Mutations in genes required for the glycosylation of α-dystroglycan lead to muscle and brain diseases known as dystroglycanopathies. However, the precise structure and biogenesis of the assembled glycan are not completely understood. Here we report that three enzymes mutated in dystroglycanopathies can collaborate to attach ribitol phosphate onto α-dystroglycan. Specifically, we demonstrate that isoprenoid synthase domain-containing protein (ISPD) synthesizes CDP-ribitol, present in muscle, and that both recombinant fukutin (FKTN) and fukutin-related protein (FKRP) can transfer a ribitol phosphate group from CDP-ribitol to α-dystroglycan. We also show that ISPD and FKTN are essential for the incorporation of ribitol into α-dystroglycan in HEK293 cells. Glycosylation of α-dystroglycan in fibroblasts from patients with hypomorphic ISPD mutations is reduced. We observe that in some cases glycosylation can be partially restored by addition of ribitol to the culture medium, suggesting that dietary supplementation with ribitol should be evaluated as a therapy for patients with ISPD mutations.

No abstract

Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

The testicans are a three-member family of secreted proteoglycans structurally related to the BM-40/secreted protein acidic and rich in cystein (SPARC) osteonectin family of extracellular calcium-binding proteins. In vitro studies have indicated that testicans are involved in the regulation of extracellular protease cascades and in neuronal function. Here, we describe the biochemical characterization and tissue distribution of mouse testican-3 as well as the inactivation of the corresponding gene. The expression of testican-3 in adult mice is restricted to the brain, where it is located diffusely within the extracellular matrix, as well as associated with cells. Brain-derived testican-3 is a heparan sulphate proteoglycan.In cell culture, the core protein is detected in the supernatant and the extracellular matrix, whereas the proteoglycan form is restricted to the supernatant. This indicates possible interactions of the testican-3 core protein with components of the extracellular matrix which are blocked by addition of the glycosaminoglycan chains. Mice deficient in testican-3 are viable and fertile and do not show an obvious phenotype. This points to a functional redundancy among the different members of the testican family or between testican-3 and other brain heparan sulphate proteoglycans. 1998]. Their modular structure is characterized by an N-terminal testican-specific domain followed by the follistatin-like (FS) and extracellular calciumbinding (EC) domains characteristic of the BM-40 family. Towards the C-terminus they contain a thyroglobulin-like domain (TY) and a novel sequence (domain V), which includes two potential glycosaminoglycan attachment sites.Testican-1 was originally isolated as a proteolytic fragment from human seminal plasma carrying chondroitin sulphate and heparan sulphate chains (Bonnet et al. 1993). In the mouse, testican-1 is predominantly expressed in the nervous system during embryonic development and its expression correlates with periods of neuronal migration and axonal growth (Charbonnier et al. 2000). In adult mice, expression is restricted to the brain where it is located at post-synaptic densities (Bonnet et al. 1996). In search for homologous members of the BM-40 family, we identified cDNAs that were most similar to testican-1 and designated the corresponding proteins testican-2 and -3 (Vannahme et al. 1999;Hartmann and Maurer 2001).In adult mice, testican-2 shows a broader tissue distribution than testican-1. Although the proteoglycan can be found

Protein O-mannosylation is an important modification in mammals, and deficiencies thereof lead to a variety of severe phenotypes. Although it has already been shown that the amount of O-mannosyl glycans in brain is very high, only very few proteins have been identified as O-mannosylated. Additionally, the functions of the O-mannose-based glycans are still speculative and only investigated for α-dystroglycan. In a previous study a cis-located peptide was identified, which controls O-mannosylation in mammals. A BLAST search on the basis of this peptidic determinant identified other potential O-mannosylated proteins. Among these neurofascin was chosen for further analysis as a recombinant probe (mucin domain) and as an endogenous protein from mouse brain. Mass spectrometric data for both proteins confirmed that neurofascin186 is indeed O-mannosylated. Glycopeptide analysis by liquid chromatography-tandem mass spectrometry allowed for the identification of some of the O-mannosylation sites, which are not restricted to the mucin domain but were found also within N-terminal IgG and Fibronectin domains of the protein.

No abstract

O-Mannosylation is an important protein modification in brain. During the last years, a few mammalian proteins have been identified as targets of the protein-O-mannosyltransferases 1 and 2. However, these still cannot explain the high content of O-mannosyl glycans in brain and the strong brain involvement of congenital muscular dystrophies caused by POMT mutations (Walker-Warburg syndrome, dystroglycanopathies). By fractionating and analyzing the glycoproteome of mouse and calf brain lysates, we could show that proteins of the perineural net, the lecticans, are O-mannosylated, indicating that major components of neuronal extracellular matrix are O-mannosylated in mammalian brain. This finding corresponds with the high content of O-mannosyl glycans in brain as well as with the brain involvement of dystroglycanopathies. In contrast, the lectican neurocan is not O-mannosylated when recombinantly expressed in EBNA-293 cells, revealing the possibility of different control mechanisms for the initiation of O-mannosylation in different cell types.

Previous studies of the mucin-type O-glycome of the fruit fly Drosophila melanogaster have revealed a restricted pattern of neutral core-type glycans corresponding to the Tn-(GalNAca) and the T-antigen (Galb1-3GalNAca). In particular, no extension of the core 1 glycan with acidic sugars, like sialic acid, was detected. Here we report on the identification of an acidic O-linked trisaccharide expressed on secreted endogenous and recombinant glycoproteins of the embryonal hemocyte-like Drosophila Schneider-2 (S2) cell line. The glycan is composed of glucuronic acid, galactose and N-acetylgalactosamine and its structure was determined as GlcA1-3Gal1-3GalNAc. The Olinked trisaccharide resembles the peripheral structures of acidic D. melanogaster glycosphingolipids. Glucuronic acid may substitute for sialic acid in this organism, however its expression on the S2 cell surface may only marginally contribute to the negative surface charge as revealed by free-flow cell electrophoresis prior to and after b-glucuronidase treatment of the cells.