MYORG is a dimeric α-galactosidase that shows distinct substrate specificity.

(a) SEC-MALLS traces of glycosylated and EndoH-treated MYORG. (b) Fluorescent activity assay of MYORG against 4MU-α-linked substrates. Data is mean from 3 technical replicates ± standard deviations. (c) Example isothermal titration calorimetry trace of DGJ binding to MYORG. (ci) Raw baseline subtracted injection profile of the ITC experiment. (cii) Titration curve with points in blue and fitted line in black. (d) Activity screening of MYORG against disaccharides. Experiment repeated twice with 3 technical repeats in each replicate. (e) Michaelis–Menten kinetics for processing of Gal-α1-4-Glc by MYORG. Data from 3 technical repeats. All raw data underlying graphs can be found in S1 Data. BGBT, blood group B trisaccharide; DGJ, deoxygalactonojirimycin; MYORG, myogenesis-regulating glycosidase.</p

Mapping PFBC disease-associated mutations in MYORG and stabilisation of MYORG by DGJ.

(a) Missense mutations of MYORG are depicted in green and R504 is shown as sticks in magenta. (b) Boltzmann fit of thermal shift data depicting difference between MYORG alone and MYORG in the presence of DGJ at a 1:20 molar ratio. Results from 3 technical replicates. All raw data underlying graphs can be found in S1 Data. DGJ, deoxygalactonojirimycin; MYORG, myogenesis-regulating glycosidase; PFBC, primary familial brain calcification.</p

Isothermal titration calorimetry results for MYORG.

Values are the mean of 2 technical repeats ± standard deviations. (PDF)</p

Data collection and refinement statistics for MYORG.

Values in parenthesis are for highest-resolution shell. Each dataset was derived from a single crystal. (PDF)</p

Mutations identified in MYORG that cause PFBC and the associated structural consequences.

(PDF)</p

Representative CE-LIF electropherograms for candidate MYORG substrates.

MYORG-active substrate (i) Gal-α1,4-Glc, MYORG-resistant substrate (ii) blood group B trisaccharide, and (iii) 2′-fucosyllactose, which was included as an internal standard in all reactions. Peaks denoted with an asterisks (*) are due to excess fluorogenic reagents. Note that monosaccharides are lost during the desalting process prior to fluorescent labelling. (PDF)</p

SDS-PAGE gel displaying both fully glycosylated MYORG and MYORG after EndoH treatment.

A clear reduction in size is seen upon digestion indicating removal of glycans. (PDF)</p

The region of MYORG used for docking simulations.

Chain B of MYORG from the MYORG-Gal-α1,4-Glc complex was used for docking. Docking region enclosed in green square. Acid/base and nucleophile residue coloured in magenta. (PDF)</p

MYORG is a membrane bound dimer that selectively binds an unusual Gal-α1,4-Glc epitope.

(a) Domain boundaries of MYORG with numbering representing the last residue of the domain. (b) Cartoon ribbon representation of MYORG with N-glycans depicted as sticks with the glycosylated Asn residues labelled. (c) The MYORG dimer arrangement showing the insert region and the expected orientation of MYORG with respect to the ER membrane based on analyses using PSIPRED [27], DISOPRED [28], and MEMSAT [29]. (d) Comparison of the active site of MYORG (blue, residue labelling in black) with that of Mmα-Glu-II (purple; PDB: 5H9O). D-glucose is bound by Mmα-Glu-II and is depicted in pink. (e) Residues involved in the positioning and binding of DGJ. (f) Residues involved in binding Gal-α1,4-Glc. Dashed lines in (e) and (f) represent hydrogen bonding. Magenta sticks are used to emphasise the catalytic acid (D520, mutated to N520 in (f)) and nucleophile (D463) residues. DGJ, deoxygalactonojirimycin; ER, endoplasmic reticulum; MYORG, myogenesis-regulating glycosidase; NTβSD, N-terminal β-sheet domain; PβSD, proximal β-sheet domain; TMD, transmembrane domain.</p

Raw data underlying graphs.

(XLSX)</p