Biochemische Untersuchungen zum Metabolismus von N-Glycolylneuraminsäure in menschlichen und tierischen Zellen
Zusammenfassung der Projektergebnisse
Mammalian cells typically decorate their surfaces with a variety of glycoconjugates, and the terminal position of their glycan chains is commonly occupied by sialic acids (Sias). The two major mammalian Sias 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 allow animal cells to adjust Neu5Gc levels to their needs. Moreover, humans are incapable of synthesizing Neu5Gc due to an inactivated CMAH gene, but exogenous Neu5Gc from dietary sources (such as red meats) can still be metabolically incorporated in the face of an anti-Neu5Gc antibody response. The aim of this DFG-funded project was to unravel the metabolic turnover of Neu5Gc, which apparently prevents human cells from continued accumulation of this immunoreactive sialic acid. We found that 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 serve as precursors for biosynthesis of endogenous Neu5Gc. The unexpected finding that known mammalian pathways for the turnover of N- acetylhexosamines seem to be tolerant towards the N-glycolyl substituent prompted a follow-up project, which aimed to study if N-glycolylated species such as GlcNGc or N-glycolylgalactosamine (GalNGc) are incorporated into mammalian cell surface glycans. After feeding GalNGc to mammalian cells, we demonstrate by mass spectrometry that the metabolic intermediates UDP-GalNGc and UDP-GlcNGc serve as substrates for assembly of most major classes of cellular glycans. We show for the first time incorporation of GalNGc and GlcNGc into Chondroitin/Dermatan sulfates and Heparan sulfates, respectively. As demonstrated by structural analysis, N-glycolylated hexosamines were found in cellular gangliosides and incorporated into known CHO-cell O-glycans. Unexpectedly, we found that exogenously added GalNGc even serves as a precursor for Neu5Gc de novo biosynthesis, presumably involving 7 distinct mammalian enzymes. Following the GalNAc salvage pathway, UDP-GalNGc is epimerized to UDP-GlcNGc, which might compete with endogenous UDP-GlcNAc for Neu5Ac biosynthetic pathway. Using GNE (UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase) deficient cells, we confirm that conversion of GalNGc into Neu5Gc depends on this key enzyme of sialic acid biosynthesis. This study demonstrates that mammalian N-acetylhexosamine pathways and glycan assembly are unexpectedly tolerant towards the N-glycolyl substituent. If this was to happen in vivo, mammalian cells might naturally harbor a so far unnoticed small subset of glycans containing GlcNGc and GalNGc as a result of cellular Neu5Gc turnover. However, just as Neu5Gc itself represents a diet-acquired “xenoautoantigen” in humans with all humans having circulating antibodies against this epitope, even low amounts of its breakdown products (GlcNGc and GalNGc) would theoretically represent additional putative diet-acquired “xeno-autoantigens” in humans. This reasoning prompted yet another project aiming at studying the natural abundance of GalNGc- and GlcNGc-containing glycoconjugates and their possible immunogenicity in humans, which I am addressing at the moment.
