sodium-borohydride and Ovarian-Neoplasms

Recent investigations have implicated aberrant glycosylations in various malignancies, including epithelial ovarian cancer (EOC). The protocol here identifies O-linked carbohydrate patterns in EOC plasma glycoproteins through chemical cleavage and purification of these glycans. Dialyzed plasma is subjected to reductive beta-elimination with alkaline borohydride to release O-linked oligosaccharides from glycoproteins. Enrichment of released glycans, as well as removal of peptide and other contaminants, is followed by carbohydrate pattern analysis with MALDI-FT-ICR-MS.

Topics: Blood Proteins; Borohydrides; Dialysis; Female; Glycosylation; Hexosamines; Hexoses; Hexuronic Acids; Humans; Middle Aged; Ovarian Neoplasms; Polysaccharides; Pronase; Solid Phase Extraction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Galactosyltransferase purified from the ascites of ovarian cancer patients can use to an equal extent N-glycosidic glycoproteins, such as asialoagalactofetuin, and O-glycosidic mucin, such as asialo bovine submaxillary mucin (BSM), as acceptors. Thermal treatment and substrate competition experiments demonstrated that the same enzyme catalyzed the transfer of galactose to both types of acceptors. Alkaline borohydride treatment showed that, while the product with asialoagalactofetuin was totally resistant, about 90% of the product with asialo BSM was hydrolyzed by this treatment. Gel filtration of the released oligosaccharides on a calibrated Biogel P-2 column showed three peaks. One major oligosaccharide (O-2) of size 5.7 glucose U and two minor peaks (O-1 and O-3) of sizes 8.7 and 3.7 glucose U, respectively, were obtained. The oligosaccharides were doubly labeled, first by incubation with uridine-diphosphate [14C]galactose, followed by alkali treatment in the presence of [3H]borohydride. The doubly labeled oligosaccharides were separately purified by gel filtration and ion-exchange chromatography and digested with various exoglycosidases. The digested products were characterized by gel filtration and paper chromatography in three different systems. From these results, the structures of these oligosaccharides were computed as follows: O-1 = beta-galactosyl-beta-N-acetylglucosamine-galactosaminitol (sialic acid); O-2 = beta-galactose-beta-N-acetylglucosamine-galactosaminitol; O-3 = beta-galactose-galactosaminitol. These results suggest that the galactosyltransferase from the ascites of ovarian cancer patients catalyzes the transfer of galactose to N-acetylglucosamine, irrespective of whether it is a part of an N-glycan or an O-glycan.

Topics: Acetylglucosamine; alpha-Fetoproteins; Ascites; Asialoglycoproteins; Binding, Competitive; Borohydrides; Chromatography; Chromatography, Gel; Female; Fetuins; Galactose; Galactosyltransferases; Glycoside Hydrolases; Hot Temperature; Humans; Mucins; Oligosaccharides; Ovarian Neoplasms; Substrate Specificity; Uridine Diphosphate Galactose

Pronase digests of cultured teratocarcinoma-derived cells (PA 1) of human origin have been previously shown to contain large-sized glycopeptides (relative mass (Mr) greater then 7400), of which 15-23% are retained by columns of concanavalin A (Con A) - Sepharose and can be eluted with 10 mM methyl alpha-D-mannopyranoside. The present data show that this fraction (A - Con A II) contains a family of glycopeptides that are degradable with anhydrous hydrazine as well as with 0.05 M NaOH - 1 M NaBH4. The cleavage products representing individual oligosaccharide chains, presumably as oligosaccharides and glycopeptides, consisted mostly of medium- (Mr 1400-6000) and small-sized (Mr less than 1400) molecules. This implies that glycopeptides bearing several oligosaccharide chains were present in A - Con A II. Most of the individual oligosaccharide chains were not bound to Con A - Sepharose, but some were retained by the lectin column in the same way as the original glycopeptides. Some of the oligosaccharides were degraded partially with endo-beta-galactosidase from Escherichia freundii suggesting the presence of GalbetaGlcNAcbeta repeats. The present findings show that A - Con A II may be different from the "embryonic" glycopeptides of mouse teratocarcinoma cells that are reportedly not cleaved by mild alkaline borohydride treatment. Instead, A - Con A II is reminiscent of the T-1 glycopeptide of glycophorin.

Topics: Borohydrides; Cell Line; Chemical Phenomena; Chemistry; Female; Humans; Hydrazines; Hydrogen-Ion Concentration; Oligosaccharides; Ovarian Neoplasms; Receptors, Concanavalin A; Teratoma