Abstract
Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. While Diplococcus pneumoniae endo-alpha-DN-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra- and trisaccharides (Gal beta 1----3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal beta 1----3 [Gal beta 1----4GlcNAc beta 1----6] GalNAc). The number of O-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of O-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggest that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal beta 1----3GalNAc to form the NeuAc alpha 2----3Gal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.
Highlights
The structure of the hexasaccharide was determined fetuin, which are predominantly of the complex triantennary to be NeuAca2+3Gal~1+3[NeuAca2+3Gal@l+ 4GlcNAc@1+6]GalNAc, onthe basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation
Leased thesialicacid-free tetra-andtrisaccharides (Gal@1+3GalNAc)t,his enzyme did not cleave the peptide attachment of the asialohexasaccharide
The absence of 0-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggests that the attachment of this sugar is a rate-limiting step; tlhime ited occurrence of the hexasaccharide may indicate that the addition
Summary
OligosaccharidesReleased by Alkaline Borohydride Treatment of Fetuin-Examination by thin layer chromatography of the acidic saccharides released from two fetuin preparations by alkaline NaB[3H]4 treatment revealed three radiolabeled components (A, B , and C) which migrated in Solvent System A with an RLecHo2f 0.10, 0.20, and 0.78, respectively (Fig. 1). Components B and C represented the 0-linked tetrasaccharide and trisaccharide unitsof fetuin Examination by thin layer chromatography of the neutral fraction obtained after alkalinNe aB[3H], treatment revealed no radiolabeledcomponentsevenwhenseveralfoldlarger amounts than required for visualizationof component A (Fig. 1)were applied to the plate (not shown). The radioactive saccharides which remained at theorigin(component N ) arebelievedtobe derived from the N-linked oligosaccharides of fetuin as glucosaminitol was identified as the only 3H-labeled monosaccharide after acid hydrolysis of this material; it has
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