Species-Specific N-Glycomes and Methylation Patterns of Oysters Crassostrea gigas and Ostrea edulis and Their Possible Consequences for the Norovirus-HBGA Interaction.

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles.

Involvement of ST6Gal I-mediated α2,6 sialylation in myoblast proliferation and differentiation.

Myogenesis is a physiological process which involves the proliferation of myoblasts and their differentiation into multinucleated myotubes, which constitute the future muscle fibers. Commitment of myoblasts to differentiation is regulated by the balance between the myogenic factors Pax7 and MyoD. The formation of myotubes requires the presence of glycans, especially N-glycans, on the cell surface. We examined here the involvement of α2,6 sialylation during murine myoblastic C2C12 cell differentiation by generating a st6gal1-knockdown C2C12 cell line; these cells exhibit reduced proliferative potential and precocious differentiation due to the low expression of Pax7. The earlier fusion of st6gal1-knockdown cells leads to a high fusion index and a drop in reserve cells (Pax7+ /MyoD- ). In st6gal1-knockdown cells, the Notch pathway is inactivated; consequently, Pax7 expression is virtually abolished, leading to impairment of the proliferation rate. All these results indicate that the decrease in α2,6 sialylation of N-glycans favors the differentiation of most cells and provokes a significant loss of reserve cells.

Analysis of N- and O-linked glycans from glycoproteins using MALDI-TOF mass spectrometry.

Glycosylation represents the most common of all known protein post-translational modifications. Carbohydrates can modulate the biological functions of a glycoprotein, protect a protein against hydrolysis via protease activity, and reduce or prevent aggregation of a protein. The determination of the carbohydrate structure and function in glycoproteins remains one of the most challenging tasks given to biochemists, as these molecules can exhibit complex branched structures that can differ in linkage and in the level of branching. In this review, we will present the approach followed in our laboratory for the elucidation of N- and O-glycan chains of glycoproteins. First, reduced/carboxamidomethylated glycoproteins are digested with a protease or a chemical reagent. N-Glycans are then released from the resulting peptides/glycopeptides via digestion with peptide N-glycosidase F (PNGase F). Oligosaccharides released by PNGase F are separated from peptides and glycopeptides using a C18 Sep-Pak, and their methylated derivatives are characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). O-Glycans are released by reductive elimination, which are permethylated, purified on a Sep-Pak C18 cartridge, and analyzed with MALDI-TOF-MS. Finally, to confirm the structures N-glycans released by PNGase F are characterized using MALDI-TOF-MS following on-plate sequential exoglycosidase digestions. The clean-up procedures of native and permethylated oligosaccharides for an efficient MALDI-TOF-MS analysis will also be described. This strategy was applied to calf fetuin and glycoproteins present in human serum.

The dual origin of Toxoplasma gondii N-glycans.

N-Linked glycosylation is the most frequent modification of secreted proteins in eukaryotic cells that plays a crucial role in protein folding and trafficking. Mature N-glycans are sequentially processed in the endoplasmic reticulum and Golgi apparatus through a pathway highly conserved in most eukaryotic organisms. Here, we demonstrate that the obligate intracellular protozoan parasite Toxoplasma gondii independently transfers endogenous truncated as well as host-derived N-glycans onto its own proteins.Therefore, we propose that the apicomplexan parasite scavenges N-glycosylation intermediates from the host cells to compensate for the rapid evolution of its biosynthetic pathway, which is primarily devoted to modification of proteins with glycosylphosphatidylinositols rather than N-glycans.

A rapid mass spectrometric strategy for the characterization of N- and O-glycan chains in the diagnosis of defects in glycan biosynthesis.

Glycosylation of proteins is a very complex process which involves numerous factors such as enzymes or transporters. A defect in one of these factors in glycan biosynthetic pathways leads to dramatic disorders named congenital disorders of glycosylation (CDG). CDG can affect the biosynthesis of not only protein N-glycans but also O-glycans. The structural analysis of glycans on serum glycoproteins is essential to solving the defect. For this reason, we propose in this paper a strategy for the simultaneous characterization of both N- and O-glycan chains isolated from the serum glycoproteins. The serum (20 microL) is used for the characterization of N-glycans which are released by enzymatic digestion with PNGase F. O-glycans are chemically released by reductive elimination from whole serum glycoproteins using 10 microL of the serum. Using strategies based on mass spectrometric analysis, the structures of N- and O-glycan chains are defined. These strategies were applied on the sera from one patient with CDG type IIa, and one patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex.

The use of mass spectrometry for the proteomic analysis of glycosylation.

Of all protein PTMs, glycosylation is by far the most common, and is a target for proteomic research. Glycosylation plays key roles in controlling various cellular processes and the modifications of the glycan structures in diseases highlight the clinical importance of this PTM. Glycosylation analysis remains a difficult task. MS, in combination with modern separation methodologies, is one of the most powerful and versatile techniques for the structural analysis of glycoconjugates. This review describes methodologies based on MS for detailed characterization of glycoconjugates in complex biological samples at the sensitivity required for proteomic work.

Discrimination between lumenal and cytosolic sites of deglycosylation in endoplasmic reticulum-associated degradation of glycoproteins by using benzyl mannose in CHO cell lines.

Recent studies demonstrated that deglycosylation step is a prerequisite for endoplasmic reticulum (ER)-associated degradation of misfolded glycoproteins. Here, we report the advantages of using benzyl mannose during pulse-chase experiments to study the subcellular location of the deglycosylation step in Chinese hamster ovary (CHO) cell lines. Benzyl mannose inhibited both the ER-to-cytosol transport of oligomannosides and the trimming of cytosolic-labeled oligomannosides by the cytosolic mannosidase in vivo. We pointed out the occurrence of two subcellular sites of deglycosylation. The first one is located in the ER lumen, and led to the formation of Man8GlcNAc2 (isomer B) in wild-type CHO cell line and Man4GlcNAc2 in Man-P-Dol-deficient cell line. The second one was revealed in CHO mutant cell lines for which a high rate of glycoprotein degradation was required. It occurred in the cytosol and led to the liberation of oligosaccharides species with one GlcNAc residue and with a pattern similar to the one bound onto glycoproteins. The cytosolic deglycosylation site was not specific for CHO mutant cell lines, since we demonstrated the occurrence of cytosolic pathway when the formation of truncated glycans was induced in wild-type cells.

Endoplasmic reticulum-associated degradation of glycoproteins bearing Man5GlcNAc2 and Man9GlcNAc2 species in the MI8-5 CHO cell line.

Endoplasmic reticulum-associated degradation of newly synthesized glycoproteins has been demonstrated previously using various mammalian cell lines. Depending on the cell type, glycoproteins bearing Man9 glycans and glycoproteins bearing Man5 glycans can be efficiently degraded. A wide variety of variables can lead to defective synthesis of lipid-linked oligosaccharides and, therefore, in mammalian cells, species derived from Man9GlcNAc2 or Man5GlcNAc2 are often recovered on newly synthesized glycoproteins. The degradation of glycoproteins bearing these two species has not been studied. We used a Chinese hamster ovary cell line lacking Glc-P-Dol-dependent glucosyltransferase I to generate various proportions of Man5GlcNAc2 and Man9GlcNAc2 on newly synthesized glycoproteins. By studying the structure of the soluble oligomannosides produced by degradation of these glycoproteins, we demonstrated the presence of a higher proportion of soluble oligomannosides originating from truncated glycans, showing that glycoproteins bearing Man5GlcNAc2 glycans are degraded preferentially.

Comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants.

We have compared the site-by-site N-glycosylation status of human lactoferrin (Lf) produced in maize, a monocotyledon, and in tobacco, used as a model dicotyledon. Maize and tobacco plants were stably transformed and recombinant Lf was purified from both seeds and leaves. N-glycopeptides were generated by trypsin digestion of recombinant Lf and purified by reverse-phase HPLC. The N-glycosylation pattern of each site was determined by mass spectrometry. Our results indicated that the N-glycosylation patterns of recombinant Lf produced in maize and tobacco share common structural features. In particular, both N-glycosylation sites of each recombinant Lf are mainly substituted by typical plant paucimannose-type N-glycans, with beta1,2-xylose and alpha1,3-linked fucose at the proximal N-acetylglucosamine. However, tobacco Lf shows a significant amount of processed N-glycans with one or two beta1,2GlcNAc linked to the trimannose core, which are weakly expressed in maize Lf. Finally, no Lewisa epitope was observed on tobacco Lf.

Lipomannan and lipoarabinomannan from a clinical isolate of Mycobacterium kansasii: novel structural features and apoptosis-inducing properties.

Although Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M. kansasii pathogenicity. Lipoarabinomannan (LAM), a major mycobacterial cell wall lipoglycan, is an important virulence factor for many mycobacteria, as it modulates the host immune response. Therefore, the detailed structures of the of M. kansasii LAM (KanLAM), as well as of its biosynthetic precursor lipomannan (KanLM), were determined in a clinical strain isolated from a human immunodeficiency virus-positive patient. Structural analyses revealed that these lipoglycans possess important differences as compared with those from other mycobacterial species. KanLAM carries a mannooligosaccharide cap but is devoid of the inositol phosphate cap present in Mycobacterium smegmatis. Characterization of the mannan core of KanLM and KanLAM demonstrated the following occurrences: 1) alpha1,2-oligo-mannopyranosyl side chains, contrasting with the single mannopyranosyl residues substituting the mannan core in all the other structures reported so far; and 2) 5-methylthiopentose residues that were described to substitute the arabinan moiety from Mycobacterium tuberculosis LAM. With respect to the arabinan domain of KanLAM, succinyl groups were found to substitute the C-3 position on 5-arabinofuranosyl residues, reported to be linked to the C-2 of the 3,5-arabinofuranose in Mycobacterium bovis bacillus calmette-guerin LAM. Because M. kansasii has been reported to induce apoptosis, we examined the possibility of the M. kansasii lipoglycans to induce apoptosis of THP-1 cells. Our results indicate that, in contrast to KanLAM, KanLM was a potent apoptosis-inducing factor. This work underlines the diversity of LAM structures among various pathogenic mycobacterial species and also provides evidence of LM being a potential virulence factor in M. kansasii infections by inducing apoptosis.

The unfolded protein response in a dolichyl phosphate mannose-deficient Chinese hamster ovary cell line points out the key role of a demannosylation step in the quality-control mechanism of N-glycoproteins.

The CHO (Chinese hamster ovary) glycosylation mutant cell line, B3F7, transfers the truncated glycan Glc(3)Man(5)GlcNAc(2) on to nascent proteins. After deglucosylation, the resulting Man(5)GlcNAc(2) glycan is subjected to two reciprocal enzymic processes: the action of an endoplasmic-reticulum (ER) kifunensine-sensitive alpha1,2-mannosidase activity to yield a Man(4)GlcNAc(2) glycan, and the reglucosylation involved in the quality-control system which ensures that only correctly folded glycoproteins leave the ER. We show that the recombinant secreted alkaline phosphatase (SeAP) produced in stably transfected B3F7 cells, is co-immunoprecipitated with the GRP78 (glucose-regulated protein 78), a protein marker of the unfolded protein response (UPR). The level of GRP78 transcription has been evaluated by reverse transcription-PCR (RT-PCR) and we demonstrate that B3F7 cells present a constitutively higher level of UPR in the absence of inductors, compared with Pro(-5) cells. Interestingly, a decrease was observed in the UPR and an increase in SeAP secretion in the kifunensine-treated B3F7 cells. Altogether, these data highlight the relationships between the glycan structure, the quality control system and the UPR. Moreover, they support the idea that a specific demannosylation step is a key event of the glycoprotein quality control in B3F7 cells.