SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function

Abstract

Phosphorylated O-mannosyl trisaccharide [N-acetylgalactosamine-β3-N-acetylglucosamine-β4-(phosphate-6-)mannose] is required for dystroglycan to bind laminin-G domain-containing extracellular proteins with high affinity in muscle and brain. However, the enzymes that produce this structure have not been fully elucidated. We found that glycosyltransferase-like domain-containing 2 (GTDC2) is a protein O-linked mannose β 1,4-N-acetylglucosaminyltransferase whose product could be extended by β 1,3-N-acetylgalactosaminyltransferase2 (B3GALNT2) to form the O-mannosyl trisaccharide. Furthermore, we identified SGK196 as an atypical kinase that phosphorylated the 6-position of O-mannose, specifically after the mannose had been modified by both GTDC2 and B3GALNT2. These findings suggest how mutations in GTDC2, B3GALNT2, and SGK196 disrupt dystroglycan receptor function and lead to congenital muscular dystrophy.

Fig. 1

GTDC2 has a protein O-linked mannose β1,4-N-acetylglucosaminyltransferase activity. (A) HEK293 cells expressing c-Myc tagged GTDC2 were stained with anti-Myc (green), ERp72 (ER marker, red), and DAPI (nuclei, blue). Bar indicates 10 µm. (B) The product of the GTDC2 in vitro assay when a DG-derived peptide modified with O-linked mannose and UDP-GlcNAc were used as substrates was analyzed by MALDI-TOF/MS. (C) Reactant of the GTDC2dTM assay using Man-α-MU and UDP-GlcNAc was separated on Superdex Peptide 10/300 columns. S, unreacted acceptor substrate. P, enzymatic product. (D) Structure of the product in (C), with the sugar subunits labeled A and B. (E–F) HMQC (E) and overlay (F) of the HMQC (black and red) and HMBC (green) spectra of the product. Assigned cross peaks are labeled with a first letter representing the subunit (as designated in D), and the rest of the label representing the position on that subunit. The red peak in (E) is the folded peak. ppm, parts per million.

Fig. 2

Mutations in GTDC2 and B3GALNT2 cause defects in the synthesis of phosphorylated α-DG. (A) The product of the B3GALNT2dTM in vitro assay using the product depicted in Fig. 1B and UDP-GalNAc as substrates was analyzed by MALDI-TOF/MS. (B) Laminin- (open circle, left) and WFA- (open circle, right) binding to DG-derived peptide modified with the GalNAc-β3-GlcNAc-β4-Mannose was measured by solid-phase assay (n = 3). The trisaccharide-modified peptide produced by the GTDC2dTM and B3GALNT2dTM reactions was conjugated to maleimide-activated plates. The peptide modified with mannose was used for background subtraction. Wild type muscle glycoproteins (solid circle) served as positive control in the laminin-binding assay. Error bars indicate standard deviation (SD). (C) Fc-tagged DGFc340 was produced in [³²P]-orthophosphate-labeled fibroblasts derived from control individual and GTDC2- or B3GALNT2-mutated patient. DGFc340 was isolated from the culture medium using protein-A agarose, separated by SDS-PAGE, stained with Coomassie Brilliant Blue (CBB), and analyzed by phosphorimaging ([³²P]). (B) Reactants of rabbit brain total membrane fraction incubated with ATP and GalNAc-β3-GlcNAc-β4-Man-α-MU at 37°C for 6 hours were separated on a C18 reverse-phase column. S, unreacted acceptor substrate. P, enzymatic product.

Fig. 3

SGK196 phosphorylates GalNAc-β3-GlcNAc-β4-Man. (A) Cell lysates from control fibroblasts and fibroblasts derived from patients with a mutation in SGK196, GTDC2, or B3GALNT2, as well as SGK196 patient-derived fibroblasts ectopically expressing SGK196- Myc-DDK, were subjected to a kinase assay using GalNAc-β3-GlcNAc-β4-Man-α-MU. Data obtained from three individual experiments are shown, with error bars indicating SD. (B) Reactants from a phosphorylation assay in which SGK196-Myc-DDK was used were separated on a C18 reverse-phase column. GalNAc-β3-GlcNAc-β4-Man-α-MU (left), GlcNAc-β4-Man-α-MU (middle), or Man-α-MU (right) was used as acceptor, in the absence (upper) or presence (lower) of ATP.

Fig. 4

SGK196 phosphorylates the 6-position of O-mannose. (A) ³¹P/¹H COSY spectrum of the product depicted in Fig. 3B when GalNAc-β3-GlcNAc-β4-Man-α-MU was used as the acceptor. Assigned cross peaks are labeled as described in Fig. 1, using the subunit designation indicated in B. (B) Structure of the phosphorylated product, with sugar subunits labeled A–C. (C) Model of α-DG glycan structures. Proposed classification of each O-mannosyl core structure is indicated at bottom. Enzymes responsible for forming the respective linkages are indicated at left; those identified as causing (POMT1/2, POMGNT1 and 2, B3GALNT2, and POMK) DG-related disorders are indicated in italics. Green circle, Man; blue square, GlcNAc; yellow circle, Gal; yellow square, GalNAc, red circle, phosphate.