted glycoform synthesized by FKTN does not serve as an acceptor substrate for FKRP and that therefore further elongation of the outer glycan chain cannot occur with this glycoform. Finally, CDP-Gro inhibited the RboP transfer activities of both FKTN and FKRP. These results suggest that CDP-Gro inhibits the synthesis of the functional O-mannosyl glycan of α-DG by preventing further elongation of the glycan chain. This is the first report of GroP transferases in mammals.
Highlights
␣-Dystroglycan (␣-DG) is a highly glycosylated cell-surface laminin receptor
Because CDP-Gro is a known donor substrate for glycerol phosphate (GroP) transferase in bacterial teichoic acid biosynthesis (13), we examined whether sFKTN has GroP transferase activity using
Because whether the GroP on the core M3–type glycan of recombinant ␣-DG (12) is glycerol 1-phosphate (Gro1P) or glycerol 3-phosphate (Gro3P) remains unclear, we first used the mixture of CDP-Gro enantiomers. sFKTN was incubated with mixCDP-Gro and the phospho-core M3 peptide, and the products were analyzed by HPLC
Summary
To determine whether FKTN has GroP transferase activity as well as RboP transferase activity to the phospho-core M3 unit, we prepared a soluble form of FKTN with a His/Myc tag at the N terminus (sFKTN) and a synthetic phospho-core M3 peptide (AT*PAPVAAIGPK) modified with phospho-core M3 at the Thr* as an acceptor substrate. sFKTN was expressed in HEK293T cells and immunoprecipitated from the culture medium using an anti-c-Myc antibody. These results indicate that the GroPphospho-core M3 peptide cannot serve as an acceptor for sFKRP Both FKTN and FKRP have RboP transfer activity using CDP-Rbo as a donor substrate, and the catalytic domain of FKRP shows significant sequence similarity to that of FKTN (16), indicating that FKRP might have GroP transfer activity using CDP-Gro. sFKRP was incubated with CDPGro and its usual acceptor, the RboP-phospho-core M3 peptide, and the products were analyzed by HPLC. As was the case with sFKTN, in the HPLC chromatograms, the product peak (indicated by an open triangle) became small as the molar ratio of CDP-Gro to CDP-Rbo increased (Fig. 7C) In accord with this observation, the RboP transfer activity decreased with an increasing molar ratio of CDP-Gro to CDP-Rbo (Fig. 7D).
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