Alg12-Cdg N-Glycome Discovers Unusual Hybrid and High Mannose Glycans

PS2 Poster session 2 Even numbers
Location (hall): 
Start/end time: 
Tuesday, July 2, 2019 - 15:45 to 17:15

Francesca Esposito1, Sebastiano Bianca2, Domenico Garozzo1, Angela Messina1, Angelo Palmigiano1, Luisa Sturiale1, Chiara Barone2, Antonio Novelli3, Jaak Jaeken4, Rita Barone1,5

1National Research Country - Institute for Polymers, Composites and Biomaterials, Catania, Italy, 2Medical Genetics, Referral Centre for Rare Genetic Diseases, ARNAS Garibaldi , Catania, Italy, 3Laboratory of Medical Genetics, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy, 4Center for Metabolic Diseases, UZ and KU Leuven, Leuven, Belgium, 5Child Neurology and Psychiatry, Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy

Congenital Disorders of Glycosylation (CDG) are genetic diseases characterized by altered glycosylation of proteins, lipids and/or both. Protein glycosylation defects are divided in N- and O-glycosylation defects. The majority of CDG involves protein N-glycosylation, commonly classified in two groups. CDG type I defects affect oligosaccharide assembling in the ER, ad are characterized by unoccupied glycosylation sites in the protein, but with structurally complete glycans. CDG type II refers to defects in the subsequent processing steps in ER and Golgi, leading to the increase of abnormal glycans.

ALG12-CDG is an inherited multisystemic disorder due to mutation of ALG12 gene. ALG12 encodes for Dol-P-Man:Man7-GlcNAc2-PP-Dol α-1,6-mannosyltransferase that normally transfers the 8th mannose to the lipid-linked oligosaccharide (LLO precursor), which is required for subsequent N-glycosylation of proteins. The synthesis of completed glycan precursor GlcNAc2-Man9-Glc3 oligosaccharide decreases in ALG12-CDG, limiting its transfer to selected asparagine residues of nascent polypeptides (CDG type I defects). 

Clinical phenotype includes severe neurological disease and recurrent infections associated with hypogammaglobulinemia and cell B dysfunctions. Nine cases of ALG12-CDG have been reported until now. 

We investigated transferrin and serum N-glycosylation including targeted IgG in a novel ALG12-CDG patient by a combination of MALDI-MS and UPLC-ESI-MS strategies. We focused on serum and IgG N-glycome change, in order to investigate the impact of ALG12 molecular defect on the immunological phenotype of ALG12-CDG.

MALDI-MS analysis of serum transferrin showed, as expected for a CDG I, peaks corresponding to the di-glycosylated transferrin and to the mono-glycosylated isoform.

MALDI-MS analysis of IgG and serum N-glycan released after PNGase F treatment revealed occurrence of increasing amounts of hybrid and high mannose N-glycans in IgG and in total serum profiles. UPLC-ESI-MS analysis revealed these accumulating N-glycans were a sum of multiple isomeric structures. Abnormal hybrid structures were detected both in total serum and in IgG N-glycan samples, while abnormal high mannoses glycans were identified only through total serum N-glycan analysis. To further characterize the hybrid glycans accumulating in ALG12-CDG, we compared serum N-glycan structures of ALG12 patient with a control (showed in the attached figure) and also with a MAN1B1-CDG (1,2-α-mannosidase deficiency) sample serum, which is characterized by an increase of unusual hybrid configuration with α-1,2-terminal mannose residue. By the comparison of Extracted Ion Chromatograms (EICs) of released N-glycans from ALG12-CDG and MAN1B1-CDG serum samples, we demonstrated that the hybrid-type glycans accumulated in ALG12-CDG shared the same isomeric structures with the abnormal N-glycans that accumulate in MAN1B1–CDG. Similar results were observed analyzing EICs of GlcNAc2-Man5-7. In ALG12-CDG all these abnormal high mannosylated structure lacked the α1,6 mannose residue at the branched 6-Man arm, according to the molecular defect. These peculiar configurations of GlcNAc2-Man5-7 were completely absent in the control serum glycoprofile and never observed before.

These results highlighted that ALG12-CDG behaves as a dual CDG (CDG-I and II), because of the occurrence of unoccupied glycosylation sites in the transferrin typical of CDG-I defects alongside with accumulation of abnormal N-glycans caused by alteration in processing chain typical of CDG-II.

UHPLC-ESI Total Ion Current Chromatograms (TICCs) of serum N-glycans in the ALG12-CDG patient (red line) and a healthy pediatric control (black line) with major assignments. Glycan symbols: GlcNAc, blue square; Man, green circle; Gal, yellow circle; NeuAc, purple lozenge, Fuc, red triangle