Glycoconjugate Journal

, Volume 33, Issue 5, pp 779–788 | Cite as

Housekeeping promoter 5’pcmah-2 of pig CMP-N-acetylneuraminic acid hydroxylase gene for NeuGc expression

  • Kwon-Ho Song
  • Choong-Hwan Kwak
  • Un-Ho Jin
  • Sun-Hyung Ha
  • Jun-Young Park
  • Fukushi Abekura
  • Young-Chae Chang
  • Seung-Hak Cho
  • Kichoon Lee
  • Tae-Wook Chung
  • Ki-Tae Ha
  • Young-Choon Lee
  • Cheorl-Ho Kim
Original Article


In the present study, we isolated pCMAH house-keeping promoter regions (Ph), which are responsible for transcriptional regulation and which are located upstream of the alternative transcript pcmah-2. Luciferase reporter assays using serial construction of each deleted promoter demonstrated that the Ph promoter was highly active in pig-derived kidney PK15. Ph promoter of pcmah lacked a TATA box, but contained three putative Sp1 binding sites. Mutations of these Sp1 binding sites always resulted in the reduction of luciferase activities in Ph-334. In addition, treatment with mithramycin A (25–100 nM) decreased the luciferase activities of the Ph promoters and NeuGc expression in a dose-dependent manner. Electrophoretic mobility shift assay analysis revealed that the probes containing each Sp1 binding site bound to Sp1. Taken together, the results indicate that Sp1 bind to their putative binding sites on the Ph promoter regions of pcmah and positively regulate the promoter activity in pig kidney cells. Interspecies comparison of 5’UTRs and 5’flanking regions shows high homology between pig and cattle, and Sp1 binding sites existing in genomic regions corresponding Ph region are evolutionally conserved.


CMP-N-acetylneuraminic acid hydroxylase (CMAH) N-glycolylneuraminic acid (NeuGc) Specific protein 1 (Sp1) 



This study has been in part supported from the Basic Science Research Program through National Research Foundation of Korea (NRF) grant (NRF-2015R1D1A1A01057153).


  1. 1.
    Angata T., Varki A.: Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem. Rev. 102, 439–469 (2002)CrossRefPubMedGoogle Scholar
  2. 2.
    Irie A., Suzuki A.: CMP-N-Acetylneuraminic acid hydroxylase is exclusively inactive in humans. Biochem. Biophys. Res. Commun. 248, 330–333 (1998)CrossRefPubMedGoogle Scholar
  3. 3.
    Schauer R., de Freese A., Gollub M., Iwersen M., Kelm S., Reuter G., Schlenzka W., Vandamme-Feldhaus V., Shaw L.: Functional and biosynthetic aspects of sialic acid diversity. Indian J. Biochem. Biophys. 34, 131–141 (1997)PubMedGoogle Scholar
  4. 4.
    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. Proc. Natl. Acad. Sci. U. S. A. 95, 11751–11756 (1998)CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Byres E., Paton A.W., Paton J.C., Lofling J.C., Smith D.F., Wilce M.C., Talbot U.M., Chong D.C., Yu H., Huang S., Chen X., Varki N.M., Varki A., Rossjohn J., Beddoe T.: Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin. Nature. 456, 648–652 (2008)CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    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. Proc. Natl. Acad. Sci. U. S. A. 105, 18936–18941 (2008)CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    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. J. Immunol. 175, 228–236 (2005)CrossRefPubMedGoogle Scholar
  8. 8.
    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. J. Biol. Chem. 270, 16458–16463 (1995)CrossRefPubMedGoogle Scholar
  9. 9.
    Muchmore E.A., Milewski M., Varki A., Diaz S.: Biosynthesis of N-glycolyneuraminic acid. The primary site of hydroxylation of N-acetylneuraminic acid is the cytosolic sugar nucleotide pool. J. Biol. Chem. 264, 20216–20223 (1989)PubMedGoogle Scholar
  10. 10.
    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-sulphur protein to be described in Eukarya. FEBS Lett. 385, 197–200 (1996)CrossRefPubMedGoogle Scholar
  11. 11.
    Shaw L., Schauer R.: Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver. Biochem. J. 263, 355–363 (1989)CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Karlsson N.G., Olson F.J., Jovall P.A., Andersch Y., Enerback L., Hansson G.C.: Identification of transient glycosylation alterations of sialylated mucin oligosaccharides during infection by the rat intestinal parasite Nippostrongylus brasiliensis. Biochem. J. 350(Pt 3), 805–814 (2000)CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Naito Y., Takematsu H., Koyama S., Miyake S., Yamamoto H., Fujinawa R., Sugai M., Okuno Y., Tsujimoto G., Yamaji T., Hashimoto Y., Itohara S., Kawasaki T., Suzuki A., Kozutsumi Y.: Germinal center marker GL7 probes activation-dependent repression of N-glycolylneuraminic acid, a sialic acid species involved in the negative modulation of B-cell activation. Mol. Cell. Biol. 27, 3008–3022 (2007)CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Portner A., Peter-Katalinic J., Brade H., Unland F., Buntemeyer H., Muthing J.: Structural characterization of gangliosides from resting and endotoxin-stimulated murine B lymphocytes. Biochemistry. 32, 12685–12693 (1993)CrossRefPubMedGoogle Scholar
  15. 15.
    Malykh Y.N., King T.P., Logan E., Kelly D., Schauer R., Shaw L.: Regulation of N-glycolylneuraminic acid biosynthesis in developing pig small intestine. Biochem. J. 370, 601–607 (2003)CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Song K.H., Kang Y.J., Jin U.H., Park Y.I., Kim S.M., Seong H.H., Hwang S., Yang B.S., Im G.S., Min K.S., Kim J.H., Chang Y.C., Kim N.H., Lee Y.C., Kim C.H.: Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-human xenotransplantation. Biochem. J. 427, 179–188 (2010)CrossRefPubMedGoogle Scholar
  17. 17.
    Chung T.W., Lee Y.C., Ko J.H., Kim C.H.: Hepatitis B Virus X protein modulates the expression of PTEN by inhibiting the function of p53, a transcriptional activator in liver cells. Cancer Res. 63, 3453–3458 (2003)PubMedGoogle Scholar
  18. 18.
    Nystedt J., Anderson H., Hirvonen T., Impola U., Jaatinen T., Heiskanen A., Blomqvist M., Satomaa T., Natunen J., Saarinen J., Lehenkari P., Valmu L., Laine J.: Human CMP-N-acetylneuraminic acid hydroxylase is a novel stem cell marker linked to stem cell-specific mechanisms. Stem Cells. 28, 258–267 (2010)PubMedGoogle Scholar
  19. 19.
    Nakamuta M., Oka K., Krushkal J., Kobayashi K., Yamamoto M., Li W.H., Chan L.: Alternative mRNA splicing and differential promoter utilization determine tissue-specific expression of the apolipoprotein B mRNA-editing protein (Apobec1) gene in mice. Structure and evolution of Apobec1 and related nucleoside/nucleotide deaminases. J. Biol. Chem. 270, 13042–13056 (1995)CrossRefPubMedGoogle Scholar
  20. 20.
    Rajagopalan S., Wan D.F., Habib G.M., Sepulveda A.R., McLeod M.R., Lebovitz R.M., Lieberman M.W.: Six mRNAs with different 5’ ends are encoded by a single gamma-glutamyltransferase gene in mouse. Proc. Natl. Acad. Sci. U. S. A. 90, 6179–6183 (1993)CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Tolner B., Roy K., Sirotnak F.M.: Structural analysis of the human RFC-1 gene encoding a folate transporter reveals multiple promoters and alternatively spliced transcripts with 5’ end heterogeneity. Gene. 211, 331–341 (1998)CrossRefPubMedGoogle Scholar
  22. 22.
    Kim S.W., Kang N.Y., Lee S.H., Kim K.W., Kim K.S., Lee J.H., Kim C.H., Lee Y.C.: Genomic structure and promoter analysis of human NeuAc alpha2,3Gal beta1,3GalNAc alpha2,6-sialyltransferase (hST6GalNAc IV) gene. Gene. 305, 113–120 (2003)CrossRefPubMedGoogle Scholar
  23. 23.
    Okuda T., Nakayama K.: Identification and characterization of the human Gb3/CD77 synthase gene promoter. Glycobiology. 18, 1028–1035 (2008)CrossRefPubMedGoogle Scholar
  24. 24.
    Kim S.W., Lee S.H., Kim K.S., Kim C.H., Choo Y.K., Lee Y.C.: Isolation and characterization of the promoter region of the human GM3 synthase gene. Biochim. Biophys. Acta. 1578, 84–89 (2002)CrossRefPubMedGoogle Scholar
  25. 25.
    Lundell K.: The porcine taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21) gene: evolution by gene duplication and gene conversion. Biochem. J. 378, 1053–1058 (2004)CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Smale S.T.: Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes. Biochim. Biophys. Acta. 1351, 73–88 (1997)CrossRefPubMedGoogle Scholar
  27. 27.
    Pugh B.F., Tjian R.: Mechanism of transcriptional activation by Sp1: evidence for coactivators. Cell. 61, 1187–1197 (1990)CrossRefPubMedGoogle Scholar
  28. 28.
    Li L., He S., Sun J.M., Davie J.R.: Gene regulation by Sp1 and Sp3. Biochem. Cell Biol. 82, 460–471 (2004)CrossRefPubMedGoogle Scholar
  29. 29.
    Blume S.W., Snyder R.C., Ray R., Thomas S., Koller C.A., Miller D.M.: Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo. J. Clin. Invest. 88, 1613–1621 (1991)CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Kwon-Ho Song
    • 1
  • Choong-Hwan Kwak
    • 1
  • Un-Ho Jin
    • 1
  • Sun-Hyung Ha
    • 1
  • Jun-Young Park
    • 1
  • Fukushi Abekura
    • 1
  • Young-Chae Chang
    • 2
  • Seung-Hak Cho
    • 3
  • Kichoon Lee
    • 4
  • Tae-Wook Chung
    • 5
  • Ki-Tae Ha
    • 5
  • Young-Choon Lee
    • 6
  • Cheorl-Ho Kim
    • 1
    • 7
    • 8
  1. 1.Molecular and Cellular Glycobiology Unit, Department of Biological ScienceSungkyunkwan UniversitySuwonSouth Korea
  2. 2.Research Institute of Biomedical Engineering and Department of MedicineCatholic University of Daegu School of MedicineDaeguSouth Korea
  3. 3.Department of Enteric Bacterial InfectionsKorea Centers for Disease Control and PreventionCheongwon-gunSouth Korea
  4. 4.Functional Genomics Laboratory, Department of Animal SciencesThe Ohio State UniversityColumbusUSA
  5. 5.Division of Applied Medicine, School of Korean MedicinePusan National UniversityYangsan CitySouth Korea
  6. 6.Faculty of Medicinal BiotechnologyDong-A UniversityBusanSouth Korea
  7. 7.Department of Medical Device Management and Research, Samsung Advanced Institute for Health Sciences & Technology (SAIHST)Sungkyunkwan UniversitySeoulSouth Korea
  8. 8.Department of Biological ScienceSungkyunkwan UniversitySuwonSouth Korea

Personalised recommendations