Metabolism of vertebrate amino sugars with N-glycolyl groups: elucidating the intracellular fate of the non-human sialic acid N-glycolylneuraminic acid

Abstract

The two major mammalian sialic acids are N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). The only known biosynthetic pathway generating Neu5Gc is the conversion of CMP-N-acetylneuraminic acid into CMP-Neu5Gc, which is catalyzed by the CMP-Neu5Ac hydroxylase enzyme. Given the irreversible nature of this reaction, there must be pathways for elimination or degradation of Neu5Gc, which would allow animal cells to adjust Neu5Gc levels to their needs. Although humans are incapable of synthesizing Neu5Gc due to an inactivated CMAH gene, exogenous Neu5Gc from dietary sources can be metabolically incorporated into tissues in the face of an anti-Neu5Gc antibody response. However, the metabolic turnover of Neu5Gc, which apparently prevents human cells from continued accumulation of this immunoreactive sialic acid, has not yet been elucidated. In this study, we show that pre-loaded Neu5Gc is eliminated from human cells over time, and we propose a conceivable Neu5Gc-degrading pathway based on the well studied metabolism of N-acetylhexosamines. We demonstrate that murine tissue cytosolic extracts harbor the enzymatic machinery to sequentially convert Neu5Gc into N-glycolylmannosamine, N-glycolylglucosamine, and N-glycolylglucosamine 6-phosphate, whereupon irreversible de-N-glycolylation of the latter results in the ubiquitous metabolites glycolate and glucosamine 6-phosphate. We substantiate this finding by demonstrating activity of recombinant human enzymes in vitro and by studying the fate of radiolabeled pathway intermediates in cultured human cells, suggesting that this pathway likely occurs in vivo. Finally, we demonstrate that the proposed degradative pathway is partially reversible, showing that N-glycolylmannosamine and N-glycolylglucosamine (but not glycolate) can serve as precursors for biosynthesis of endogenous Neu5Gc.

FIGURE 1.

CMAH-deficient human and mouse cells do not synthesize Neu5Gc de novo. A, murine Cmah^−/− fibroblasts were cultivated under Neu5Gc-free conditions using 5% human serum. Thereafter, the feeding media were supplemented with either 5 mm Neu5Gc (positive control; shaded dark gray) or human serum was substituted by 10% fetal calf serum (FCS; dotted gray line) to analyze if cells are capable of incorporating exogenous Neu5Gc from the medium supplement FCS. In parallel, cells were kept in 5% human serum without feeding (negative control; black line). After 3 days of feeding, cells were harvested and analyzed by flow cytometry using the polyclonal chicken anti-Neu5Gc antibody (anti-Neu5Gc IgY (25)) for sensitive detection of cell-surface glycosidically bound Neu5Gc. As an additional negative control, cells fed 5 mm Neu5Gc were also stained with control chicken IgY antibody (control IgY; shaded light gray) to verify the absence of Neu5Gc on Cmah^−/− fibroblasts. B, to investigate whether increased glycolic acid levels will result in de novo biosynthesis of Neu5Gc in Neu5Gc-deficient Cmah^−/− fibroblasts, cells were also fed with 10 mm glycolic acid in the media. Cells were treated and detected for Neu5Gc by flow cytometry in parallel to all controls as described above. Also, human THP-I cells (C), M-21 cells (D), and human B-cell lymphoma cells BJA-B K20 (E) were analyzed for their ability to express cell-surface Neu5Gc after feeding with 10 mm glycolic acid by flow cytometry as described above.

FIGURE 2.

Elimination of Neu5Gc in Neu5Gc-loaded cells over time. A, human THP-I cells were cultivated under Neu5Gc-free conditions using 5% human serum and confirmed to be devoid of detectable Neu5Gc. Thereafter, the feeding medium was supplemented with 5 mm Neu5Gc. After 3 days, the feeding media were removed, and cells were washed well, split equally into 10 aliquots, and grown under Neu5Gc-free conditions for up to 28 days. Additional media were added at day 7 and later as necessary. At different time points of the chase, all cells grown in one of the aliquots were harvested and pellets stored at −20 °C. DMB-HPLC analysis was performed once all time points were collected. For DMB-HPLC analysis, total cell lysates were prepared, and the Neu5Gc contents were determined after mild acid hydrolysis. The total Neu5Gc content of each cell pellet was calculated and is depicted as pmol of Neu5Gc over time. B, proposed pathway for the metabolic turnover of excess Neu5Gc in mammalian cells. Neu5Gc is known be a substrate for the pyruvate lyase, which results in the formation of ManNGc (a) (41, 42). Following epimerization from ManNGc toward GlcNGc, which is potentially catalyzed by the GlcNAc-2′-epimerase (b) (73, 74, 90), phosphorylation of GlcNGc in the 6 position might occur by action of the GlcNAc kinase (c) (75, 76) resulting in the formation of GlcNGc-6-P. Thereafter, the N-glycolyl group might be irreversibly removed from GlcNGc-6-P by the GlcNAc-6-P deacetylase (d) (76, 77), which would result in GlcNH₂-6-P, a precursor e.g. for glycolysis, and glycolate, a molecule able to enter the citric acid cycle via glyoxylate.

FIGURE 3.

Evidence for the predicted Neu5Gc-degrading pathway in murine tissue cytosolic extracts. To study the putative degradative pathway of Neu5Gc, all predicted intermediates harboring a radiolabel in the N-glycolyl group were prepared. A, separation of [³H]ManNGc, [¹⁴C]GlcNGc, [³H]GlcNGc-6-P, and [³H]glycolate and commercially available [¹⁴C]glycolate was achieved by HPLC under alkaline conditions using a PA-1 column. Fractions (0.5 ml) were collected, and radioactivity was determined by scintillation counting. The elution profile of ³H radioactivity refers to the left y axis (black symbols and line) and ¹⁴C radioactivity (gray symbols and line) is depicted on the right y axis. B, freshly prepared mouse liver cytosolic extracts were incubated with [³H]ManNGc for 6 h at 37 °C. The reaction was quenched by addition of ethanol (70% final) and subsequent precipitation at −20 °C overnight. The supernatant was supplemented with [¹⁴C]GlcNGc as an internal standard, followed by HPLC analysis and subsequent scintillation counting as described above. The elution profile reveals ³H radioactivity (black symbols and line) derived from the [³H]ManNGc in the sample on the left y axis, and ¹⁴C radioactivity (gray symbols and line) from the internal standard is shown on the right y axis. C, freshly prepared mouse liver cytosolic extracts were incubated with [³H]ManNGc for 6 h at 37 °C. To ensure the presence of ATP to allow the putative kinase reaction (GlcNGc → GlcNGc-6-P), 5 mm ATP, 10 mm MgCl₂, and additional lysate were added at 6 h, and the reaction mixture was incubated for another 6 h at 37 °C. Thereafter, the reaction mixture was analyzed and depicted as described for B. [¹⁴C]GlcNGc and [¹⁴C]glycolate were added as internal standards. Notably, the predicted pathway intermediate GlcNH₂-6-P cannot be detected in this assay as the radiolabels are placed in the N-glycolyl groups exclusively. D, freshly prepared mouse liver cytosolic extracts were incubated with [³H]GlcNGc in the presence of 5 mm ATP and 10 mm MgCl₂ for 6 h at 37 °C. The sample was worked up, analyzed, and presented as mentioned above in B. E, freshly prepared mouse liver cytosolic extracts were incubated with [³H]GlcNGc-6-P for 6 h at 37 °C. The reaction mixture was analyzed and depicted as described in B. F, freshly prepared mouse liver cytosolic extracts were incubated with [³H]Neu5Gc for 6 h at 37 °C. Thereafter, 5 mm ATP, 10 mm MgCl₂, and additional lysate were added as described in C, and the reaction mixture was incubated for another 6 h at 37 °C. Thereafter, the reaction mixture was analyzed and depicted as described for B. The displayed data are representative of at least three independent experiments.

FIGURE 4.

Purified recombinant human GlcNAc kinase NagK phosphorylates GlcNGc. Purified recombinant human GlcNAc kinase NagK was incubated with either 1 mm commercially available GlcNAc or 1 mm synthesized GlcNGc in the presence of 50 mm Tris-HCl pH 7.5, 10 mm MgCl₂, 5 mm ATP, and 5 mm DTT for 1 h at 37 °C. Reactions were quenched with ethanol (70% final), and the protein was precipitated overnight. The filtered supernatants were analyzed by HPAEC-PAD HPLC under alkaline conditions using a PA-1 column. A, 1 μg of GlcNAc-6-P standard. B, NagK reaction mixture with GlcNAc as the substrate. C, 1 μg of GlcNGc-6-P standard. D, NagK reaction mixture with GlcNGc as the substrate. E, control reaction lacking substrates to determine the background arising from the NagK enzyme preparation.

FIGURE 5.

Purified recombinant human GlcNAc-6-P deacetylase was purified and shown to remove the N-glycolyl group of GlcNGc-6-P. A, human GlcNAc-6-P deacetylase was cloned into pET22b vector, expressed in E. coli BL21(DE3) carrying a C-terminal hexahistidine tag, and isolated using immobilized metal affinity chromatography followed by dialysis. Steps of the purification process were analyzed by 10% SDS-PAGE followed by Coomassie staining (upper panel) or Western blot detection using anti-His antibody (lower panel). Shown are aliquots of the bacterial lysate (lane 1), run-through (lane 2), wash fraction (lane 3), enzyme-containing fractions eluted from the immobilized metal affinity chromatography column (lanes 4–9), and the pool of enzyme-containing fractions before (lane 10) and after (lane 11) dialysis. B, activity assays to demonstrate enzymatic activity of the purified recombinant human GlcNAc-6-P deacetylase were set up with either commercially available GlcNAc-6-P or synthesized GlcNGc-6-P as substrates. Aliquots of the reaction mixtures at 0 and 120 min were analyzed by descending paper chromatography followed by silver staining. As a standard (STD), the expected reaction product GlcNH₂-6-P was analyzed as well as GlcNGc. C, purified recombinant human GlcNAc-6-P deacetylase was incubated with either 2 mm commercial GlcNAc-6-P or ∼2 mm synthesized GlcNGc-6-P in the presence of 25 mm Tris-HCl pH 7.5, and 1 mm DTT at 37 °C. At different time points, aliquots were removed; the enzymatic reaction was quenched by addition of ethanol (70% final), and the protein was precipitated overnight. The supernatants were dried down, resuspended in water, and analyzed by HPAEC-PAD HPLC under alkaline conditions using a PA-1 column. The areas under the substrate and product peaks were determined, and the conversion into GlcNH₂-6-P was calculated for all time points. The graph represents the conversion of the substrates, GlcNAc-6-P (black symbols and line) and GlcNGc-6-P (gray symbols and line), into the product GlcNH₂-6-P over time in percent. To control for nonenzymatic decay of the substrates over time, equivalent reactions were set up using heat-inactivated enzyme. No detectable decomposition of the substrates was found when incubating GlcNAc-6-P (black dotted line) or GlcNGc-6-P (gray dotted line).

FIGURE 6.

Proposed degradative pathway occurs in human cells. Human THP-I cells were cultivated under Neu5Gc-free conditions using 5% human serum. Thereafter, the culture medium was supplemented with either [³H]ManNGc or [³H]GlcNGc. After 3 days, the feeding medium was removed, and the cells were washed intensively with PBS. Subsequently, the cell pellets were resuspended in hypotonic 10 mm Tris-HCl pH 7.5, and lysed by repetitive freeze-thaw. Ethanol was added to a final concentration of 70%, and precipitation occurred at −20 °C overnight. The supernatant was supplemented with internal standards and subsequently analyzed by HPLC under alkaline conditions using a PA-1 column. Fractions (0.5 ml) were collected, and radioactivity was determined by scintillation counting. A, THP-I cells fed with [³H]ManNGc were prepared and analyzed as described above. [¹⁴C]GlcNGc and [¹⁴C]glycolate were added as internal standards prior to HPLC analysis. The elution profile reveals ³H radioactivity (black symbols and line) derived from the [³H]ManNGc in the sample on the left y axis, and ¹⁴C radioactivity (gray symbols and line) from the internal standards is shown on the right y axis. B, THP-I cells fed with [³H]GlcNGc were treated as described above. [³H]Mannose (peak marked ***) was added as internal standard prior to HPLC analysis.

FIGURE 7.

Proposed Neu5Gc-degrading pathway is partially reversible. A, synthesis of GlcNGc and peracetylated GlcNGc from a commercial source of glucosamine. a, acetoxyacetyl chloride and aqueous sodium bicarbonate (yield = 94%). b, pyridine and acetic anhydride (yield = 90%). B, human THP-I cells and murine Cmah^−/− fibroblasts were cultivated under Neu5Gc-free conditions using 5% human serum. Thereafter, the feeding media were supplemented with either 5 mm Neu5Gc (positive control; shaded dark gray) or synthesized ManNGc and GlcNGc derivatives. In parallel, cells were kept in 5% human serum without feeding (negative control; black line). After 3 days of feeding, cells were harvested and analyzed by flow cytometry using anti-Neu5Gc IgY (25) for sensitive detection of cell-surface glycosidically bound Neu5Gc. As an additional negative control, cells fed 5 mm Neu5Gc were also stained with control chicken IgY antibody (control IgY; shaded light gray). Histograms of THP-I cells (left panel) and Cmah^−/− fibroblasts (right panel) are shown. Cells were fed either 100 μm ManNGc (gray line), 10 mm ManNGc (dotted black line), or 100 μm per-O-acetylated ManNGc (per-O-acetyl-ManNGc) in addition to the controls described above. C, flow cytometry analysis of THP-I cells (left panel) and Cmah^−/− fibroblasts (right panel). Cells were incubated with either 100 μm GlcNGc (gray line), 10 mm GlcNGc (dotted black line), or 100 μm peracetylated GlcNGc (per-O-acetyl-GlcNGc) in the feeding media. Experiments were done in parallel with the control feedings explained above.