Biochemical correlation of activity of the α-dystroglycan-modifying glycosyltransferase POMGnT1 with mutations in muscle-eye-brain disease

Abstract

Congenital muscular dystrophies have a broad spectrum of genotypes and phenotypes and there is a need for a better biochemical understanding of this group of diseases in order to aid diagnosis and treatment. Several mutations resulting in these diseases cause reduced O-mannosyl glycosylation of glycoproteins, including α-dystroglycan. The enzyme POMGnT1 (protein-O-mannose N-acetylglucosaminyltransferase 1; EC 2.4.1.-) catalyses the transfer of N-acetylglucosamine to O-linked mannose of α-dystroglycan. In the present paper we describe the biochemical characterization of 14 clinical mutants of the glycosyltransferase POMGnT1, which have been linked to muscle-eye-brain disease or similar conditions. Truncated mutant variants of the human enzyme (recombinant POMGnT1) were expressed in Escherichia coli and screened for catalytic activity. We find that three mutants show some activity towards mannosylated peptide substrates mimicking α-dystroglycan; the residues affected by these mutants are predicted by homology modelling to be on the periphery of the POMGnT1 surface. Only in part does the location of a previously described mutated residue on the periphery of the protein structure correlate with a less severe disease mutant.

Golgi Phosphoprotein 3 Mediates the Golgi Localization and Function of Protein O-Linked Mannose β-1,2-N-Acetlyglucosaminyltransferase 1

“…These mutations occurred within the stem domain of POMGnT1. In vitro data showed that E223K displayed complete inactivity (32), whereas R265H and C269Y resulted in a 5-fold decrease in enzymatic activity (32). The subcellular localization of these mutant proteins, however, has not been examined.…”

Dissecting the Molecular Basis of the Role of the O‐Mannosylation Pathway in Disease: α‐Dystroglycan and Forms of Muscular Dystrophy

“…[63–64] MEB displays a range of severities due to differences in residual enzyme activity, [65–66] and in some instances has been correlated with particular point mutations in POMGNT1. [67] Further examination of the impacts of these specific mutations on the glycosylation pattern of α-dystroglycan may reveal the relative importance of the sequential locations at which particular O -Man glycans are displayed. Interestingly, the reduced molecular weight of α-dystroglycan in MEB is consistent with the absence of the laminin binding structure, even though this entity is not associated with the GlcNAcβ(1,2)-Man linkage.…”

“…[27] The observation that severity of disease varies depending on the POMGNT1 mutation, [89] has prompted the analysis of specific mutations on enzyme activity. [67] This effort was accomplished by assaying recombinant forms of the mutant enzyme with a series of synthetic O -mannosyl containing glycopeptide substrates derived from α-dystroglycan sequences.…”

Clinical Features and Molecular Characterization of a Patient With Muscle-Eye-Brain Disease

“…Indeed, the recent biochemical characterization of various POMGNT1 mutants containing Cys490Tyr mutation and nonsense mutations affecting residues within the catalytic domain such as Trp590X and Arg580X revealed no enzymatic activity, which allows postulating that the two alterations reported may strongly impair the enzyme activity. 22 We have characterized a severe dystroglycanopathy patient with brain and eye abnormalities typical of MEB but with very mild histological muscular changes. Severe muscle weakness, hypotonia and elevated serum CK levels were detected, along with the absence of glycosylated α-DG.…”