The primary familial brain calcification-associated protein MYORG is an α-galactosidase with restricted substrate specificity

Abstract

Primary familial brain calcification (PFBC) is characterised by abnormal deposits of calcium phosphate within various regions of the brain that are associated with severe cognitive impairments, psychiatric conditions, and movement disorders. Recent studies in diverse populations have shown a link between mutations in myogenesis-regulating glycosidase (MYORG) and the development of this disease. MYORG is a member of glycoside hydrolase (GH) family 31 (GH31) and, like the other mammalian GH31 enzyme α-glucosidase II, this enzyme is found in the lumen of the endoplasmic reticulum (ER). Though presumed to act as an α-glucosidase due to its localization and sequence relatedness to α-glucosidase II, MYORG has never been shown to exhibit catalytic activity. Here, we show that MYORG is an α-galactosidase and present the high-resolution crystal structure of MYORG in complex with substrate and inhibitor. Using these structures, we map detrimental mutations that are associated with MYORG-associated brain calcification and define how these mutations may drive disease progression through loss of enzymatic activity. Finally, we also detail the thermal stabilisation of MYORG afforded by a clinically approved small molecule ligand, opening the possibility of using pharmacological chaperones to enhance the activity of mutant forms of MYORG.

Structure–function analysis of bacterial GH31 α‐galactosidases specific for α‐(1→4)‐galactobiose

“…While MYORG also dimerizes through the inserted region, the GH31_19 proteins associate via loops between Aβ2 to Aα2 and Aβ1 to Aα1 in addition to the insertion region. Therefore, the interface area of BsGH31_19 (chains A and B) and FpGH31_19 is 936 and 1101 Å 2 , respectively, which is greater than that of MYORG (639 Å 2 ) [25].…”

“…Next, we examined the enzymatic activity of BsGH31_19 and FpGH31_19 toward various oligosaccharides and polysaccharides. Interestingly, they demonstrated hydrolytic activity against Gb3 and a-Gal-(1?4)-Gal disaccharides, which are not substrates for MYORG and PsGal31A [19,25]. However, their activity against a-Gal-(1?4)-Glc, melibiose, blood group B antigen triose (a-Gal-(1?3)-[a-L-Fuc-(1?2)]-Gal), and guar gum was weak, with less than 1% of the activity observed for a-Gal-(1?4)-Gal (Table 2).…”

“…The model for BsGH31_19 was C-domain is absent in Bacteroides thetaiotaomicron aglucosidase BT_0339 (GH31_3) [17], Aspergillus sp. axylosidases (GH31_5) [30], cycloalternan-degrading enzymes (GH31_7) [31], sulfoquinovosidases (GH31_13) [20,32], and MYORG (GH31_20) [25]. A structural homology search using the Dali server [33] indicated that FpGH31_19 has the highest Z score (37.4) for MYORG (PDB 5HPO) among known GH31 enzymes and a lower Z score (27.5) for bacterial agalactosidase PsGal31A (PDB 4XPO).…”

“…This suggests that their natural substrate might be polysaccharides containing Gal and L-fucose residues [19]. In 2022, the myogenesisregulating glycosidase (MYORG) which was found in Homo sapiens and belongs to GH31 subfamily 20 (GH31_20), was identified as an a-galactosidase specific for the a-Gal-(1?4)-Glc disaccharide [25]. Initially identified as a protein involved in myogenic differentiation, MYORG mutations have been associated with primary familial brain calcification disease [26,27].…”

T-cell infiltration in the central nervous system and their association with brain calcification in Slc20a2-deficient mice

“…Inexplicably, the EC permeability was higher in the noncalcification-prone than in the calcification-prone brain areas; this region-specific heterogeneity in permeability was not associated with cell junction proteins or pericyte loss (Moura et al, 2017;Villasenor et al, 2017). MYORG encodes an endoplasmic reticulum-localized α-glucosidase that is specifically expressed in astrocytes, and its functional defects also cause brain calcification (Vanlandewijck et al, 2018;Yao et al, 2018;Meek et al, 2022). This evidence suggested that pericytes and astrocytes, or the differences in protein glycosylation in their supporting BBB-ECs, might affect the pathogenesis of brain calcification by regulating endocytosis or transcytosis functions (Keller et al, 2013;Villasenor et al, 2017).…”