Journal
JOURNAL OF PROTEOME RESEARCH
Volume 12, Issue 3, Pages 1173-1187Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr300806b
Keywords
Dictyostelium; fucose; sulfate; methylphosphate; mannosyltransferase; glycan
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Funding
- Austrian Fonds zur Forderung der wissenschaftlichen Forschung [P19615, P21946]
- Austrian Science Fund (FWF) [P 21946] Funding Source: researchfish
- Austrian Science Fund (FWF) [P19615, P21946] Funding Source: Austrian Science Fund (FWF)
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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 alpha 1,3-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.
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