Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INCORPORATION OF N-GLYCOLYLHEXOSAMINES INTO MAMMALIAN GLYCANS BY FEEDING N-GLYCOLYLGALACTOSAMINE

Anne K Bergfeld,Oliver M.T Pearce,Sandra L Diaz,Roger Lawrence,David J Vocadlo,Biswa Choudhury,Jeffrey D Esko,Ajit Varki

doi:10.1074/jbc.m112.363499

Anne K Bergfeld, Oliver M.T Pearce + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m112.363499

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Abstract

The outermost positions of mammalian cell-surface glycans are predominantly occupied by the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). To date, hydroxylation of CMP-Neu5Ac resulting in the conversion into CMP-Neu5Gc is the only known enzymatic reaction in mammals to synthesize a monosaccharide carrying an N-glycolyl group. In our accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, jbc.M112.363549), we report a metabolic pathway for degradation of Neu5Gc, demonstrating that N-acetylhexosamine pathways are tolerant toward the N-glycolyl substituent of Neu5Gc breakdown products. In this study, we show that exogenously added N-glycolylgalactosamine (GalNGc) serves as a precursor for Neu5Gc de novo biosynthesis, potentially involving seven distinct mammalian enzymes. Following the GalNAc salvage pathway, UDP-GalNGc is epimerized to UDP-GlcNGc, which might compete with the endogenous UDP-GlcNAc for the sialic acid biosynthetic pathway. Using 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. Furthermore, 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 Chinese hamster ovary cell O-glycans. Remarkably, GalNAc derivatives altered the overall O-glycosylation pattern as indicated by the occurrence of novel O-glycan structures. This study demonstrates that mammalian N-acetylhexosamine pathways and glycan assembly are surprisingly tolerant toward the N-glycolyl substituent.

Highlights

N-Acetylhexosamines are precursors of all major vertebrate glycan types
The majority of recovered counts from [3H]GalNGc-fed cells co-eluted with UDP-GalNGc/UDP-GlcNGc standards, whereas the majority of counts aligned with UDP-GalNAc/ UDP-GlcNAc in samples from cells incubated in the presence of [3H]GalNAc (Fig. 4B). This finding showed that human cells are capable of incorporating GalNGc and converting it into UDP-GalNGc as well as UDP-GlcNGc. This observation is consistent with separate observations made in the preceding accompanying papers, wherein we showed that GlcNGc is a metabolite of the Neu5Gc-degrading pathway [1] and that exogenously added GlcNGc can be activated to UDP-GlcNGc and UDP-GalNGc to serve as a substrate to O-GlcNAc transferase [5]
We show for the first time that exogenously added GalNGc can be successfully incorporated into cellular GAGs via the GalNAc-salvage pathway, as GalNGc and as GlcNGc

Summary

Background

Results: Mammalian cells can incorporate N-glycolylgalactosamine (GalNGc) and N-glycolylglucosamine (GlcNGc) into most major cellular glycans and utilize GalNGc for de novo biosynthesis of Neu5Gc. Conclusion: N-Acetylhexosamine biosynthetic pathways and glycan assembly are mostly tolerant toward the N-glycolyl substituent. Therein, we show that mammalian cells can convert Neu5Gc into ManNGc, followed by epimerization to GlcNGc and phosphorylation into GlcNGc 6-phosphate, with final de-acylation of the latter, resulting in formation of the ubiquitous cellular metabolites glycolate and glucosamine 6-phosphate [1] This finding already suggests a certain level of tolerance of basic metabolic enzymes toward the N-glycolyl substituent and raises the question of whether the N-glycolylated breakdown products of Neu5Gc are able to be salvaged. The GalNAc salvage pathway has already been described to be very efficient in introducing artificial GalNAc derivatives, resulting in modified O-glycans on glycoproteins and nucleocytoplasmic proteins

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Conclusion and Future