Functional Roles of -Glycosylation.

-Glycosylation in general has impact on a diversity of biological processes covering cellular aspects (targeted transport of glycoproteins), molecular aspects (protein conformation, resistance to proteolysis), and aspects involved in cellular communication (cell-cell and cell-matrix interaction). [..

ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto α-dystroglycan.

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.

Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

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.

Functional Analysis of the Glucuronyltransferases GlcAT-P and GlcAT-S of Drosophila melanogaster: Distinct Activities towards the O-linked T-antigen.

The Drosophila melanogaster glucuronyltransferases dGlcAT-S and dGlcAT-P were reported to be expressed ubiquitously and results of in vitro activity assays indicate a functional redundancy. We analyzed both transferases in vivo and in vitro and could show significant differences in their activity towards N-and O-glycoproteins in vivo. While GlcAT-P is able to use N-linked N-acetyllactosamine chains and the O-linked T-antigen as a substrate to form non-sulfated HNK1- (GlcAβ1-3Galβ1-4GlcNAcβ1-) and glucuronyl-T-antigens in vivo, GlcAT-S adds glucuronic acid only to N-linked chains, thereby synthesizing only the non-sulfated HNK1-antigen.

The lecticans of mammalian brain perineural net are O-mannosylated.

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).

Testican-3: a brain-specific proteoglycan member of the BM-40/SPARC/osteonectin family.

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.

A sensitive gel-based global O-glycomics approach reveals high levels of mannosyl glycans in the high mass region of the mouse brain proteome.

We developed a gel-based global O-glycomics method applicable for highly complex protein mixtures entrapped in discontinuous gradient gel layers. The protocol is based on in-gel proteolysis with pronase followed by (glyco)peptide elution and off-gel reductive β-elimination. The protocol offers robust performance with sensitivity in the low picomolar range, is compatible with gel-based proteomics, and shows superior performance in global applications in comparison with workflows eliminating glycans in-gel or from electroblotted glycoproteins.

Neurofascin 186 is O-mannosylated within and outside of the mucin domain.

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.

O-linked N,N'-diacetyllactosamine (LacdiNAc)-modified glycans in extracellular matrix glycoproteins are specifically phosphorylated at subterminal N-acetylglucosamine.

The terminal modification of glycans by β4 addition of N-acetylgalactosamine to N-acetylglucosamine with formation of the N,N-diacetyllactosediamine (LacdiNAc) moiety has been well documented for a number of N-linked glycoproteins and peptides, like neurohormones. Much less is known about O-glycoproteins in this regard because only human zona pellucida glycoprotein 3 (ZP3) and bovine proopiomelanocortin were reported to be LacdiNAc-modified. In searching for mammalian proteins modified with O-linked LacdiNAc we identified six positive species among nine endogenous and recombinant O-glycoproteins, which were extracellular matrix, or matrix-related proteins.

O-glycomics: profiling and structural analysis of mucin-type O-linked glycans.

The great variability of O-glycan structures makes their analysis a challenging task, which can be solved by the use of several complementary methods. While chromatographic analysis of the fluorescently labeled oligosaccharides shows the quantitative amount of the different glycans in comparison to a standard, mass spectrometry analysis of permethylated oligosaccharides allows identification of new or uncommon glycan structures. In combination with liquid chromatography, all structures present in one sample can be identified.

Protein-specific glycosylation: signal patches and cis-controlling peptidic elements.

The term 'protein-specific glycosylation' refers to important functional implications of a subset of glycosylation types that are under direct control of recognition determinants on the protein. Examples of the latter are found in the formation of the mannose-6-phosphate receptor ligand on lysosomal hydrolases, and in polysialylation of NCAM, which are regulated via conformational signal patches on the protein. Distinct from these examples, the beta4-GalNAc modification of N-linked glycans on a selected panel of proteins, such as carbonic anhydrase or glycodelin, was demonstrated recently to require specific protein (sequence) determinants proximal to the glycosylation site that function as cis-regulatory elements.

Initiation of mammalian O-mannosylation in vivo is independent of a consensus sequence and controlled by peptide regions within and upstream of the alpha-dystroglycan mucin domain.

To reveal insight into the initiation of mammalian O-mannosylation in vivo, recombinant glycosylation probes containing sections of human alpha-dystroglycan (hDG) were expressed in epithelial cell lines. We demonstrate that O-mannosylation within the mucin domain of hDG occurs preferentially at Thr/Ser residues that are flanked by basic amino acids. Protein O-mannosylation is independent of a consensus sequence, but strictly dependent on a peptide region located upstream of the mucin domain.

Glucuronic acid can extend O-linked core 1 glycans, but it contributes only weakly to the negative surface charge of Drosophila melanogaster Schneider-2 cells.

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-(GalNAcalpha) and the T-antigen (Galbeta1-3GalNAcalpha). 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.