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THE INCORPORATION OF LABELLED AMINO SUGARS BY BACILLUS SUBTILIS.

ABSTRACT: 1. N-Acetyl[1-(14)C]glucosamine (10mum-5mm) is incorporated by cells of Bacillus subtilis at a constant rate (0.2-2mmumoles/mg. dry wt./hr.). The rate of [1-(14)C]glucosamine (2.5mum-5mm) incorporation is proportional to the concentration; it approaches that of N-acetyl[1-(14)C]glucosamine at 5mm. 2. Label from N-acetyl-[1-(14)C]glucosamine and [1-(14)C]glucosamine is incorporated predominantly into the ;hot-trichloroacetic acid-soluble' and ;residue' fractions of cells. Acid hydrolysis of the hot-trichloroacetic acid-soluble fraction yields mainly [(14)C]glucosamine; hydrolysis of the residue fraction yields [(14)C]glucosamine and [(14)C]muramic acid. The label from N-[1-(14)C]acetylglucosamine and sodium [1-(14)C]acetate enters most cell fractions. Incorporation of N-[1-(14)C]acetylglucosamine is inhibited by the addition of unlabelled acetate. 3. Glucose competes with [1-(14)C]glucosamine for incorporation. N-Propionylglucosamine and N-formylglucosamine compete with N-acetyl[1-(14)C]glucosamine. 4. Cells pregrown on N-acetylglucosamine or glucosamine incorporate up to ten times as much N-acetyl[1-(14)C]glucosamine or [1-(14)C]glucosamine in a given time as cells pregrown on glucose.

SUBMITTER: BATES CJ

PROVIDER: S-EPMC1206916 | biostudies-other | 1965 Jul

REPOSITORIES: biostudies-other

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Project description: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.

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THE INCORPORATION OF LABELLED AMINO SUGARS BY BACILLUS SUBTILIS.

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