Functional Analysis of the Glucuronyltransferases GlcAT-P and GlcAT-S of Drosophila melanogaster: Distinct Activities towards the O-linked T-antigen

Abstract

The Drosophila melanogaster glucuronyltransferases dGlcAT-S and dGlcAT-P were reported to be expressed ubiquitously and results of in vitro activity assays indicate a functional redundancy. We analyzed both transferases in vivo and in vitro and could show significant differences in their activity towards N-and O-glycoproteins in vivo. While GlcAT-P is able to use N-linked N-acetyllactosamine chains and the O-linked T-antigen as a substrate to form non-sulfated HNK1- (GlcAβ1-3Galβ1-4GlcNAcβ1-) and glucuronyl-T-antigens in vivo, GlcAT-S adds glucuronic acid only to N-linked chains, thereby synthesizing only the non-sulfated HNK1-antigen.

Keywords: Drosophila melanogaster; N-glycans; O-glycans; glucuronyltransferases; glycomics; mass spectrometry.

Figure 1

(A) Recombinant glucuronyltransferase fusion proteins expressed in Drosophila cells. Plasmids encoding intracellular (dGlcAT-P, dGlcAT-S) and secreted (dGlcAT-P sol, dGlcAT-S sol) fusion proteins with C-terminal V5 and 6 × His-Tags were generated. BiP, Hsc70-3 signal sequence; dGlcAT, Drosophila β1-3-glucuronyltransferase; V5, Paramyxovirus SV5-antigen. (B) Western blot analysis of cell supernatants and cell lysates of transfected Drosophila S2 cells with mAb anti-V5. 1, cell culture supernatant of S2-cells expressing dGlcAT-P sol; 2, culture supernatant of S2-dGlcAT-S sol; 3, cell lysate of S2-dGlcAT-P; 4, cell lysate S2-dGlcAT-S.

Figure 2

MALDI-MS of permethylated O-glycans from S2-cell lysates. In comparison to wt cells (lower panel), S2 cells overexpressing dGlcAT-P (upper panel) show an increase in non-sulfated glucuronyl-T-antigen expression (HN-GlcA + Na, m/z 752) relative to the precursor T-antigen (HN+Na, m/z 534), while no difference is observed in dGlcAT-S overexpressing cells (middle panel). The arrows indicate signals corresponding to mono-undermethylated proton adducts (MNa-36). Other mass signals in the range up to 1000 Da refer to matrix-derived ions. Short notation of glycan structures: H, hexose, N, N-acetylhexosamine, GlcA, glucuronic acid.

Figure 3

Western blot of S2 cell lysates, immunostained with non-sulfated HNK1-epitope specific mAb M6749. The signals appear exclusively after prolonged exposure times and only minor differences can be observed between dGlcAT-S (S), dGlcAT-P (P) transfected and wildtype (wt) cells. All signals disappear completely after PNGaseF digestion (+) confirming the localisation of the non-sulfated HNK1-epitope on N-glycan chains.

Figure 4

Immunostaining of Western blots with mAb 114-2G11-A (anti-GlcA) of cell lysates from S2-wt and dGlcAT-P (P) or dGlcAT-S (S) overexpressing cells reveals a strong increase of GlcA-epitopes in cells overexpressing GlcAT-P in the mass range around 60–70 kDa. A silver-stained SDS-PAGE of proteins immunoprecipitated with anti-GlcA specific mAb 144-2G11-A (lane 1) from S2-dGlcAT-P cell lysates (IP-P) shows a comparable pattern with most intense protein staining in the mass range of 60–70 kDa (arrow). Immunoprecipitation with the anti-T antibody HH8 (lane 2) leads to a partial overlap of precipitated proteins, whereas a nonrelevant IgM antibody (lane 3) did not reveal comparable protein enrichment in the respective mass range, indicating that most of the immunoprecipitated protein in lanes 1 and 2 had specifically bound to the carbohydrate-specific antibodies.

Figure 5

Post-Source-Decay MALDI-MS/MS spectrum of the glycopeptide EGFQLNESEKSK (1395.7 Da) from the molecular chaperone glycoprotein 93 modified with an N-glycan chain (Hex5HexNAc2, H5N2). We identified a series of y-ions, partially modified with N-glycans linked to asparagine within the consensus site NES. The unglycosylated peptide was identified by a triplet of signals representing the peptide mass as sodium adduct accompanied by signals at −17 Da and + 83 Da.

Figure 6

Analysis of the non-sulfated HNK1-antigen produced by in vitro glucuronylation of asialofetuin with dGlcAT-P sol. The MALDI-MS spectrum of the permethylated N-glycans shows a mono-glucuronylated sugar at m/z 2737. This was immunochemically verified by applying GlcA-fetuin (3 µg/lane) on a Western blot using the anti-HNK1 antibody M6749 (insert). The signal disappears after PNGaseF and β-glucuronidase digestion, verifying β1-3 linked GlcA on N-glycans. 1: GlcA-modified fetuin + PNGaseF; 2: GlcA-modified fetuin; 3: GlcA-modified fetuin + β-glucuronidase. Short notation of glycan structures: H, hexose; N, N-acetylhexosamine; F, fucose; GlcA, glucuronic acid.

Figure 7

(a) MALDI mass spectrometric analysis of methylated O-glycan alditols derived from in vitro glucuronylated asialo-fetuin (dGlcAT-P sol). The mass spectrum reveals glucuronylated mucin-type glycan chains at m/z 752 (HN-GlcA, core 1) and 1201 (H2N2-GlcA, core 2). (b) MALDI mass spectrometric analysis of methylated O-glycan alditols derived from in vitro glucuronylated asialo-fetuin (dGlcAT-S sol). The mass spectrum shows the same pattern of glycan alditols as in (a), but the ratio of glucuronylated O-glycan chains vs. non-glucuronylated chains is significantly lower. (*: matrix signals and non-identified contaminants) The insert shows a coomassie-stained SDS-Gel of asialofetuin glucuronylated in vitro by dGlcAT-P (lane P) or dGlcAT-S (lane S). The difference in the apparent molecular masses corresponds to the higher activity of dGlcAT-P towards O-glycans as shown in (a,b). Short notation of glycan structures: H, hexose; N, N-acetylhexosamine; GlcA, glucuronic acid.

Figure 8

MALDI-MS spectrum of the permethylated O-glycan alditols derived by reductive β-elimination from hDG5 coexpressed with dGlcAT-P in CHO-Lec2 cells. An intense signal at m/z 752 (HN-GlcA + Na⁺) revealed a glucuronylation of the T-antigen (HN + Na⁺). Non-labeled signals in the mass range up to m/z 1000 are matrix-derived signals. Short notation of glycan structures: H, hexose; N, N-acetylhexosamine.

Figure 9

MALDI-MS/MS spectrum of a permethylated N-glycan chain bearing the non-sulfated HNK1-epitope at m/z 2430 (M + Na⁺), derived by PNGAseF-digestion of Nid1 coexpressed with dGlcAT-P in CHO-Lec2 cells. The glucuronylated glycan as well as part of the fragments were detected with a mass incremental loss of 32 Da, corresponding to the elimination of methanol. The presence of terminal GlcA is indicated by B series ions (B3α and B3α-32) and by double fragmentation ions (B3αY5α).

Figure 10

MALDI-MS spectrum of the permethylated O-glycan alditols derived by reductive β-elimination from hDG5 coexpressed with dGlcAT-S in CHO-Lec2 cells. No glucuronylation of O-glycans was observed. Non-labeled signals in the mass range up to m/z 1000 are matrix-derived signals or derived from a polyhexose impurity. Short notation of glycan structures: H, hexose; N, N-acetylhexosamine.

Figure 11

MALDI-MS spectrum of the permethylated N-glycans of Nid1, coexpressed with dGlcAT-S in CHO-Lec2 cells. The signals were detected as molecular ions M+Na⁺ -54, corresponding to a loss of sodium-methylate. The non-sulfated HNK1-epitope carrying glycan (H5N4F-GlcA) was detected at m/z 2408. The structure was verified by MS/MS. Short notation of glycan structures: H, hexose; N, N-acetylhexosamine; F, fucose; GlcA, glucuronic acid.