N‐glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by ‘‘off‐line’’ liquid chromatography and mass spectrometry

Abstract

In this study, we have performed the first mass spectrometric analysis of N-glycans of the M31 mutant strain of the cellular slime mould Dictyostelium discoideum, previously shown to have a defect in glucosidase II. Together with glucosidase I, this enzyme mediates part of the initial processing of N-glycans; defects in either glucosidase are associated with human diseases and result in an accumulation of incorrectly-processed oligosaccharides which are not, or only poor, substrates for a range of downstream enzymes. To examine the effect of the glucosidase II mutation in Dictyostelium, we employed off-line LC-MALDI-TOF-MS in combination with chemical and enzymatic treatments and MS/MS to analyse the neutral and anionic N-glycans of the mutant as compared to the wild-type. The major neutral species were, as expected, of the composition Hex 10-11 HexNAc 2-3 with one or two terminal glucose residues. Consistent with the block in processing of neutral N-glycans caused by the absence of glucosidase II, fucose was apparently absent from the N-glycans and bisecting N-acetylglucosamine was rare. The major anionic oligosaccharides were sulphated and/or methylphosphorylated forms of Hex 8-11 HexNAc 2-3 , many of which surprisingly lacked glucose residues entirely. As anionic Nglycans are considered to be mostly associated with lysosomal enzymes in Dictyostelium, we hypothesise that glycosidases present in the acidic compartments may act on the oligosaccharides attached to such slime mould proteins. Furthermore, our chosen analytical approach enabled us, via observation of diagnostic negative-mode MS/MS fragments, to determine the fine structure of the methylphosphorylated and sulphated N-glycans of the M31 glucosidase mutant in their native state.

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

“…). High‐mannose glycans with bisected 6‐ antenna (e.g., 81 ) and several sulfated and methyl‐phosphated high‐mannose glycans have been identified from Dictyostelium discoideum (Hykollari et al, ). Bern et al () using permethylated samples, have detected very large N ‐glycans in mouse lung tissue with masses exceeding 13 kDa.…”

Hydrophilic interaction anion exchange for separation of multiply modified neutral and anionic Dictyostelium N‐glycans

“…The M31 (glucosidase II mutant) strain, obtained from the Dictyostelium Stock Centre, was grown in HL5 liquid medium according to standard techniques . Cellular material (2–3 g wet weight) was heat‐treated in water, homogenized by sonication and proteolysed with pepsin in 5% formic acid, prior to purification of the (glyco)peptides and release of N‐glycans with PNGase A and F (Roche) as previously published . After Dowex AG50 chromatography, the flow‐through was subject to solid‐phase extraction using nonporous graphitized carbon (NPGC; ≈ 300 μl ENVI™ Carb, Sigma‐Aldrich), with 40% acetonitrile and then 40% acetonitrile containing 0.1% trifluoroacetic acid (each thrice 300 μL) to elute respectively the neutral and anionic N‐glycans; the pools were then separately labeled with 2‐aminopyridine at the reducing terminus prior to subsequent HPLC and MALDI‐TOF MS/MS analysis .…”

“…Dictyostelium glycomutants tend to modify their N‐glycans with fewer anionic groups (methylphosphate and sulphate) on average as compared to the "wild‐type" AX3, due to the absence or blockage of acceptor sites on the glycans for these modifications . In the course of our previous study on the M31 glucosidase mutant , we were under the impression that more glycan structures were present than we could confidently assign and that we had reached the sensitivity limit with the mass spectrometers we were using, especially in negative mode MS/MS. However, by using instruments of increased sensitivity and performing further digests, we could extend our analysis of the neutral and anionic N‐glycans of the M31 strain as fractionated by HIAX chromatography.…”

“…Subsequently, the neutral and anionic‐enriched PA‐labeled pools were subject to hydrophilic interaction anion exchange (HIAX) HPLC, which separates glycans according to their size/charge, resulting in fractions containing less complex mixtures so facilitating the use of MALDI‐TOF MS together with glycosidase or chemical treatments; the fractionation prior to MS also reduces suppression effects. This type of procedure has been performed with the HL241/ alg9 and M31/ modA strains deficient in two conserved enzymes of N‐glycosylation in the endoplasmic reticulum , respectively, the ALG9 α1,2‐mannosyltransferase (EC 2.4.1.59) and the α1,3‐glucosidase II (EC 3.2.1.84; see also Fig. A).…”

Evolutionary diversity of social amoebae N-glycomes may support interspecific autonomy

“…For example, a global fucosylation mutant exhibits slow growth and forms abnormal cell aggregates in suspension [13], mucin-type O-glycosylation mutants exhibit abnormal sorting of prespore and prestalk cells [14] and abnormal spore coat assembly [15, 16], anionic N-glycan processing mutants exhibit altered kinetics of protein compartmentalization [17, 18], and cytoplasmic glycosylation mutants exhibit abnormal O 2 -sensing [19, 20]. Studies from our laboratory and others have begun to use MS to explore the N-glycomes of two cellular slime molds [6, 3, 7, 8, 21, 22] used as model organisms for interspecific relations, Dd and Dictyostelium purpureum ( Dp ). Recently, the genomes of additional social amoebae have been sequenced [23, 2], opening the door to apply genomics to glycogene identification to enable a genetic approach to addressing these questions.…”