Frédéric Chirat

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 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.

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.

Determination of the sialylation level and of the ratio α-(2→3)/α-(2→6) sialyl linkages of N-glycans by methylation and GC/MS analysis

Abstract A methodology for the determination of the sialylation pattern of N -glycans, extent of sialylation and the ratio between α -(2→3) and α -(2→6) sialyl linkages, is presented based on the labelling of the C-3 and C-6 hydroxyl groups of Gal residues obtained after permethylation, saponification, selective desialylation of sialylated oligosaccharides and methanolysis. Deuteromethylation and GC/MS analysis of Gal derivatives allow to determine the sialylation level of glycans. O -Ethyl ether labelling followed by GC analysis of the resulting Gal derivatives allows to obtain the ratio between α -(2→3) and α -(2→6) sialyl linkages. The method was applied to LNT (LcOse 4 : β - d -Gal p -(1→3)- β - d -Glc p NAc-(1→3)- β - d -Gal p -(1→4)- d -Glc p ), LSTa (IV 3 NeuAcLcOse 4 : α -Neu p 5Ac-(2→3)- β - d -Gal p -(1→3)- β - d -Glc p NAc-(1→3)- β - d -Gal p -(1→4)- d -Glc p ), LSTc (IV 6 NeuAcnLcOse 4 : α -Neu p 5Ac-(2→6)- β - d -Gal p -(1→4)- β - d -Glc p NAc-(1→3)- β - d -Gal p -(1→4)- d -Glc p ) and a bisialylated biantennary N -glycan in which sialic acid is bound to Gal residues via an α -(2→6) linkage. Using this method, it was found that 92.8% of N -glycans in bovine fetuin is sialylated and that the ratio of α -(2→6) versus α -(2→3) sialyl linkages was 31:19.

A chondroitin-sulfate chain is located on serine-10 of the urinary trypsin inhibitor.

1. The glycopeptide carrying the glycosaminoglycan chain of the urinary trypsin inhibitor (immunologically and structurally related to inter-alpha-trypsin inhibitor) was isolated. 2. The data from amino acid composition and part sequencing of this glycopeptide unambiguously demonstrate that the glycosaminoglycan is covalently linked to serine-10 of the peptide chain of UTI.