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Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups

ELUCIDATING THE INTRACELLULAR FATE OF THE NON-HUMAN SIALIC ACID N-GLYCOLYLNEURAMINIC ACID*
  • Anne K. Bergfeld
    Affiliations
    From the Departments of Medicine and Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California San Diego, La Jolla, California 92093-0687
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  • Oliver M.T. Pearce
    Affiliations
    From the Departments of Medicine and Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California San Diego, La Jolla, California 92093-0687
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  • Sandra L. Diaz
    Affiliations
    From the Departments of Medicine and Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California San Diego, La Jolla, California 92093-0687
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  • Tho Pham
    Footnotes
    Affiliations
    From the Departments of Medicine and Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California San Diego, La Jolla, California 92093-0687
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  • Ajit Varki
    Correspondence
    To whom correspondence should be addressed: Glycobiology Research and Training Center, Depts. of Medicine and Cellular and Molecular Medicine, University of California San Diego, 9500 Gilman Dr., University of California San Diego, La Jolla, CA 92093-0687. Tel.: 858-534-2214; Fax: 858-534-5611;
    Affiliations
    From the Departments of Medicine and Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California San Diego, La Jolla, California 92093-0687
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01GM32373 and R01CA38701 (to A. V.). This work was also supported by Deutsche Forschungsgemeinschaft Research Fellowship BE 4643/1-1 (to A. K. B.) and a Cancer Research Institute/Samuel and Ruth Engelberg Fellowship (to O. M. T. P.). A. V. is a co-founder of and consultant for Sialix Inc., a biotech startup company interested in the practical implications of Neu5Gc uptake in humans.
    This article contains supplemental Figs. S1–S5.
    1 Present address: Stanford School of Medicine, Dept. of Pathology, 300 Pasteur Dr., Stanford, CA 94304.
Open AccessPublished:June 12, 2012DOI:https://doi.org/10.1074/jbc.M112.363549
      The two major mammalian sialic acids are N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). The only known biosynthetic pathway generating Neu5Gc is the conversion of CMP-N-acetylneuraminic acid into CMP-Neu5Gc, which is catalyzed by the CMP-Neu5Ac hydroxylase 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. Although 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 this study, we show that pre-loaded Neu5Gc is eliminated from human cells over time, and we 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, N-glycolylglucosamine, and N-glycolylglucosamine 6-phosphate, whereupon irreversible de-N-glycolylation of the latter results in 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 N-glycolylmannosamine and N-glycolylglucosamine (but not glycolate) can serve as precursors for biosynthesis of endogenous Neu5Gc.

      Introduction

      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 (
      • Schauer R.
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      ,
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      The sialome. Far more than the sum of its parts.
      ). Among the >50 naturally occurring sialic acid derivatives, mammalian cells predominantly synthesize and express N-acetylneuraminic acid (Neu5Ac)
      The abbreviations used are: Neu5Ac
      N-acetylneuraminic acid
      Neu5Gc
      N-glycolylneuraminic acid
      CMAH
      CMP-Neu5Ac hydroxylase
      CV
      column volume
      DMB
      1,2-diamino-4,5-methylenedioxybenzene
      GlcNGc
      N-glycolylglucosamine
      HPAEC-PAD
      high performance anion exchange chromatography-pulsed amperometric detection
      ManNAc
      N-acetylmannosamine
      ManNGc
      N-glycolylmannosamine
      P
      phosphate
      EDC
      ethyl-3-(3-dimethylaminopropyl)-carbodiimide
      sulfo-NHS
      N-hydroxysulfosuccinimide
      Sia
      sialic acid.
      and N-glycolylneuraminic acid (Neu5Gc). Activation of sialic acids occurs in the nucleus, and the resulting CMP-activated sialic acids are transported to the Golgi apparatus, where they are transferred onto underlying glycan chains by action of >20 sialyltransferases (
      • Harduin-Lepers A.
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      The human sialyltransferase family.
      ). The only known biosynthetic pathway yielding Neu5Gc takes place at the level of activated sugars and results in conversion of CMP-Neu5Ac into CMP-Neu5Gc, which is catalyzed by the cytosolic CMP-Neu5Ac hydroxylase (CMAH) (
      • Shaw L.
      • Schauer R.
      The biosynthesis of N-glycoloylneuraminic acid occurs by hydroxylation of the CMP-glycoside of N-acetylneuraminic acid.
      ,
      • Kozutsumi Y.
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      Participation of cytochrome b5 in CMP-N-acetylneuraminic acid hydroxylation in mouse liver cytosol.
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      ,
      • Kawano T.
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      • Suzuki A.
      Regulation of biosynthesis of N-glycolylneuraminic acid-containing glycoconjugates. Characterization of factors required for NADH-dependent cytidine 5′-monophosphate-N-acetylneuraminic acid hydroxylation.
      ,
      • Shaw L.
      • Schneckenburger P.
      • Schlenzka W.
      • Carlsen J.
      • Christiansen K.
      • Jürgensen D.
      • Schauer R.
      CMP-N-acetylneuraminic acid hydroxylase from mouse liver and pig submandibular glands. Interaction with membrane-bound and soluble cytochrome b5-dependent electron transport chains.
      ,
      • Takematsu H.
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      • Suzuki A.
      • Kawasaki T.
      Reaction mechanism underlying CMP-N-acetylneuraminic acid hydroxylation in mouse liver. Formation of a ternary complex of cytochrome b5, CMP-N-acetylneuraminic acid, and a hydroxylation enzyme.
      ,
      • Kawano T.
      • Koyama S.
      • Takematsu H.
      • Kozutsumi Y.
      • Kawasaki H.
      • Kawashima S.
      • Kawasaki T.
      • Suzuki A.
      Molecular cloning of cytidine monophospho-N-acetylneuraminic acid hydroxylase. Regulation of species- and tissue-specific expression of N-glycolylneuraminic acid.
      ,
      • Schlenzka W.
      • Shaw L.
      • Kelm S.
      • Schmidt C.L.
      • Bill E.
      • Trautwein A.X.
      • Lottspeich F.
      • Schauer R.
      CMP-N-acetylneuraminic acid hydroxylase. The first cytosolic Rieske iron-sulfur protein to be described in Eukarya.
      ). In humans, the single copy CMAH gene was found inactivated (
      • Chou H.H.
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      • Diaz S.
      • Iber J.
      • Nickerson E.
      • Wright K.L.
      • Muchmore E.A.
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      A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence.
      ,
      • Irie A.
      • Koyama S.
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      • Kawasaki T.
      • Suzuki A.
      The molecular basis for the absence of N-glycolylneuraminic acid in humans.
      ) by an Alu-mediated replacement of a 92-bp exon resulting in a frameshift mutation (
      • Chou H.H.
      • Takematsu H.
      • Diaz S.
      • Iber J.
      • Nickerson E.
      • Wright K.L.
      • Muchmore E.A.
      • Nelson D.L.
      • Warren S.T.
      • Varki A.
      A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence.
      ,
      • Hayakawa T.
      • Aki I.
      • Varki A.
      • Satta Y.
      • Takahata N.
      Fixation of the human-specific CMP-N-acetylneuraminic acid hydroxylase pseudogene and implications of haplotype diversity for human evolution.
      ,
      • Varki A.
      N-Glycolylneuraminic acid deficiency in humans.
      ). The complete absence of Neu5Gc in Cmah−/− mice carrying the human-like mutation (
      • Hedlund M.
      • Tangvoranuntakul P.
      • Takematsu H.
      • Long J.M.
      • Housley G.D.
      • Kozutsumi Y.
      • Suzuki A.
      • Wynshaw-Boris A.
      • Ryan A.F.
      • Gallo R.L.
      • Varki N.
      • Varki A.
      N-Glycolylneuraminic acid deficiency in mice. Implications for human biology and evolution.
      ) suggests that humans completely lost the ability for de novo Neu5Gc biosynthesis. Despite the lack of an alternative pathway for Neu5Gc synthesis, the presence of Neu5Gc has been conclusively demonstrated on various human carcinomas and in fetal meconium (
      • Higashi H.
      • Hirabayashi Y.
      • Fukui Y.
      • Naiki M.
      • Matsumoto M.
      • Ueda S.
      • Kato S.
      Characterization of N-glycolylneuraminic acid-containing gangliosides as tumor-associated Hanganutziu-Deicher antigen in human colon cancer.
      ,
      • Miyoshi I.
      • Higashi H.
      • Hirabayashi Y.
      • Kato S.
      • Naiki M.
      Detection of 4-O-acetyl-N-glycolylneuraminyl lactosylceramide as one of tumor-associated antigens in human colon cancer tissues by specific antibody.
      ,
      • Hirabayashi Y.
      • Higashi H.
      • Kato S.
      • Taniguchi M.
      • Matsumoto M.
      Occurrence of tumor-associated ganglioside antigens with Hanganutziu-Deicher antigenic activity on human melanomas.
      ,
      • Kawachi S.
      • Saida T.
      • Uhara H.
      • Uemura K.
      • Taketomi T.
      • Kano K.
      Heterophile Hanganutziu-Deicher antigen in ganglioside fractions of human melanoma tissues.
      ,
      • Devine P.L.
      • Clark B.A.
      • Birrell G.W.
      • Layton G.T.
      • Ward B.G.
      • Alewood P.F.
      • McKenzie I.F.
      The breast tumor-associated epitope defined by monoclonal antibody 3E1.2 is an O-linked mucin carbohydrate containing N-glycolylneuraminic acid.
      ,
      • Malykh Y.N.
      • Schauer R.
      • Shaw L.
      N-Glycolylneuraminic acid in human tumors.
      ). With the development of additional sensitive detection methods, low levels of Neu5Gc have been found on normal human tissues such as endothelia of blood vessels (
      • Diaz S.L.
      • Padler-Karavani V.
      • Ghaderi D.
      • Hurtado-Ziola N.
      • Yu H.
      • Chen X.
      • Brinkman-Van der Linden E.C.
      • Varki A.
      • Varki N.M.
      Sensitive and specific detection of the non-human sialic acid N-glycolylneuraminic acid in human tissues and biotherapeutic products.
      ,
      • Tangvoranuntakul P.
      • Gagneux P.
      • Diaz S.
      • Bardor M.
      • Varki N.
      • Varki A.
      • Muchmore E.
      Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid.
      ) and apparently originate from Neu5Gc-rich dietary sources such as red meats (
      • Tangvoranuntakul P.
      • Gagneux P.
      • Diaz S.
      • Bardor M.
      • Varki N.
      • Varki A.
      • Muchmore E.
      Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid.
      ,
      • Bardor M.
      • Nguyen D.H.
      • Diaz S.
      • Varki A.
      Mechanism of uptake and incorporation of the non-human sialic acid N-glycolylneuraminic acid into human cells.
      ). Our companion paper (
      • Banda K.
      • Gregg C.J.
      • Chow R.
      • Varki N.
      • Varki A.
      Metabolism of vertebrate amino sugars with N-glycolyl groups. Mechanisms underlying gastrointestinal incorporation of the non-human sialic acid xeno-autoantigen N-glycolylneuraminic acid.
      ) shows for the first time that Cmah−/− mice incorporate dietary Neu5Gc with a human-like bodily distribution after feeding with Neu5Gc-containing glycoconjugates. Indeed, human cells were also shown to take up exogenous Neu5Gc from the culture medium and incorporate it into cell-surface glycoconjugates, demonstrating that intrinsic biochemical pathways are compatible with Neu5Gc and recognize it biochemically as “self” (
      • Tangvoranuntakul P.
      • Gagneux P.
      • Diaz S.
      • Bardor M.
      • Varki N.
      • Varki A.
      • Muchmore E.
      Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid.
      ,
      • Bardor M.
      • Nguyen D.H.
      • Diaz S.
      • Varki A.
      Mechanism of uptake and incorporation of the non-human sialic acid N-glycolylneuraminic acid into human cells.
      ). In striking contrast, the human immune system regards Neu5Gc-containing glycan structures as “foreign,” resulting in a polyclonal humoral response, with circulating and varying titers of anti-Neu5Gc antibodies among all humans (
      • Padler-Karavani V.
      • Yu H.
      • Cao H.
      • Chokhawala H.
      • Karp F.
      • Varki N.
      • Chen X.
      • Varki A.
      Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: potential implications for disease.
      ,
      • Nguyen D.H.
      • Tangvoranuntakul P.
      • Varki A.
      Effects of natural human antibodies against a nonhuman sialic acid that metabolically incorporates into activated and malignant immune cells.
      ). The incorporation of dietary Neu5Gc in the face of an ongoing immune response against this non-human epitope makes Neu5Gc the first known example of a “xeno-autoantigen” (
      • Varki N.M.
      • Strobert E.
      • Dick Jr., E.J.
      • Benirschke K.
      • Varki A.
      Biomedical differences between human and nonhuman hominids. Potential roles for uniquely human aspects of sialic acid biology.
      ,
      • Padler-Karavani V.
      • Hurtado-Ziola N.
      • Pu M.
      • Yu H.
      • Huang S.
      • Muthana S.
      • Chokhawala H.A.
      • Cao H.
      • Secrest P.
      • Friedmann-Morvinski D.
      • Singer O.
      • Ghaderi D.
      • Verma I.M.
      • Liu Y.T.
      • Messer K.
      • Chen X.
      • Varki A.
      • Schwab R.
      Human xeno-autoantibodies against a non-human sialic acid serve as novel serum biomarkers and immunotherapeutics in cancer.
      ). Interestingly, the major sites of Neu5Gc accumulation in humans (endothelia of blood vessels and epithelia lining the hollow organs) are also the locations where predominantly human-specific diseases involving chronic inflammation seem to occur (
      • Varki N.M.
      • Strobert E.
      • Dick Jr., E.J.
      • Benirschke K.
      • Varki A.
      Biomedical differences between human and nonhuman hominids. Potential roles for uniquely human aspects of sialic acid biology.
      ). Notably, such chronic inflammation-mediated human health issues (e.g. atherosclerosis of medium and large vessels or certain epithelial carcinomas) are also associated with the consumption of Neu5Gc-rich foods such as red meats (
      • Willett W.C.
      Diet and cancer.
      ,
      • Norat T.
      • Lukanova A.
      • Ferrari P.
      • Riboli E.
      Meat consumption and colorectal cancer risk. Dose-response meta-analysis of epidemiological studies.
      ,
      • Zhang J.
      • Kesteloot H.
      Milk consumption in relation to incidence of prostate, breast, colon, and rectal cancers. Is there an independent effect?.
      ,
      • Wiseman M.
      The second World Cancer Research Fund/American Institute for Cancer Research expert report. Food, nutrition, physical activity, and the prevention of cancer. A global perspective.
      ). Thus, “xenosialitis” has been proposed to be a novel human-specific mechanism, which could exacerbate vascular pathologies such as arteriosclerosis (
      • Pham T.
      • Gregg C.J.
      • Karp F.
      • Chow R.
      • Padler-Karavani V.
      • Cao H.
      • Chen X.
      • Witztum J.L.
      • Varki N.M.
      • Varki A.
      Evidence for a novel human-specific xeno-auto-antibody response against vascular endothelium.
      ), and was found to stimulate tumor growth in a human-like mouse model (
      • Hedlund M.
      • Padler-Karavani V.
      • Varki N.M.
      • Varki A.
      Evidence for a human-specific mechanism for diet and antibody-mediated inflammation in carcinoma progression.
      ). Investigating the intracellular fate of Neu5Gc may help explain the underlying mechanisms of xenosialitis in humans.
      As the CMAH reaction (CMP-Neu5Ac → CMP-Neu5Gc) is irreversible, all mammalian cells must have pathways to adjust cellular Neu5Gc levels to their needs to avoid continued accumulation. Such a metabolic pathway for the turnover of Neu5Gc has not been reported in any system. However, several enzymes involved in sialic acid biosynthesis were previously shown to accommodate both the N-acetyl substituent as well as the N-glycolyl moiety. Examples include the CMP-sialic acid synthetase from various animals, including humans (
      • Kean E.L.
      • Roseman S.
      The sialic acids. X. Purification and properties of cytidine 5′-monophosphosialic acid synthetase.
      ) and mammalian sialyltransferases (
      • Higa H.H.
      • Paulson J.C.
      Sialylation of glycoprotein oligosaccharides with N-acetyl-, N-glycolyl-, and N-O-diacetylneuraminic acids.
      ). Moreover, mammalian N-acetylneuraminate pyruvate-lyase was found to release pyruvate from Neu5Ac and Neu5Gc to give ManNAc and ManNGc, respectively (
      • Bulai T.
      • Bratosin D.
      • Artenie V.
      • Montreuil J.
      Characterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes.
      ,
      • Schauer R.
      • Sommer U.
      • Krüger D.
      • van Unen H.
      • Traving C.
      The terminal enzymes of sialic acid metabolism. Acylneuraminate pyruvate-lyases.
      ). In this study, we hypothesized that additional enzymes involved in further breakdown of ManNAc and ManNGc may also tolerate the N-glycolyl group. Based on the well known metabolism of N-acetylhexosamines in mammalian cells, we thus propose a conceivable pathway for the degradation of excess Neu5Gc. We show that the predicted reactions occur in murine tissue cytosolic extracts, demonstrating that mammalian N-acetylhexosamine pathways can accommodate the N-glycolyl group to some extent allowing degradation of excess Neu5Gc into the two ubiquitous metabolites glycolate and glucosamine 6-phosphate. Further analysis of recombinant human enzymes and studies on the fate of radiolabeled pathway intermediates in cultured human cells indicate that this pathway likely also persists in humans. The identification of the first Neu5Gc-degrading metabolic pathway in mammals has fundamental implications for sialic acid biochemistry and biology, and it may also have significant impact on the development of strategies to manage Neu5Gc accumulation in humans and its potentially deleterious consequence xenosialitis.

      EXPERIMENTAL PROCEDURES

      Materials

      [1,2-14C]Glycolic acid was received from ICN Biomedicals, Inc. All chemicals were purchased at HPLC grade from Fisher or Sigma unless otherwise stated.

      Generation of Expression Plasmids

      The sequence of all primers used in this study can be found in supplemental Fig. S5. Human NAGK gene (accession number BC0010029), cloned EcoRI/XhoI in pOTB7 vector was purchased from Thermo Scientific (clone ID 3347484). The gene was amplified by PCR using the primer pair AKB20/AKB21 and the above plasmid as template. The PCR product was cloned via EcoRI/XhoI sites into a modified pGEX-2T expression vector (GE Healthcare, harboring the additional sequence 5′-CCGGGTCGACTCGAGCGGCCGC-3′ inserted 3′ of EcoRI), resulting in the plasmid pGEX-hNAGK to express NagK with an N-terminal GST fusion tag. Human AMDHD2 gene (accession number BC018734), cloned EcoRI/XhoI in pOTB7 vector was purchased from Thermo Scientific (clone ID 4869721). The gene was amplified by PCR using the primer pair AKB3/AKB9 and the above plasmid as template. The pET22b expression vector (Novagen) was modified by adapter ligation via XbaI/BamHI of primers AKB11/AKB12 to remove the pelB leader sequence. The PCR product was subcloned via NdeI/XhoI sites into the modified pET22b expression vector described above resulting in plasmid pET22b-hAMDHD2-His to express Amdhd2 with a C-terminal hexahistidine tag. The identity of all plasmids was confirmed by sequencing.

      Expression and Purification of Recombinant Human GlcNAc Kinase

      Plasmid pGEX-hNAGK was transformed into BL21(DE3) bacteria, and the protocol for expression and purification was modified from Yamada-Okabe et al. and Weihofen et al. (
      • Yamada-Okabe T.
      • Sakamori Y.
      • Mio T.
      • Yamada-Okabe H.
      Identification and characterization of the genes for N-acetylglucosamine kinase and N-acetylglucosamine-phosphate deacetylase in the pathogenic fungus Candida albicans.
      ,
      • Weihofen W.A.
      • Berger M.
      • Chen H.
      • Saenger W.
      • Hinderlich S.
      Structures of human N-acetylglucosamine kinase in two complexes with N-acetylglucosamine and with ADP/glucose. Insights into substrate specificity and regulation.
      ). Freshly transformed bacteria were cultivated at 37 °C in 500 ml of LB medium containing 200 μg/ml carbenicillin. At A600 nm = 0.6, expression was induced by adding 0.3 mm isopropyl β-d-1-thiogalactopyranoside. Thereafter, bacteria were grown at 37 °C for 4 h, pelleted at 6000 × g for 10 min at 4 °C, and washed with 20 ml of PBS, and the pellet was kept on ice. Bacteria were resuspended in 20 ml of PBS, lysed by sonication (in ice-water, five times for 30 s pulsed with 0.5 s ON and 0.5 s OFF and 2 min breaks in-between; level 4 on a 550 sonic dismembrator, Fisher), and the membrane fraction was removed at 4 °C/27,000 × g/1 h. The supernatant containing soluble expressed GST-NagK was loaded onto a 2-ml glutathione-Sepharose 4B column (GE Healthcare) pre-equilibrated with 10 ml of PBS. After loading, the column was washed successively with 5 ml of PBS and 10 ml of buffer A (20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm dithiothreitol (DTT)). Bound proteins were eluted with 3× 600 μl of 10 mm glutathione in buffer A. Eluted fractions were pooled and dialyzed against buffer A using Spectra/Por Dialysis Membrane 6 (MWCO 10,000; Spectrum Laboratories). The enzyme was stored at −80 °C and remained active for at least 1 year.

      Expression and Purification of Recombinant Human GlcNAc-6-P Deacetylase

      Plasmid pET22b-hAMDHD2-His was transformed into BL21(DE3) bacteria. Freshly transformed bacteria were cultivated at 37 °C in 500 ml of LB medium containing 200 μg/ml carbenicillin. At A600 nm = 0.6, expression was induced by adding 1 mm isopropyl β-d-1-thiogalactopyranoside, and the medium was supplemented with 1 mm ZnCl2 as described previously for the Escherichia coli analog NagA (
      • Hall R.S.
      • Xiang D.F.
      • Xu C.
      • Raushel F.M.
      N-Acetyl-d-glucosamine-6-phosphate deacetylase. Substrate activation via a single divalent metal ion.
      ). Thereafter, bacteria were grown at 15 °C for 16 h, pelleted at 6000 × g for 10 min at 4 °C, and washed with 20 ml of PBS, and the pellet was kept on ice. Bacteria were resuspended in loading buffer (50 mm Tris-HCl pH 7.5, 100 mm NaCl, 20 mm imidazole, 1 mm DTT) supplemented with 100 μg/ml phenylmethanesulfonyl fluoride (PMSF) and lysed by sonication (on ice-water, five times for 30 s pulsed with 0.5 s ON and 0.5 s OFF and 2 min breaks in between; level 4 on a 550 sonic dismembrator, Fisher), and the membrane fraction was removed at 4 °C/27,000 × g/1 h. The supernatant containing soluble expressed Amdhd2-His was loaded onto a 1-ml HisTrapHP column (GE Healthcare) pre-equilibrated with loading buffer. Bound proteins were eluted with a 20 ml linear imidazole gradient (20–500 mm imidazole in loading buffer; flow rate 1 ml/min) by FPLC. Enzyme-containing fractions were pooled and dialyzed against storage buffer (50 mm Tris-HCl pH 7.5, 100 mm NaCl, 1 mm DTT) using Spectra/Por dialysis membrane 6 (MWCO 10,000; Spectrum Laboratories). The enzyme was either stored flash-frozen at −80 °C or in 50% glycerol at −20 °C and remained active for at least 1 year.

      SDS-PAGE, Coomassie Staining, and Immunoblotting

      SDS-PAGE was performed under reducing conditions using 2.5% (v/v) β-mercaptoethanol. Coomassie staining and Western blot analysis with anti-penta-His antibody (Qiagen) were performed as described previously (
      • Bergfeld A.K.
      • Claus H.
      • Lorenzen N.K.
      • Spielmann F.
      • Vogel U.
      • Mühlenhoff M.
      The polysialic acid-specific O-acetyltransferase OatC from Neisseria meningitidis serogroup C evolved apart from other bacterial sialate O-acetyltransferases.
      ).

      HPLC Analysis Using HPAEC-PAD Followed by Scintillation Counting

      Monosaccharides were separated by HPLC (Dionex DX-600 BioLC system equipped with an ED50 Electrochemical Detector, Dionex) using a CarboPac PA-1 column (Dionex) under alkaline conditions. Samples (100 μl) were injected, and the developed gradient described in supplemental Fig. S2 was applied at a flow rate of 1 ml/min. Following HPAEC-PAD detection, 0.5-ml fractions were collected directly into scintillation vials whenever radiolabeled material was analyzed. To each vial, 5 ml of scintillation mixture was added (Scinti Verse BD, Fisher) prior to measuring on a scintillation counter. Because of the lack of qualified hydroxyl groups, glycolic acid cannot be detected by HPAEC-PAD. However, co-elution of commercial [14C]glycolic acid and synthesized [3H]glycolic acid is observed at 13.5 ml using the gradient described in supplemental Fig. S2.

      Activity Assay for Human GlcNAc Kinase

      Assay conditions were modified from Yamada-Okabe and co-workers (
      • Yamada-Okabe T.
      • Sakamori Y.
      • Mio T.
      • Yamada-Okabe H.
      Identification and characterization of the genes for N-acetylglucosamine kinase and N-acetylglucosamine-phosphate deacetylase in the pathogenic fungus Candida albicans.
      ). The assay contained 50 mm Tris-HCl pH 7.5, 10 mm MgCl2, 5 mm DTT, 5 mm ATP, 1 mm of either GlcNAc or GlcNGc, and 0.22 nmol of purified enzyme in a total volume of 50 μl. As a background control, the reaction was set up without the substrates GlcNAc or GlcNGc. After 1 h of incubation at 37 °C, the reaction mixtures were spun through Amicon Ultra-0.5 ml 10K Ultracel filter units (Millipore) to remove the enzyme prior to analysis by HPLC following HPAEC-PAD.

      Activity Assays for Human GlcNAc-6-P Deacetylase

      1) Enzyme activity assays were carried out in 30 μl volumes containing 25 mm Tris-HCl pH 7.5, 1 mm DTT, either 5 mm GlcNAc-6-P or 5 mm GlcNGc-6-P, and 0.3 nmol of purified recombinant human deacetylase. Before and after incubation at 37 °C for 1 h, 10 μl of the reaction mixtures were spotted on 3MM CHR Whatman chromatography paper. Subsequently, separation was achieved by descending paper chromatography using running buffer (3 volumes ethanol and 7 volumes of 1 m ammonium acetate, pH 3.9) as described previously (
      • Paladini A.C.
      • Leloir L.F.
      Studies on uridine-diphosphate-glucose.
      ). The separated sugars on the chromatogram were detected by silver staining using a modified version of the protocol from Trevelyan et al. (
      • Trevelyan W.E.
      • Procter D.P.
      • Harrison J.S.
      Detection of sugars on paper chromatograms.
      ). In brief, the dried paper chromatogram was quickly and evenly dipped through a trough containing solution A (0.1 ml of saturated aqueous silver nitrate solution in 20 ml of acetone). After air-drying, the paper was dipped through solution B (20 mg of NaOH in just enough water to dissolve it, and methanol was added to a final volume of 1 liter). Once color has developed sufficiently, the staining was fixed by passing through solution C (31.8 mg of sodium thiosulfate first dissolved in 475 ml of water followed by addition of 475 ml of methanol). 2) An additional activity assay was set up in a 210-μl final volume containing 25 mm Tris-HCl pH 7.5, 1 mm DTT, either 2 mm GlcNAc-6-P or 2 mm GlcNGc-6-P, and 0.9 nmol of purified recombinant human deacylase at 37 °C. Control reactions were set up by adding heat-inactivated enzyme (5 min/95 °C) to the reaction mixtures. Aliquots (30 μl) were removed from the reaction mixtures after 30 s, 1, 5, 15, and 30 min, and 1 h and transferred into tubes harboring 70 μl of pre-cooled ethanol to quench the reaction. Precipitation was allowed to occur overnight at −20 °C. Thereafter, samples were spun at 20,000 × g/15 min/4 °C, and the supernatants were transferred into clean tubes and dried. For subsequent HPLC analysis, samples were resuspended in 110 μl of water, filtered, and analyzed as described above.

      Mice

      WT C57BL/6 mice were purchased from Harlan Laboratories. Cmah−/− mice have been described previously (
      • Hedlund M.
      • Tangvoranuntakul P.
      • Takematsu H.
      • Long J.M.
      • Housley G.D.
      • Kozutsumi Y.
      • Suzuki A.
      • Wynshaw-Boris A.
      • Ryan A.F.
      • Gallo R.L.
      • Varki N.
      • Varki A.
      N-Glycolylneuraminic acid deficiency in mice. Implications for human biology and evolution.
      ). Mice were fed standard chow (PicoLab Rodent Diet 20; LabDiet) and water ad libitum and maintained on a 12-h light/dark cycle. All animal work was performed in accordance with The Association for Assessment and Accreditation of Laboratory Animal Care under protocol S01227 approved by The Institutional Animal Care and Use Committee of the University of California San Diego.

      Preparation of Cmah−/− Mouse Embryonic Fibroblasts

      To generate Cmah−/− fibroblasts, 14- to 17-day-old embryos of a Cmah−/− mouse (
      • Hedlund M.
      • Tangvoranuntakul P.
      • Takematsu H.
      • Long J.M.
      • Housley G.D.
      • Kozutsumi Y.
      • Suzuki A.
      • Wynshaw-Boris A.
      • Ryan A.F.
      • Gallo R.L.
      • Varki N.
      • Varki A.
      N-Glycolylneuraminic acid deficiency in mice. Implications for human biology and evolution.
      ) were dissected by removing the amniotic sac and the placenta from the embryo. Blood was removed by repetitive washing in PBS, and thereafter, the carcasses were minced with scissors. Per embryo, 3–5 ml of 0.05% trypsin/EDTA solution (Invitrogen) were added and incubated at 4 °C overnight to allow diffusion of the trypsin into the tissue. The next day, without disturbing the pellet of tissue, most of the trypsin solution was removed, and the tissue pellet was incubated at 37 °C for 60 min. Thereafter, the pellet was carefully resuspended in 50 ml of fresh Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. The sample was spun at 1000 rpm for 1 min to let the remaining tissue clumps accumulate at the bottom of the tube. The supernatant was transferred to a clean tube and spun for 5 min at 1000 rpm to pellet cells. The supernatant was aspirated, and the cell pellet was resuspended in fresh media. Cells derived from 3 to 4 embryos were plated in a T-175 flask, and the media were changed the next day. The cells were passaged upon reaching confluency.
      Production of the retrovirus was conducted based on previously described methods (
      • Wazer D.E.
      • Liu X.L.
      • Chu Q.
      • Gao Q.
      • Band V.
      Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7.
      ). The packaging cell line PA317 LXSN16E6E7 developed by transfection of the retroviral vector pLXSN16E6E7, which contains the human papilloma virus as well as a gene controlling resistance to neomycin, was purchased from ATCC. These cells were grown in DMEM with 4 mm l-glutamine adjusted to contain 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, and 10% fetal bovine serum. The cells were grown to 70–80% confluency, and the supernatant was collected after 16 h. The supernatant was filter-sterilized through a 0.22-μm filter and stored at −80 °C. The supernatant was then used for transduction to immortalize Cmah−/− fibroblasts based on previous protocols (
      • Choo A.
      • Padmanabhan J.
      • Chin A.
      • Fong W.J.
      • Oh S.K.
      Immortalized feeders for the scale-up of human embryonic stem cells in feeder and feeder-free conditions.
      ). Briefly, Cmah−/− fibroblast cells were seeded at 3 × 105 cells per T-75 flask and allowed to adhere overnight. Transduction of the cells was accomplished by using 1 ml of the sterile-filtered supernatant containing the human papilloma virus in the presence of 8 μg/ml Polybrene in a total volume of 10 ml for 8 h at 37 °C in a 5% CO2-humidified incubator. The media containing the virus were removed and replaced with fresh medium (DMEM high glucose supplemented with 10% FCS, 25 units/ml penicillin, 25 units/ml streptomycin) and grown for 3 days. Transduced cells were selected by adding 100 μg/ml G418 for 14 days.

      Tissue Culture

      Human acute monocytic leukemia cell line THP-I (
      • Auwerx J.
      The human leukemia cell line, THP-1. A multifaceted model for the study of monocyte-macrophage differentiation.
      ) and murine Cmah−/− fibroblasts were cultivated in DMEM (high glucose, Invitrogen); human M-21 cells (
      • Sjoberg E.R.
      • Manzi A.E.
      • Khoo K.H.
      • Dell A.
      • Varki A.
      Structural and immunological characterization of O-acetylated GD2. Evidence that GD2 is an acceptor for ganglioside O-acetyltransferase in human melanoma cells.
      ) were grown in α-minimal essential medium, and human B lymphoma cell line BJA-B (Burkitt's lymphoma-like (
      • Menezes J.
      • Leibold W.
      • Klein G.
      • Clements G.
      Establishment and characterization of an Epstein-Barr virus (EBC)-negative lymphoblastoid B cell line (BJA-B) from an exceptional, EBV-genome-negative African Burkitt's lymphoma.
      ), subclones K88 and K20 (
      • Keppler O.T.
      • Hinderlich S.
      • Langner J.
      • Schwartz-Albiez R.
      • Reutter W.
      • Pawlita M.
      UDP-GlcNAc 2-epimerase. A regulator of cell surface sialylation.
      )) were kept in RPMI 1640 medium. All media were supplemented with 5% Neu5Gc-free human serum (heat-inactivated and sterile-filtered, Valley Biomedical Inc.) and 2 mm glutamine. Cells were cultivated in a humidified 5% CO2 atmosphere at 37 °C.

      Feeding of Animal Cells

      For feedings of suspension cells (THP-I or BJA-B), 1 × 106 cells were set up in a well of a 6-well dish in a final volume of 2 ml of feeding media as described below. Adherent cells (Cmah−/− fibroblasts, M-21 cells) were split into P-100 dishes in a manner to reach confluency after 4 days again and allowed to attach overnight. The next day, the media were removed; cells were washed with PBS, and 5 ml of feeding media were added as described below. The feeding media were supplemented with compounds as indicated using the following pH neutral and sterile-filtered stock solutions either prepared in PBS, pH 7.4 (100 mm Neu5Gc, 1 m glycolic acid, 1 m GABA, 1 m ManNGc, and 1 m GlcNGc), or dissolved in DMSO (100 mm GlcNGc, 100 mm per-O-acetyl-GlcNGc, 100 mm ManNGc, and 100 mm per-O-acetyl-ManNGc). Regarding cell feedings with ManNGc and GlcNGc, the DMSO stock was used when aiming for a final concentration of 100 μm in the media. The PBS stock was used to set up feeding media with a 10 mm final concentration of the compounds. Cells were kept in the feeding media for 3 days. Adherent cells were detached with 20 mm EDTA in PBS first, and all cells were washed three times with PBS. Cells were analyzed by flow cytometry as described below.
      For the pulse-chase experiment (Fig. 2A), 1 × 107 THP-I cells were incubated in 10 ml of fresh media supplemented with 5 mm Neu5Gc for 3 days. Thereafter, the cell pellet was washed three times with PBS, resuspended in 20 ml of fresh media, and split equally into 10 wells of 6-well dishes (2 ml/well). All cells of a well were harvested at days 0 and 1–6. At day 6 of the chase, all remaining wells were diluted 1:3 in fresh media individually and transferred into T-75 flasks. At day 14, all cells of one T-75 flask were harvested, and the remaining two T-75 flasks were further diluted 1:3. At day 21, all cells of one T-75 flask were harvested, and the last T-75 flask was again diluted 1:3, and all cells were harvested on day 28. Cell pellets were washed three times with PBS and frozen at −80 °C. After all time points were collected, Neu5Gc content of total cell lysates was determined by DMB-HPLC as described below.
      Figure thumbnail gr2
      FIGURE 2Elimination of Neu5Gc in Neu5Gc-loaded cells over time. A, human THP-I cells were cultivated under Neu5Gc-free conditions using 5% human serum and confirmed to be devoid of detectable Neu5Gc. Thereafter, the feeding medium was supplemented with 5 mm Neu5Gc. After 3 days, the feeding media were removed, and cells were washed well, split equally into 10 aliquots, and grown under Neu5Gc-free conditions for up to 28 days. Additional media were added at day 7 and later as necessary. At different time points of the chase, all cells grown in one of the aliquots were harvested and pellets stored at −20 °C. DMB-HPLC analysis was performed once all time points were collected. For DMB-HPLC analysis, total cell lysates were prepared, and the Neu5Gc contents were determined after mild acid hydrolysis. The total Neu5Gc content of each cell pellet was calculated and is depicted as pmol of Neu5Gc over time. B, proposed pathway for the metabolic turnover of excess Neu5Gc in mammalian cells. Neu5Gc is known be a substrate for the pyruvate lyase, which results in the formation of ManNGc (a) (
      • Bulai T.
      • Bratosin D.
      • Artenie V.
      • Montreuil J.
      Characterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes.
      ,
      • Schauer R.
      • Sommer U.
      • Krüger D.
      • van Unen H.
      • Traving C.
      The terminal enzymes of sialic acid metabolism. Acylneuraminate pyruvate-lyases.
      ). Following epimerization from ManNGc toward GlcNGc, which is potentially catalyzed by the GlcNAc-2′-epimerase (b) (
      • Ghosh S.
      • Roseman S.
      The sialic acids. V. N-Acyl-d-glucosamine 2-epimerase.
      ,
      • Luchansky S.J.
      • Yarema K.J.
      • Takahashi S.
      • Bertozzi C.R.
      GlcNAc 2-epimerase can serve a catabolic role in sialic acid metabolism.
      ,
      • Takahashi S.
      • Takahashi K.
      • Kaneko T.
      • Ogasawara H.
      • Shindo S.
      • Kobayashi M.
      Human renin-binding protein is the enzyme N-acetyl-d-glucosamine 2-epimerase.
      ), phosphorylation of GlcNGc in the 6 position might occur by action of the GlcNAc kinase (c) (
      • Hinderlich S.
      • Berger M.
      • Schwarzkopf M.
      • Effertz K.
      • Reutter W.
      Molecular cloning and characterization of murine and human N-acetylglucosamine kinase.
      ,
      • Weidanz J.A.
      • Campbell P.
      • Moore D.
      • DeLucas L.J.
      • Rodén L.
      • Thompson J.N.
      • Vezza A.C.
      N-Acetylglucosamine kinase and N-acetylglucosamine-6-phosphate deacetylase in normal human erythrocytes and Plasmodium falciparum.
      ) resulting in the formation of GlcNGc-6-P. Thereafter, the N-glycolyl group might be irreversibly removed from GlcNGc-6-P by the GlcNAc-6-P deacetylase (d) (
      • Weidanz J.A.
      • Campbell P.
      • Moore D.
      • DeLucas L.J.
      • Rodén L.
      • Thompson J.N.
      • Vezza A.C.
      N-Acetylglucosamine kinase and N-acetylglucosamine-6-phosphate deacetylase in normal human erythrocytes and Plasmodium falciparum.
      ,
      • Campbell P.
      • Laurent T.C.
      • Rodén L.
      Assay and properties of N-acetylglucosamine-6-phosphate deacetylase from rat liver.
      ), which would result in GlcNH2-6-P, a precursor e.g. for glycolysis, and glycolate, a molecule able to enter the citric acid cycle via glyoxylate.

      Radiolabeled Cell Feedings

      THP-I cells (1 × 106 cells, 2 ml of culture medium in a well of a 6-well dish) were incubated for 3 days in the presence of 370 kBq [3H]ManNGc, 370 kBq [3H]GlcNGc, or 74 kBq [3H]glycolic acid. Cmah−/− fibroblasts were cultured in a P-100 dish, and 3 days prior to reaching confluency, the media were removed; cells were washed, and 5 ml of fresh medium supplemented with 110 kBq [3H]glycolic acid was added. After 3 days, cells were harvested using 20 mm EDTA in PBS. All radiolabeled cells were washed three times with PBS, and pellets were stored at −20 °C. For analysis, cell pellets were resuspended in 30 μl of 10 mm Tris-HCl, pH 7.5, and lysed by repetitive freeze-thaw. Proteins and membrane debris were precipitated by addition of 70 μl of ethanol (70% final) at −20 °C overnight. Mixtures were spun for 20 min at 20,000 × g at 4 °C; the supernatants were transferred into fresh tubes, and samples were dried down. For HPLC analysis, samples were resuspended in a final volume of 110 μl of water (supplemented with internal standards whenever mentioned), filtered, and analyzed as described above.

      Flow Cytometry

      For staining, cells were resuspended in blocking solution (PBS pH 7.4, supplemented with 0.5% gelatin from cold water fish skin (Sigma) and 2 mm EDTA) containing either a 1:10,000 dilution of polyclonal monospecific chicken anti-Neu5Gc antibody (anti-Neu5Gc IgY (
      • Diaz S.L.
      • Padler-Karavani V.
      • Ghaderi D.
      • Hurtado-Ziola N.
      • Yu H.
      • Chen X.
      • Brinkman-Van der Linden E.C.
      • Varki A.
      • Varki N.M.
      Sensitive and specific detection of the non-human sialic acid N-glycolylneuraminic acid in human tissues and biotherapeutic products.
      )) or control chicken IgY (Jackson ImmunoResearch). Cells were incubated for 1 h on ice. Thereafter, cells were pelleted and resuspended in blocking solution containing a 1:4000 dilution of Cy5-conjugated donkey anti-chicken IgY antibody (Jackson ImmunoResearch). Cells were incubated for 1 h on ice, pelleted, and resuspended in blocking solution for subsequent analysis. Data were collected on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using the Flowjo software (Tree Star).

      DMB-HPLC

      DMB-HPLC was performed as described in the companion paper (
      • Banda K.
      • Gregg C.J.
      • Chow R.
      • Varki N.
      • Varki A.
      Metabolism of vertebrate amino sugars with N-glycolyl groups. Mechanisms underlying gastrointestinal incorporation of the non-human sialic acid xeno-autoantigen N-glycolylneuraminic acid.
      ). As starting material, ∼5% of cells from a confluent P-100 dish or 100 μl of a dense suspension cell culture were used in the protocol. Cell lysates were prepared by repetitive freeze-thaw followed by sonication. For the pulse-chase experiment with THP-I cells (Fig. 2A), 90% (days 0–6), 45% (days 14 and 21), and 22.5% (day 28) of total cell lysate, respectively, were used for DMB-HPLC analysis. Resulting sialic acid amounts were corrected for the dilution factor thereafter.

      Preparation of Cytosolic Extracts from Mouse Liver

      A whole mouse liver (C57/BL6, ∼0.8 g) was harvested and minced with scissors in pre-cooled 2.4 ml of PBS pH 7.4, on ice. The tissue was homogenized using a Polytron (Brinkmann Instruments) for 30 s at medium speed on ice and subsequently spun at 100,000 × g for 1.5 h at 4 °C. The supernatant was recovered and represents the cytosolic extract. Beforehand, 50,000 cpm (1.31 kBq) of radiolabeled monosaccharides were dried down in tubes and now resuspended in 50 μl of the fresh cytosolic liver extract. Remaining tissue extract was kept on ice. Where mentioned, 5 mm ATP (along with 10 mm MgCl2) was added to the sample, and all reaction mixtures were incubated at 37 °C for 6 h. Thereafter, reactions were either quenched with ethanol immediately or supplemented with 5 mm ATP (along with 10 mm MgCl2) and extra 50 μl of cytosolic extracts for an additional 6 h incubation at 37 °C if mentioned in the text. To quench reactions, pre-cooled ethanol was added to a final concentration of 70%, and precipitation was allowed to occur at −20 °C overnight. Thereafter, samples were spun at 20,000 × g for 15 min at 4 °C. The supernatant was transferred into clean tubes and dried down. For subsequent HPLC analysis, samples were resuspended in a final volume of 110 μl of water (supplemented with internal standards where it applied), filtered, and analyzed as described above.

      Chemical Methods

      General

      “Brine” refers to a saturated aqueous solution of sodium chloride. Proton nuclear magnetic resonance spectra (dH) were recorded on a Jeol ECA 500 (500 MHz). 500 MHz spectra were assigned using COSY. All chemical shifts are quoted on the δ-scale in ppm, using residual solvent as the internal standard. Low resolution mass spectra, obtained at the University of California San Diego, Chemistry and Biochemistry Molecular MS Facility, were recorded on a Micromass Platform 1 spectrometer using electron spray (ES) ionization with methanol as carrier solvent. Flash column chromatography was performed using Sorbsil C60 40/60 silica gel. Thin layer chromatography (tlc) was performed using Merck Kieselgel 60F254 pre-coated aluminum backed plates. Plates were visualized using 5% sulfuric acid in methanol.

      Generalized Method to Prepare 2-Deoxy-2-[(hydroxyacetyl)amino]-d-pyranose (ManNGc and GlcNGc)

      2-Amino-2-deoxy-d-glycopyranoside hydrochloride salt (1.4 g, 6.5 mmol) was dissolved in water (60 ml) with sodium bicarbonate (10.8 g, 130 mmol) and cooled in an ice bath. Acetoxyacetyl chloride (4.2 ml, 39 mmol) was added slowly to the reaction mixture. After 30 min tlc (EtOAc/MeOH, 7:3) showed complete consumption of the starting material (Rf 0.0) and the formation of the product (approximate Rf 0.3). The reaction mixture was neutralized with mixed bed resin. Finally, the product was purified through silica column and concentrated under vacuum to yield a white gum (1.4 g, 5.9 mmol, 91%).

      2-Deoxy-2-[(hydroxyacetyl)amino]-d-mannopyranose (ManNGc)

      Spectral data are in good agreement with a published report (
      • Pearce O.M.
      • Varki A.
      Chemo-enzymatic synthesis of the carbohydrate antigen N-glycolylneuraminic acid from glucose.
      ).

      2-Deoxy-2-[(hydroxyacetyl)amino]-d-glucopyranose (GlcNGc) (
      • Sinay P.
      Synthé du 3-O-(d-1-carboxyéthyl)-2-désoxy-2-glycolamido-d-glucose (acide N-glycolylmuramique).
      )

      For 1H NMR (D2O, 500 MHz) (assigned for the major anomer), δ = 3.31 (t, J = 3.6 Hz, 1H), 3.34 (t, J = 9.6 Hz, 1H), 3.64 (t, J = 9.7 Hz, 1H), 3.67 (t, J = 3.4 Hz, 1H), 3.72 (m, 1H), 3.78 (dd, J1,2 = 3.7 Hz, J2,3 = 10.9 Hz, 1H), 3.96 (s, 2H), 5.04 (d, J1,2 = 3.7 Hz, 1H). m/z (ESI+) was 260 (M + Na+, 100%). The HRMS m/z (ES+) calculated for C8H15NO7Na (M + Na+) was 260.0741 and found was 260.0743.

      Generalized Method to Make 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-[(acetoxyacetyl)amino]-d-glycopyranose (Per-O-acetyl-ManNGc and Per-O-acetyl-GlcNGc)

      2-Deoxy-2-[(hydroxyacetyl)amino]-d-glycopyranose (200 mg, 0.80 mmol) was slurried in acetic anhydride (5 ml) and pyridine (5 ml). After 24 h, tlc (EtOAc/petrol, 1:9) showed complete consumption of the starting material (Rf = 0.0). The clear/colorless solution was dried under reduced pressure to yield a sticky straw-colored gum. The residue was dissolved in chloroform and washed with sodium bicarbonate, water, and finally brine. The solution was then dried with sodium sulfite, filtered, and dried under vacuum to yield a white gum. The crude product was purified through silica column and dried under vacuum to yield a clear colorless gum. (320 mg, 0.71 mmol, 90%).

      1,3,4,6-Tetra-O-acetyl-2-deoxy-2-[(acetoxy-acetyl)amino]-d-mannopyranose (Per-O-acetyl-ManNGc) (
      • Sampathkumar S.G.
      • Li A.V.
      • Jones M.B.
      • Sun Z.
      • Yarema K.J.
      Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology.
      )

      1H NMR (CD3Cl, 500 MHz) (assigned for the major anomer) was as follows: δ = 1.99 (s, 3H, CH3), 2.05 (s, 3H, CH3), 2.09 (s, 3H, CH3), 2.18 (s, 3H, CH3), 2.20 (s, 3H, CH3), 4.03–4.07 (m, 1H), 4.24 (dd, J = 12.8, J = 4.8, 1H), 4.60 (s, 2H), 4.76 (ddd, J = 9.1, J = 3.7, J = 1.7, 1H), 5.16 (t, J = 10.1, 1H), 5.31 (dd, J = 4.6, J = 10.4, 1H), 6.03 (d, J = 2.0, 1H), 6.41 (d, J = 9.1, 1H). m/z (ESI+) was 470 (M + Na+, 100%), 465 (M + NH4+, 10%). The HRMS m/z (ES+) calculated for C18H25NO12Na (M + Na+) was 470.1269 and found was 470.1270.

      1,3,4,6-Tetra-O-acetyl-2-deoxy-2-[(acetoxy-acetyl)amino]-d-glucopyranose (per-O-acetyl-GlcNGc) (
      • Sinay P.
      Synthé du 3-O-(d-1-carboxyéthyl)-2-désoxy-2-glycolamido-d-glucose (acide N-glycolylmuramique).
      )

      1H NMR (CD3Cl, 500 MHz) (assigned for the major anomer) is as follows: δ = 2.04 (s, 3H, CH3), 2.04 (s, 3H, CH3), 2.08 (s, 3H, CH3), 2.16 (s, 3H, CH3), 2.19 (s, 3H, CH3), 3.97–4.01 (m, 1H), 4.04 (dd, J = 2.3, J = 12.6, 1H), 4.23 (dd, J = 12.6, J = 4.0, 1H), 4.43 (m, 2H), 4.57 (s, 2H), 5.19–5.28 (m, 1H), and 6.22 (d, J = 3.7, 1H, H-1). m/z (ESI+) was 470 (M + Na+, 100%), 465 (M + NH4+, 10%). The HRMS m/z (ES+) calculated for C18H25NO12Na (M + Na+) was 470.1269 and found was 470.1271.

      Method to Prepare 2-Deoxy-2-[(hydroxyacetyl)amino]-6-phosphate-d-glucopyranose (GlcNGc-6-P) (
      • Jourdian G.W.
      • Roseman S.
      The sialic acids. II. Preparation of N-glycolylhexosamines, N-glycolylhexosamine 6-phosphates, glycolyl coenzyme A, and glycolyl glutathione.
      )

      To glucosamine 6-phosphate in phosphate-buffered saline (pH 7.4, 50 mm, 1 ml) was added (final concentration) glycolic acid (250 mm), N-hydroxysulfosuccinimide (sulfo-NHS, 5 mm), and ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, 100 mm). The reaction was incubated at RT for 30 min, after which water (10 ml) was added. The product was purified by ion-exchange column (Dowex 50 H+ form, 2 ml; Bio-Rad). The column was washed with water (5 ml). The flow-through and the wash were collected and passed through a second ion-exchange column (AG 50W-X8, 2 ml; Bio-Rad). The column was washed with water (10 ml) and formic acid (1 m, 10 ml). The product was eluted from the column using formic acid (5 m, 10 ml). The eluate was dried under reduced pressure to yield a clear colorless gum. 1H NMR (D2O, 500 MHz) (assigned for the major anomer) is as follows: δ = 3.47 (t, J 9.5 Hz, 1H,), 3.52 (dd, J 4.0 Hz, J 9. 8 Hz, 1H), 3.64 (t, J 9.7 Hz, 1H), 3.69 (t, J 9.4 Hz, 1H), 3.85 (dd, J 4.0 Hz, J 10.6 Hz, 1H), 3.93 (dd, J 4.1 Hz, 10.9 Hz, 1H), 4.00 (s, 2H), 5.08 (d, J1,2 3.6 Hz, 1H). m/z (ESI) 316 (M − H+, 100%), 338 (M + Na-2H+, 30%). The HRMS m/z (ES+) calculated for C8H16NO10PNa (M + Na+) was 340.0404 and found was 340.0406.

      Preparation of [3H]Glycolic Acid

      [3H]Glycolic acid was synthesized from [3H]NaBH4 and glyoxylate. Commercial solid [3H]NaBH4 (PerkinElmer Life Sciences, 37 GBq, 2.442 GBq/μmol) was resuspended in 250 μl of 1 mm NaOH, whereupon 100 μl (14.8 GBq) were used in the following. A 500 mm glyoxylate stock solution was prepared (pH 8.0), and 69 μl were added to the [3H]NaBH4. The reaction mixture was incubated at room temperature for 1.5 h and thereafter quenched by adding 3.8 μl of acetone. After incubation for another hour at room temperature, the sample was diluted to 2.5 ml with water and then purified by polyethyleneimine (PEI) chromatography to remove excess glyoxylate. The diluted sample was loaded onto a 3 ml column of PEI-cellulose, pre-equilibrated in water. The column was washed with 30× 6 ml of water, washed with 3 × 6 ml of 40 mm NaCl, and eluted with 3 × 6 ml of 100 mm NaCl. The recovery was monitored by scintillation counting of diluted aliquots of each of the wash and eluate fractions. The pooled eluate of 18 ml was stored frozen. The final yield was ∼85% (12.6 GBq), and the identity of synthesized [3H]glycolic acid was confirmed by HPLC, observing co-elution with commercial [14C]glycolic acid.

      Preparation of [glycolyl-3H]ManNGc and [glycolyl-3H]GlcNGc

      To synthesize [glycolyl-3H]ManNGc, 4 GBq of prepared [3H]glycolic acid were incubated with 50 mm mannosamine in the presence of 50 mm EDC and 2.5 mm sulfo-NHS in 50 mm MOPS buffer pH 7.5, in a final volume of 8 ml. Synthesis of [glycolyl-3H]GlcNGc was achieved in the same manner using 0.63 GBq of prepared [3H]glycolic acid and 50 mm glucosamine. The reaction mixtures were incubated overnight at room temperature with gentle mixing, subsequently diluted to 20 ml with water, and applied to 5 ml Dowex-50 columns (AG 50W-X2, Bio-Rad) pre-equilibrated with water. The columns were washed with 5 CV water; the washes were pooled with the run-through, and the pools were applied onto 5 ml of AG3X4A (OH form, Bio-Rad) columns pre-equilibrated with water. The columns were washed with 5 CV water, and the washes were pooled with the run-through. The pools containing the synthesized [glycolyl-3H]ManNGc and [glycolyl-3H]GlcNGc, respectively, were adjusted to 50% ethanol and stored at −20 °C. The preparations were diluted to prevent autoradiolysis. The recovery was monitored by scintillation counting, and the final yields of [glycolyl-3H]ManNGc and [glycolyl-3H]GlcNGc were 0.84 GBq (21.0%) and 0.27 GBq (43.2%), respectively. The identity of synthesized [glycolyl-3H]ManNGc and [glycolyl-3H]GlcNGc was confirmed by HPLC, demonstrating co-elution with their nonlabeled chemically well characterized counterparts.

      Preparation of [14C]GlcNGc

      To synthesize [14C]GlcNGc, 1.85 MBq of d-[U-14C]glucosamine hydrochloride (Amersham Biosciences, in 3% ethanol) were dried down on a shaker evaporator. The dry material was then incubated with 50 mm glycolic acid in the presence of 50 mm EDC and 2.5 mm sulfo-NHS in 50 mm MOPS, pH 7.5, in a final volume of 100 μl. The sample was processed as described above for preparation of [glycolyl-3H]GlcNGc, but a 1-ml Dowex-50 column was used here. The final yield of [14C]GlcNGc was 0.98 MBq (53.0%), and the identity was confirmed by HPLC, demonstrating co-elution with [glycolyl-3H]GlcNGc.

      Preparation of [glycolyl-3H]GlcNGc-6-P

      The [glycolyl-3H]GlcNGc-6-P was prepared from [3H]glycolic acid and GlcNH2-6-P. In a 200-μl final volume, 3.7 MBq of [3H]glycolic acid were incubated in the presence of 10 mm GlcNH2-6-P, 100 mm EDC, and 5 mm sulfo-NHS in PBS, pH 7.5. The reaction mixture was incubated for 15 min at room temperature and thereafter diluted to 1 ml with water. Subsequently, the sample was applied onto a 1 ml Dowex-50 (AG-50W-X2) column pre-equilibrated with water. The column was washed with 5 CV of water, and the run-through was pooled with the water washes. This pool was applied onto a 1 ml Dowex-1 column (AG-1-X8), pre-equilibrated with water. The column was washed with 5 CV water followed by 5 CV of 1 m formic acid. The [glycolyl-3H]GlcNGc-6-P was subsequently eluted with 5 CV of 5 m formic acid. The formic acid was driven off by using a shaker evaporator. The final yield of synthesized [glycolyl-3H]GlcNGc-6-P was 0.392 MBq (10.6%), and the identity of the compound was confirmed by HPLC, revealing co-elution with chemically well characterized nonlabeled GlcNGc-6-P.

      Preparation of [glycolyl-3H]Neu5Gc

      Previously described protocols for synthesis of radiolabeled Neu5Gc (
      • Nöhle U.
      • Beau J.M.
      • Schauer R.
      Uptake, metabolism, and excretion of orally and intravenously administered, double-labeled N-glycoloylneuraminic acid and single-labeled 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in mouse and rat.
      ) have been modified to achieve a compound exclusively labeled in the N-glycolyl group. The [glycolyl-3H]Neu5Gc was prepared from [glycolyl-3H]ManNGc by enzymatic conversion using the N-acetylneuraminate pyruvate-lyase (EC 4.1.3.3) from Pasteurella multocida. A His6-tagged version of the enzyme was recombinantly expressed and purified as described previously (
      • Li Y.
      • Yu H.
      • Cao H.
      • Lau K.
      • Muthana S.
      • Tiwari V.K.
      • Son B.
      • Chen X.
      Pasteurella multocida sialic acid aldolase. A promising biocatalyst.
      ). In a total volume of 100 μl, 456 MBq [glycolyl-3H]ManNGc were incubated with 33.67 μg of purified N-acetylneuraminate pyruvate-lyase in the presence of 100 mm Tris-HCl, pH 7.5 and 200 mm sodium pyruvate. The reaction was incubated at 37 °C for 20 h with mixing and thereafter quenched by diluting to 5 ml with water. The [glycolyl-3H]Neu5Gc was purified from nonreacted [glycolyl-3H]ManNGc by passing the sample through a 2 ml column of AG-50-X2 (H+ form). The flow-through and 5 CV water washes were collected and combined. This effluent was then loaded onto a 2 ml column of AG-1-X8 (formate form) equilibrated in water. The column was washed with 5 CV of water followed by 7 ml of 10 mm formic acid and finally eluted into a separate tube with 10 ml of 1 m formic acid. The eluate was dried down to remove formic acid. The final [glycolyl-3H]Neu5Gc preparation was dissolved in water. Recovery was followed by counting an aliquot of each fraction at each step of the purification process. Final yield was found to be 70 MBq (15.4%). The identity of [glycolyl-3H]Neu5Gc was confirmed by co-elution with commercial Neu5Gc (Inalco) by HPLC as described above.

      Acknowledgments

      We thank Bradley Hayes, Natasha Naidu, and Biswa Choudhury from University of California San Diego, Glycotechnology Core Resource for their help with the development of some methods for this study. We also thank Xi Chen for kindly providing the plasmid for recombinant expression of N-acetylneuraminate pyruvate-lyase from P. multocida.

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