N-linked oligosaccharide changes with oncogenic transformation require sialylation of multiantennae.
Glycopeptides derived from NIH 3T3 fibroblasts and these cells transformed by transfection with human DNA containing oncogene H-ras were analyzed by 500-MHz 1H-NMR spectroscopy and binding to immobilized lectins. The cells were metabolically labeled with D-[3H]glucosamine or L-[3H]fucose and the glycopeptides included in Bio-Gel P-10 (Mr 5000-3500) were separated into neutral and charged fractions on DEAE-cellulose. The major portion (80%) of these [3H]fucose glycopeptides from the non-transformed NIH 3T3 fibroblasts were neutral or contained one or two charged residues, whereas 90% of the glycopeptides from the transformed cells contained two or more charged residues. The structure of the predominant neutral glycopeptide from the non-transformed NIH 3T3 cells was determined by 1H-NMR spectroscopy to be tetraantennary containing terminal Gal alpha 1----3. (formula; see text) This structure was verified by binding to the immobilized alpha-Gal-specific lectin, Griffonia simplicifolia I and leukoagglutinating phytohemagglutinin from Phaseolus vulgaris (L-PHA), which binds certain tri- or tetraantennary glycopeptides. In contrast, the structure derived by NMR spectroscopy of one of the predominant charged glycopeptides from the transformed cells was triantennary containing terminal NeuNAc alpha 2----3 in addition to Gal alpha 1----3. (formula; see text) In attempting to verify this structure by lectin-binding properties it was found that removal of NeuNAc alpha 2----3 reduced the affinity to L-PHA - agarose. The other major glycopeptides of the transformed cells which were more charged also cotained NeuNAc alpha 2----3 but no NeuNAc alpha 2----6 or Gal alpha 1----3. A tentative structure was proposed for the major glycopeptide of the first charged class from NIH 3T3 cells on the basis of lectin-binding properties and the NMR spectrum which showed, in addition to NeuNAc alpha 2----3, the presence of NeuNAc alpha 2----6 and Gal alpha 1----3. On the basis of the NMR spectrum and other results, it is concluded that the presence of tetraantennary oligosaccharides are not sufficient for the transformed oligosaccharide phenotype. Rather, the tri- or tetraantennae must be sialylated in alpha 2----3 linkage, on more than one antennae, when properties of transformation are expressed in NIH 3T3 cells. Prior to transformation the tetraantennary oligosaccharides of these cells are terminated in alpha-Gal residues, whereas after transformation alpha-Gal residues appear to be replaced by NeuNAc alpha 2----3.(ABSTRACT TRUNCATED AT 400 WORDS)
Santer
U V
UV
Joseph Stokes Jr Research Institute, Children's Hospital, Philadelphia.
DeSantis
R
R
Hård
K J
KJ
van Kuik
J A
JA
Vliegenthart
J F
JF
Won
B
B
Glick
M C
MC
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002850:Chromatography, Gel; D005643:Fucose / Q000378:metabolism; D011905:Genes, ras; D005944:Glucosamine / Q000378:metabolism; D006020:Glycopeptides / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D012794:Sialic Acids / Q000378:metabolism*
D006020:Glycopeptides; D009844:Oligosaccharides; D012794:Sialic Acids; D005643:Fucose; D005944:Glucosamine