AglJ adds the first sugar of the N-linked pentasaccharide decorating the Haloferax volcanii S-layer glycoprotein

Abstract

Like the Eukarya and Bacteria, the Archaea also perform N glycosylation. Using the haloarchaeon Haloferax volcanii as a model system, a series of Agl proteins involved in the archaeal version of this posttranslational modification has been identified. In the present study, the participation of HVO_1517 in N glycosylation was considered, given its homology to a known component of the eukaryal N-glycosylation pathway and because of the genomic proximity of HVO_1517 to agl genes encoding known elements of the H. volcanii N-glycosylation process. By combining the deletion of HVO_1517 with mass spectrometric analysis of both dolichol phosphate monosaccharide-charged carriers and the S-layer glycoprotein, evidence was obtained showing the participation of HVO_1517, renamed AglJ, in adding the first hexose of the N-linked pentasaccharide decorating this reporter glycoprotein. The deletion of aglJ, however, did not fully prevent the attachment of a hexose residue to the S-layer glycoprotein. Moreover, in the absence of AglJ, the level of only one of the three monosaccharide-charged dolichol phosphate carriers detected in the cell was reduced. Nonetheless, in cells lacking AglJ, no further sugar subunits were added to the remaining monosaccharide-charged dolichol phosphate carriers or to the monosaccharide-modified S-layer glycoprotein, pointing to the importance of the sugar added through the actions of AglJ for proper N glycosylation. Finally, while aglJ can be deleted, H. volcanii surface layer integrity is compromised in the absence of the encoded protein.

FIG. 1.

Deletion of HVO_1517 does not affect cell viability but enhances S-layer glycoprotein SDS-PAGE migration. (A, left and middle) PCR amplification was performed by using a forward primer directed at the 5′ HVO_1517 flanking region and a reverse primer directed at a sequence within the HVO_1517 coding region (yielding primer pair a) or using a forward primer directed at a sequence within the trpA sequence and the same reverse primer as that described above (yielding primer pair b), together with genomic DNA from cells of the parent strain (left) or from cells that had replaced the HVO_1517 gene with the trpA sequence (middle) as a template. (Right) PCR amplification was performed by using primers directed against the HVO_1517 coding region (i.e., primer pair c), together with genomic DNA from cells of the parent strain (parent) or the HVO_1517-deleted strain (ΔHVO_1517). Schematic diagrams showing the relative positions of the forward and reverse primers in each primer pair appear below the panels. Note that primer pairs a and b share the same reverse primer, while primer pairs a and c share the same forward primer. (B) RT-PCR was performed by using primers directed at HVO_1517 (primer pair c) and genomic DNA from parent strain cells or cDNA or RNA from HVO_1517-deleted cells as a template. (C) Deletion of H. volcanii HVO_1517 affects the apparent molecular weight of the S-layer glycoprotein. Equivalent aliquots of H. volcanii cells lacking HVO_1517 (ΔHVO_1517), cells of the parent strain (parent), or cells of the same strain lacking aglD (ΔaglD) were separated by 7.5% SDS-PAGE and Coomassie stained. Only the gel region containing the S-layer glycoprotein is shown.

FIG. 2.

Matrix-assisted laser desorption ionization (MALDI)-TOF analysis of an Asn-13-containing H. volcanii S-layer glycoprotein-derived glycopeptide. The MALDI-TOF spectra of the Asn-13-containing tryptic peptides derived from the S-layer glycoprotein from cells of the parent strain (top) or of the HVO_1517-deleted strain (ΔHVO_1517) (bottom) are shown. The components of the glycopeptide-associated sugar residues, as well as the glycopeptide amino acid sequence, are shown in the inset box, while the glycan moieties decorating the peptide peaks are marked on the MALDI-TOF spectra accordingly.

FIG. 3.

The absence of HVO_1517 affects the level of monosaccharide-modified dolichol phosphate. (A) Normal-phase LC-ESI/MS analysis in the negative-ion mode of the total lipid extracts from cells of the parent strain (parent) (top) and the HVO_1517-deleted strain (ΔHVO_1517) (bottom). amu, atomic mass units. The mass spectra were averaged from the spectra obtained between the 15.5- and 17.5-min retention times. The inset in each panel represents a 5-fold expansion of the hexose-(C₆₀)dolichol phosphate region of the profile. (B) MS/MS verification of dolichylphosphate-hexose by collision-induced dissociation of its [M − H]⁻ ion at m/z 1,079.9.

FIG. 4.

Only one of three monosaccharide-modified dolichol phosphates is affected by a lack of AglJ. Normal-phase LC-extracted ion chromatograms (EIC) of the dolichylphosphate-hexose [M − H]⁻ ion at m/z 1,079.8 from the parent strain (top) and the ΔaglJ strain (bottom) are shown. The peaks at different retention times suggest the existence of three different dolichylphosphate-hexose species. Note the different scales used on the ordinates of the two graphs, highlighting that the 16.2-min peak is reduced 7.3-fold in the mutant compared with the parent strain, suggesting that AglJ is specific for the formation of this particular monosaccharide-modified dolichol phosphate species.

FIG. 5.

HVO_1613, a Dpm1 homologue, is responsible for generating one of three H. volcanii monosaccharide-modified dolichol phosphates. Normal-phase LC EICs of the dolichylphosphate-hexose [M − H]⁻ ion at m/z 1,079.8 from the parent strain (top) and from ΔaglJ (middle) and ΔHVO_1613 cells (bottom) are shown. In the absence of HVO_1613, the first dolichylphosphate-hexose species (16.06 min) is absent.

FIG. 6.

The S layer surrounding H. volcanii cells is protease sensitive in cells deleted of aglJ. Shown are data for cells of the parent strain (parent) (top) or cells of the same strain lacking AglJ (ΔaglJ) or that were challenged with 1 mg/ml proteinase K (pK) at 37°C (bottom). Aliquots were removed immediately prior to incubation with the protease and at subsequent intervals up to 3 h and examined by 7.5% SDS-PAGE.