Mass Spectrometric Analysis of Neutral and Anionic N‑Glycans from a Dictyostelium discoideum Model for Human Congenital Disorder of Glycosylation CDG IL

• Published in: Journal of proteome research Vol. 12; no. 3; pp. 1173 - 1187
• Main Authors: Hykollari, Alba, Balog, Crina I. A, Rendić, Dubravko, Braulke, Thomas, Wilson, Iain B. H, Paschinger, Katharina
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.03.2013
• Subjects: Base Sequence; Blotting, Western; Chromatography, High Pressure Liquid; congenital abnormalities; Congenital Disorders of Glycosylation - metabolism; Dictyostelium - chemistry; Dictyostelium discoideum; DNA Primers; enzymatic treatment; glycosylation; Humans; mass spectrometry; Models, Biological; mutants; N-acetylglucosamine; phosphates; polysaccharides; Polysaccharides - analysis; reversed-phase high performance liquid chromatography; solid phase extraction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; stop codon; sulfates; Tandem Mass Spectrometry - methods; transferases; fucose; mannosyltransferase; glycan; Dictyostelium; sulfate; methylphosphate
• ISSN: 1535-3893; 1535-3907; 1535-3907
• DOI: 10.1021/pr300806b

The HL241 mutant strain of the cellular slime mold Dictyostelium discoideum is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its alg9 mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting N-acetylglucosamine, bisecting N-acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism.