Isabelle Breloy

Initiation of Mammalian O-Mannosylation in Vivo Is Independent of a Consensus Sequence and Controlled by Peptide Regions within and Upstream of the α-Dystroglycan Mucin Domain

To reveal insight into the initiation of mammalian O-mannosylation in vivo, recombinant glycosylation probes containing sections of human α-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. This peptide region cannot be replaced by other N-terminal peptides, however, it is not sufficient to induce O-mannosylation on a structurally distinct mucin domain in hybrid constructs. The presented in vivo evidence for a more complex regulation of mammalian O-mannosylation contrasts with a recent in vitro study of O-mannosylation in human α-dystroglycan peptides indicating the existence of an 18-meric consensus sequence. We demonstrate in vivo that the entire region p377–417 is necessary and sufficient for O-mannosylation initiation of hDG, but not of MUC1 tandem repeats. The feature of a doubly controlled initiation process distinguishes mammalian O-mannosylation from other types of O-glycosylation, which are largely controlled by structural properties of the substrate positions and their local peptide environment.

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. 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.

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. 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.

O-Linked N,N′-Diacetyllactosamine (LacdiNAc)-modified Glycans in Extracellular Matrix Glycoproteins Are Specifically Phosphorylated at Subterminal N-Acetylglucosamine

Background: LacdiNAc and sulfo-LacdiNAc modification of N-glycoproteins/peptides is well documented. Results: O-Linked LacdiNAc and the novel phospho-LacdiNAc modification were detected and structurally characterized on core 2-glycans of six ECM-related proteins. Conclusion: LacdiNAc and phospho-LacdiNAc are expressed on ECM proteins. Significance: This novel modification opens new aspects in the posttranslational control of protein function. 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. These are ZP3 and the five novel LacdiNAc-positive species ECM1, AMACO, nidogen-1, α-dystroglycan, and neurofascin. The mass spectrometric analyses revealed a core 2-based tetrasaccharide as the common structural basis of O-linked LacdiNAc that could be further modified, similar to the type 2 LacNAc termini, with fucose, sialic acid, or sulfate. Here, we provide structural evidence for a novel type of mucin-type O-glycans that is strictly specific for LacdiNAc termini: sugar phosphorylation with formation of GalNAcβ1–4(phospho-)GlcNAc. The structural details of the phosphatase-labile compound were elucidated by MS2 analysis of tetralysine complexes and by MSn measurements of the permethylated glycan alditols. Phospho-LacdiNAc was detected in human HEK-293 as well as in mouse myoblast cells and in bovine brain tissue.

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). 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.

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‐(GalNAcα) and the T‐antigen (Galβ1‐3GalNAcα). 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 O‐linked 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 β‐glucuronidase treatment of the cells.

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. 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.

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

Abstract 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. By applying this method, we analyzed the O-glycome of human myoblasts and of the mouse brain O-glycoproteome. After semipreparative separation of mouse brain proteins by one-dimensional SDS gel electrophoresis, the O-glycans from proteins in different mass ranges were characterized with a focus on O-mannose-based glycans. The relative proportion of the latter, which generally represent a rare modification, increases to comparatively high levels in the mouse brain proteome in dependence of increasing protein masses.

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

Abstract 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 β4-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. Another example of such a cis-regulatory element was described for the control of mammalian O-mannosylation. In this case, the structural features of substrate sites within the mucin domain of α-dystroglycan are necessary, but not sufficient for determining the transfer of mannose to Ser/Thr. Evidence has been provided that an upstream-located peptide is also essential. Such cis-controlling elements provide a higher level of protein specificity, because a putative glycosylation site cannot result from a single point mutation. Here, we highlight recent work on protein-specific glycosylation with particular emphasis on the above-cited examples and we will try to link protein-specific glycosylation to function.

O-Glycomics: Profi ling 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. The linkage of the monosaccharides can be analyzed by GC-MS after further derivatization of the permethylated glycans.