Presence of galactosylated core fucose on N‐glycans in the planaria Dugesia japonica

Katharina Paschinger,Ebrahim Razzazi‐Fazeli,Iain B H Wilson,Kiyoshi Furukawa

doi:10.1002/jms.1925

Abstract

Planarial species are of especial interest to biologists due to the phenomenon of pluripotency and, in comparison to other developmental processes, it can be hypothesised that glycan–lectin interactions may play a role. In order to examine the N-glycans of one of these organisms, Dugesia japonica, peptide:N-glycosidase A was employed and the released glycans were subject to pyridylamination, HPLC and mass spectrometric analysis. A range of oligomannosidic glycans was observed with a trimethylated Man5GlcNAc2 structure being the dominant species. Three glycans were also observed to contain deoxyhexose; in particular, a glycan with the composition Hex4HexNAc2Fuc1Me2 was revealed by exoglycosidase digestion, in combination with MS/MS, to contain a galactosylated core α1,6-fucose residue, whereas this core modification was found to be capped with a methylhexose residue in the case of a Hex5HexNAc2Fuc1Me3 structure. This is the first report of these types of structures in a platyhelminth and indicates that the ‘GalFuc’ modification of N-glycans is not just restricted to molluscs and nematodes. Copyright © 2011 John Wiley & Sons, Ltd.

Highlights

Glycans cover the surfaces of all cells and, it is expected that many cell–cell interactions, whether between the cells of an individual organism or between symbionts or pathogens and their hosts, are glycan dependent.[1]
The basic procedures for the analysis of the N-glycans of the planaria D. japonica were performed as with other organisms; as initial studies indicated that a polyhexose series of unknown origin was present, a subsequent preparation was subject to reduction prior to the release of the N-glycans, so that only the released glycans and not the polyhexose compounds could be later labelled by the reductive pyridylamination method
As shown by MALDI-TOF MS analyses of RP-HPLC fractions, D. japonica expresses an unusual range of glycans including standard oligomannosidic, methylated oligomannosidic and at least three galacto-fucosylated N-glycans

Summary

INTRODUCTION

Glycans cover the surfaces of all cells and, it is expected that many cell–cell interactions, whether between the cells of an individual organism or between symbionts or pathogens and their hosts, are glycan dependent.[1]. We examined the N-glycans of one of them, Dugesia japonica; such data are a pre-requisite before embarking on studies examining whether the glycome has a role in the special regenerative properties of planarian species. The final pellet suspended in water was subjected to lyophilisation. Glycopeptides were prepared from the lyophilised material (6 mg) using pepsin, basically as previously described[8]; after initial ion-exchange (Dowex 50 W × 8, Sigma–Aldrich) and gel filtration (Sephadex G25, GE Healthcare) chromatography, the sample was dissolved in 50 μl of 5% (v/v) ammonia. Thereafter, the sample was subject to Dowex 50 W × 8 cation exchange chromatography; the unbound fraction was pyridylaminated at pH 7[9] and excess reagent was removed by gel filtration (Sephadex G15, GE Healthcare)

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION