N‐linked oligosaccharide changes with oncogenic transformation require sialylation of multiantennae

Abstract

Glycopeptides derived from NIH 3T3 fibroblasts and these cells transformed by transfection with human DNA containin? om'i ~"~m c H-ras were analyzed by 500-MHz 'H-NMR spectroscopy and binding to immobilized lectins. The cells were mc1;iholically labeled with ~-[~H]glucosamine or ~- [~H]fucose and the glycopeptides included in Bio-Gel P-10 ( M , 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 'H-NMR spectroscopy to be tetraantennary containing terminal Galal -+ 3.This structure was verified by binding to the immobilized a-Gal-specific lectin, Grijfonia simplicijolia 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 NeuNAca2+3 in addition to Galal+3. Fucal-thIn attempting to verify this structure by lectin-binding properties it was found that removal of NeuNAca2 + 3 reduced the affinity to L-PHA -agarose. The other major glycopeptides of the transformed cells which were more charged also cotained NeuNAca2+3 but no NeuNAca2-+6 or Galal + 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 NeuNAca2+3, the presence of NeuNAca2-6 and Galal +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 a2-3 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 aGal residues, whereas after transformation a-Gal residues appear to be replaced by NeuNAca2+ 3. Thus, in this case, transformation may be accompanied by a change in the glycosyltransferases responsible for termination of the branches rather than those conferring branch configurations.

Glycosylation of the envelope glycoprotein from a polytropic murine retrovirus in two different host cells

“…Glycans derived from gp70(NIH3T3) contained zero to four sialic acid residues, the linkage positions of which, however, were not determined due to the small amounts of material available. Data reported by Santer et al [38] on the structures of NIH3T3-derived glycopeptides as well as glycosylation analyses of the envelope glycoprotein from an ecotropic F-MuLV propagated on the same cell line (Kempf and Geyer, unpublished results) demonstrated that NIH3T3 cells contain both (a2 -6)-and (a2 -3)-sialyltransferases. Therefore, it can be assumed that the sialylated glycans obtained here are similarly substituted by (a2 -3)-and/or (a2 -6)-linked sialic acid residues.…”

The Galα1,3Galβ1,4GlcNAc-R (α-Gal) epitope: A carbohydrate of unique evolution and clinical relevance

“…Studies on freshly isolated cells or cell lines from mouse [23][24][25][26], bovine [27] and porcine origin [28] further demonstrated the presence of α-gal epitopes in large amounts on cell surface glycoconjugates. More recently, Kim et al carried out a detailed analysis of N-linked glycans from pig kidney [29].…”

Transcriptional Regulation ofN-Acetylglucosaminyltransferase V by the srcOncogene

“…An induced alteration in the glycosylation of cell surface glycoproteins that has been documented for many years concerns the significant increase in oligosaccharide size caused by oncogenic transformation using a variety of agents (6 -14). This increase in size was found to result mainly from an increase in the levels of asparagine-linked oligosaccharides containing N-acetylglucosamine linked ␤1,6 to the ␣(1,6)-linked mannose in the trimannosyl core, (GlcNAc␤(1,6)Man), and in many cases these oligosaccharides express polylactosamine that can be sialylated (15)(16)(17)(18). The (GlcNAc␤(1,6)Man) branch is synthesized by N-acetylglucosaminyltransferase V (GlcNAc-T V), 1 the enzyme whose activity is significantly and selectively increased after transformation by tumor viruses or isolated oncogenes (16, 19 -22).…”