Catalytic deficiency of O-GlcNAc transferase leads to X-linked intellectual disability.

O-GlcNAc transferase (OGT) is an X-linked gene product that is essential for normal development of the vertebrate embryo. It catalyses the O-GlcNAc posttranslational modification of nucleocytoplasmic proteins and proteolytic maturation of the transcriptional coregulator Host cell factor 1 (HCF1). Recent studies have suggested that conservative missense mutations distal to the OGT catalytic domain lead to X-linked intellectual disability in boys, but it is not clear if this is through changes in the O-GlcNAc proteome, loss of protein-protein interactions, or misprocessing of HCF1. Here, we report an OGT catalytic domain missense mutation in monozygotic female twins (c. X:70779215 T > A, p. N567K) with intellectual disability that allows dissection of these effects. The patients show limited IQ with developmental delay and skewed X-inactivation. Molecular analyses revealed decreased OGT stability and disruption of the substrate binding site, resulting in loss of catalytic activity. Editing this mutation into the Drosophila genome results in global changes in the O-GlcNAc proteome, while in mouse embryonic stem cells it leads to loss of O-GlcNAcase and delayed differentiation down the neuronal lineage. These data imply that catalytic deficiency of OGT could contribute to X-linked intellectual disability.

APC/CFzr regulates cardiac and myoblast cell numbers, and plays a crucial role during myoblast fusion.

Somatic muscles are formed by the iterative fusion of myoblasts into muscle fibres. This process is driven by the recurrent recruitment of proteins to the cell membrane to induce F-actin nucleation at the fusion site. Although several proteins involved in myoblast fusion have been identified, knowledge about their subcellular regulation is rather elusive. We identified the anaphase-promoting complex (APC/C) adaptor Fizzy related (Fzr) as an essential regulator of heart and muscle development. We show that APC/CFzr regulates the fusion of myoblasts as well as the mitotic exit of pericardial cells, cardioblasts and myoblasts. Surprisingly, overproliferation is not causative for the observed fusion defects. Instead, fzr mutants exhibit smaller F-actin foci at the fusion site and display reduced membrane breakdown between adjacent myoblasts. We show that lack of APC/CFzr causes accumulation and mislocalisation of Rols and Duf, two proteins involved in the fusion process. Duf seems to serve as direct substrate of the APC/CFzr and its destruction depends on the presence of distinct degron sequences. These novel findings indicate that protein destruction and turnover constitute major events during myoblast fusion.

Biosynthesis and assembly of the Collagen IV-like protein Pericardin in Drosophila melanogaster.

In Drosophila, formation of the cardiac extracellular matrix (ECM) starts during embryogenesis. Assembly and incorporation of structural proteins such as Collagen IV, Pericardin, and Laminin A, B1, and B2 into the cardiac ECM is critical to the maintenance of heart integrity and functionality and, therefore, to longevity of the animal. The cardiac ECM connects the heart tube with the alary muscles; thus, the ECM contributes to a flexible positioning of the heart within the animal's body. Moreover, the cardiac ECM holds the larval pericardial nephrocytes in close proximity to the heart tube and the inflow tract, which is assumed to be critical to efficient haemolymph clearance. Mutations in either structural ECM constituents or ECM receptors cause breakdown of the ECM network upon ageing, with disconnection of the heart tube from alary muscles becoming apparent at larval stages. Finally, the heart becomes non-functional. Here, we characterised existing and new pericardin mutants and investigated biosynthesis, secretion, and assembly of Pericardin in matrices. We identified two new pericardin alleles, which turned out to be a null (pericardin3-548) and a hypomorphic allele (pericardin3-21). Both mutants could be rescued with a genomic duplication of a fosmid coding for the pericardin locus. Biochemical analysis revealed that Pericardin is highly glycosylated and forms redox-dependent multimers. Multimer formation is remarkably reduced in animals deficient for the prolyl-4 hydroxylase cluster at 75D3-4.