Serum N-glycomics of a novel CDG-IIb patient reveals aberrant IgG glycosylation

Abstract

Rare genetic mutations of the MOGS gene affecting the function of the mannosyl-oligosaccharide glucosidase (glucosidase I) are the cause of the congenital disorder of glycosylation IIb (CDG-IIb). Glucosidase I specifically removes the distal α1,2-linked glucose from the protein bound precursor N-glycan Glc3Man9GlcNAc2 which is the initial step of N-glycan maturation. Here, we comparatively analyzed N-glycosylation of the whole serum proteome, serum-derived immunoglobulin G (IgG), transferrin (TF), and α-1-antitrypsin (AAT) of a female patient who is compound-heterozygous for two novel missense mutations in the MOGS gene, her heterozygous parents, and a sibling with wildtype genotype by multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) at unprecedented depth. Thereby, we detected the CDG-IIb-characteristic non-de-glucosylated N-glycans Glc3Man7-9GlcNAc2 as well as the free tetrasaccharide Glc3-Man in whole serum of the patient but not in the other family members. The N-glycan analysis of the serum proteome further revealed that relative intensities of IgG-specific complex type di-antennary N-glycans with core-fucosylation were considerably reduced in the patient’s serum whereas TF- and AAT-characteristic sialylated di- and tri-antennary N-glycans were increased. This finding reflected the hypogammaglobulinemia diagnosed in the patient. We further detected aberrant oligo-mannose (Glc3Man7GlcNAc2) and hybrid type N-glycans on patient-derived IgGs and we attributed this defective glycosylation to be the reason for an increased IgG clearance. This mechanism can explain the hypogammaglobulinemia that is associated with CDG-IIb.

Reliable N-Glycan Analysis–Removal of Frequently Occurring Oligosaccharide Impurities by Enzymatic Degradation

“…They influence, for example, the structure, stability, and binding properties of secreted and membrane-bound proteins. Therefore, numerous investigations focus on N -glycans to identify biomarkers, e.g., of diseases [ 1 , 2 , 3 , 4 , 5 , 6 ] or stem cells [ 7 , 8 , 9 , 10 ]. Furthermore, N -glycan analysis is part of biopharmaceutical protein characterization, e.g., as a critical quality parameter of therapeutic monoclonal antibodies [ 11 , 12 ] or their biosimilarity assessment [ 13 , 14 ].…”

Plasma markers of COVID-19 severity: a pilot study

“…Sample preparation for CGE-LIF N-glycan analysis was performed as described previously [ 25 , 26 ] with slight modifications. Briefly, N-glycans from whole blood serum were dissolved in SDS (2%, w/v) in phosphate buffer, the remaining SDS was neutralized with IGEPAL CA-630 (8%, v/v, Sigma-Aldrich), and glycoproteins were digested in solution with peptide-N-glycosidase F (PNGaseF from Elizabethkingia meningoseptica ; BioReagent grade, Sigma-Aldrich) to release attached N-glycans.…”

MOGS‐CDG: Quantitative analysis of the diagnostic Glc3Man tetrasaccharide and clinical spectrum of six new cases

“…Dried samples were reconstituted in 100 μl MiliQ in which 75 nM 13 C 6 -labeled L4 and 150 nM 13 C 6 -labeled T4 were dissolved. To 6 μl of this mixture, 50 μl of 0.5% acetic acid in AcN was added.…”

“…A onepoint calibration coefficient was based on this curve. Using the following formula, the concentration of T4 in patient samples was determined: Concentration 12 C 6 2AA-labeled T4 (μmol/mmol creatinine) = area 12 C 6 2AA-labeled T4/area 13 C 6 2AA-labeled IS T4 (intensity)/ calibration coefficient x IS concentration 13 C 6 2AAlabeled T4 (μmol/mmol creatinine). This formula is also applied to labeling control L4.…”

“…This formula is also applied to labeling control L4. After these calculations, the concentration of 12 C 6 2AA-labeled T4 was corrected with the use of L4 via formula: Corrected concentration 12 C 6 2AA-labeled T4 (μmol/mmol creatinine) = calculated concentration 12 C 6 2AA-labeled T4 (μmol/mmol creatinine)/calculated concentration 12 C 6 2AA-labeled L4 (μmol/mmol creatinine) Â added 13 C 6 2AA-labeled IS L4 (μmol/mmol creatinine).…”