Metabolism of vertebrate amino sugars with N-glycolyl groups: incorporation of N-glycolylhexosamines into mammalian glycans by feeding N-glycolylgalactosamine.
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.
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093-0687, USA.
Anne K
AK
Bergfeld
Oliver M T
OM
Pearce
Sandra L
SL
Diaz
Roger
R
Lawrence
David J
DJ
Vocadlo
Biswa
B
Choudhury
Jeffrey D
JD
Esko
Ajit
A
Varki
D000818:Animals; D016466:CHO Cells; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003871:Dermatan Sulfate / Q000235:genetics / Q000378:metabolism; D006497:Heparan Sulfate / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D019158:N-Acetylneuraminic Acid / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000235:genetics / Q000378:metabolism
D002238:Carbohydrate Epimerases; D002809:Chondroitin Sulfates; D003871:Dermatan Sulfate; D006497:Heparan Sulfate; D019158:N-Acetylneuraminic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); D014539:Uridine Diphosphate Sugars; C018765:N-acylmannosamine kinase; C100033:UDP acetylglucosamine-2-epimerase