Abstract
GM3-synthase, also known as sialyltransferase I (ST-I), catalyzes the transfer of a sialic acid residue from CMP-sialic acid onto lactosylceramide to form ganglioside GM3. In order to clone this enzyme, as well as other sialyltransferases, we developed an approach that we termed combinatorial PCR. In this approach, degenerate primers were designed on the basis of conserved sequence motifs of the ST3 family of sialyltransferases (STs). The nucleotide sequence of the primers was varied to cover all amino acid variations occurring in each motif. In addition, in some primers the sequence was varied to cover possible homologous substitutions that are absent in the available motifs. A panel of cDNA from 12 mouse and 8 human tissues was used to enable cloning of tissue- and stage-specific sialyltransferases. Using this approach, the fragments of 11 new putative sialyltransferases were isolated and sequenced so far. Analysis of the expression pattern of a particular sialyltransferase across the panel of cDNA from the different tissues provided information about the tissue specificity of ST expression. We chose two new ubiquitously expressed human and mouse STs to clone full-length copies and to assay for GM3-synthase activity. One of the STs, which exhibited the highest homology to ST3 Gal III, showed activity toward lactosylceramide (LacCer) and was termed ST3 Gal V according to the suggested nomenclature [1]. The other ubiquitously expressed sialyltransferase was termed ST3Gal VI. All isolated sialyltransferases were screened for alternatively spliced forms (ASF). Such forms were found for both human ST3Gal V and ST3Gal VI in human fetal brain cDNA library. The detailed cloning strategy, functional assay, and full length cDNA and protein sequences of GM3 synthase (ST3Gal V, or ST-I) are presented.
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Tsuji S, Datta AK, Paulson JC (1996) [letter]. Glycobiology 6: v–vii.
Bremer EG, Hakomori S, Bowen-Pope DF, Raines E, Ross R (1984) J Biol Chem 259: 6818–25.
Bremer EG, Schlessinger J, Hakomori S (1986) J Biol Chem 261: 2434–40.
Nojiri H, Takaku F, Ohta M, Miura Y, Saito M (1985) Cancer Res 45: 6100–106.
Nojiri H, Takaku F, Terui Y, Miura Y, Saito M (1986) Proc Natl Acad Sci USA 83: 782–86.
Nojiri H, Kitagawa S, Nakamura M, Kirito K, Enomoto Y, Saito M. (1988) J Biol Chem 263: 7443–46.
Yada Y, Okano Y, Nozawa Y (1991) Biochem J 279: 665–70.
Rahmann H, Rosner H, Kortje KH, Beitinger H, Seybold V (1994) Brain Res 101: 127–45.
Ferrari G, Greene LA (1998) Ann NY Acad Sci 845: 263–73.
Hakomori S (1990) J Biol Chem 265: 18713–16.
Hakomori S (1997) Sphingolipid-mediated Signal Transduction. R.G.Landes Company and Chapman & Hall.
Hakomori S, Igarashi Y (1995) J Biochem (Tokyo) 118: 1091–103.
Hynds DL, Burry RW, Yates AJ (1997) Neurosci Res 47: 617–25.
Katoh N (1995) Toxicology 104: 73–81.
Yates AJ, Rampersaud A (1998) Ann NY Acad Sci 845: 57–71.
Suarez Pestana E, Greiser U, Sanchez B, Fernandez LE, Lage A, Perez R, Bohmer FD (1997) Br J Cancer 75: 213–20.
Goldenring JR, Otis LC, Yu RK, DeLorenzo RJ (1985) J Neurochem 44: 1229–34.
Kreutter D, Kim JY, Goldenring JR, Rasmussen H, Ukomadu C, DeLorenzo RJ, Yu R K (1987) J Biol Chem 262: 1633–37.
Kim JY, Goldenring JR, DeLorenzo RJ, Yu RK (1986) J Neurosci Res 15: 159–66.
Matecki A, Stopa M, Was A, Pawelczyk T (1997) Int J Biochem Cell Biol 29: 815–28.
Yang FY, Wang LH, Yang XY, Tsui ZC, Tu YP (1997) Biophys Chem 68: 137–46.
Misasi R, Sorice M, Garofalo T, Griggi T, Campana W, Giammatteo M, Pavan A, Hiraiwa M, Pontieri M, O'Brien J (1998) J Neurochem 71: 2313–21.
Hakomori S, Yamamura S, Handa AK (1998) Ann NY Acad Sci 845: 1–10.
Kojima N, Hakomori S (1991) J Biol Chem 266: 17552–58.
Kojima N, Shiota M, Sadahira Y, Handa K, Hakomori S (1992) J Biol Chem 267: 17264–70.
Hammache D, Yahi N, Pieroni G, Ariasi F, Tamalet C, Fantini J (1998) Biochem Biophys Res Commun 246: 117–22.
Hammache D, Pieroni G, Yahi N, Delezay O, Koch N, Lafont H, Tamalet C, Fantini J (1998) J Biol Chem 273: 7967–71.
Paulson JC, Beranek WE, Hill RL (1977) J Biol Chem 252: 2356–62.
Gillespie W, Kelm S, Paulson JC (1992) J Biol Chem 267: 21004–10.
Haraguchi M, Yamashiro S, Yamamoto A, Furukawa K, Takamiya K, Lloyd KO, Shiku H (1994) Proc Natl Acad Sci USA 91: 10455–59.
Sasaki K, Watanabe E, Kawashima K, Sekine S, Dohi T, Oshima M, Hanai N, Nishi T, Hasegawa M (1993) J Biol Chem 268: 22782–87.
Kim YJ, Kim KS, Do S, Kim CH, Kim SK, Lee YC (1997) Biochem Biophys Res Commun 235: 327–30.
Nakayama J, Fukuda MN, Hirabayashi Y, Kanamori A, Sasaki K, Nishi T, Fukuda M (1996) J Biol Chem 271: 3684–91.
Lee YC, Kurosawa N, Hamamoto T, Nakaoka T, Tsuji S (1993) Eur J Biochem 216: 377–85.
Kapitonov D, Yu RK (1997) [dissertation] Medical College of Virginia of Virginia Commonwealth University, Richmond
Gu X, Preuss U, Gu T, Yu RK (1995) J Neurochem 64: 2295–302.
Sipos L, von Heijne G (1993) Eur J Biochem 213: 1333–40.
Nakashima H, Nishikawa K (1992) FEBS Lett 303: 141–46.
Hartmann E, Rapoport TA, Lodish HF (1989) Proc Natl Acad Sci USA 86: 5786–90.
Nigam SK, Blobel G (1989) J Biol Chem 264: 16927–32.
Ou WJ, Thomas DY, Bell AW, Bergeron JJ. (1992) J Biol Chem 267: 23789–96.
Sfeir C, Veis A (1995) J Bone Miner Res 10: 607–15.
Ishii A, Ohta M, Watanabe Y, Matsuda K, Ishiyama K, Sakoe K, Nakamura M, Inokuchi J, Sanai Y, Saito M (1998) J Biol Chem 273: 31652–55
Kono M, Takashima S, Liu H, Inoue M, Kojima N, Lee YC, Hamamoto T, Tsuji S (1998) Biochem Biophys Res Commun 253: 170–75.
Kapitonov D, Yu RK (1999) Glycobiology (in press).
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Kapitonov, D., Bieberich, E. & Yu, R.K. Combinatorial PCR approach to homology-based cloning: Cloning and expression of mouse and human GM3-synthase. Glycoconj J 16, 337–350 (1999). https://doi.org/10.1023/A:1007091926413
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DOI: https://doi.org/10.1023/A:1007091926413
- PCR
- GM3-synthase
- sialyltransferase
- molecular cloning of glycosyltransferases
- cDNA
- ST, sialyltransferase
- ST-I, CMP-NeuAc:lactosylceramide α2-3 sialyltransferase
- ST3, a family of sialyltransferases that transfers a sialic acid residue from CMP-sialic acid to the third carbon of a sugar acceptor molecule, forming an α2-3 bond
- LacCer, lactosylceramide or Galβ1-4Glcβ1-1′Cer
- Gg3, GalNAcβ1-4Galβ1-4Glcβ1-1′Cer
- GM3, NeuAcα2-3Galβ1-4Glcβ1-1′Cer
- GM1, Galβ1-3GalNAcβ1-4Gal(3-2α NeuAc)β1-4Glcβ1-1′Cer
- GD1a, NeuAcα2-3Galβ1-3GalNAcβ1-4Gal(3-2α NeuAc)β1-4Glcβ1-1′Cer
- ASF, alternatively spliced form