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 (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). The only known biosynthetic pathway generating Neu5Gc is the conversion of CMP-Neu5Ac into CMP-Neu5Gc, which is catalyzed by the CMAH 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. While 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 the present study, we show that pre-loaded Neu5Gc is eliminated from human cells over time and 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 (ManNGc), N-glycolylglucosamine (GlcNGc), and N-glycolylglucosamine 6-phosphate, whereupon irreversible de-N-glycolylation of the latter results 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 ManNGc and GlcNGc (but not glycolate) can serve as precursors for biosynthesis of endogenous Neu5Gc.