Mass spectrometric analysis of neutral and anionic N-glycans from a Dictyostelium discoideum model for human congenital disorder of glycosylation CDG IL.

TY - JOUR

T1 - Mass spectrometric analysis of neutral and anionic N-glycans from a Dictyostelium discoideum model for human congenital disorder of glycosylation CDG IL.

AU - Hykollari, Alba

AU - Balog, Crina I A

AU - Rendić, Dubravko

AU - Braulke, Thomas

AU - Wilson, Iain B H

AU - Paschinger, Katharina

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

KW - Base Sequence

KW - Blotting, Western

KW - Chromatography, High Pressure Liquid

KW - Congenital Disorders of Glycosylation

KW - DNA Primers

KW - Dictyostelium

KW - Humans

KW - Models, Biological

KW - Polysaccharides

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Tandem Mass Spectrometry

U2 - 10.1021/pr300806b

DO - 10.1021/pr300806b

M3 - SCORING: Journal article

C2 - 23320427

VL - 12

SP - 1173

EP - 1187

JO - J PROTEOME RES

JF - J PROTEOME RES

SN - 1535-3893

IS - 3

M1 - 3

ER -