Synthesis of cell-wall glycoproteins and their characterization in oat coleoptiles

Abstract

d-[U- 14C]Glucosamine was rapidly taken up by oat coleoptile segments and metabolized to radioactive UDP- N-acetylglucosamine, which acted as specific glycosyl donor for the synthesis of glycolipids and cytosolic, membrane-bound and cell-wall glycoproteins. Cell-wall glycoproteins were solubilized from the walls by either cell-wall-degrading enzymes or chemical extractants. The solubilized cell-wall glycoproteins in the presence of peptide N-glycosidase F released oligosaccharide chains higher than seven glycosidic residues. The combined action of peptide N-glycosidase F and N-acetyl-β- d-glucosaminidase on cell-wall glycoproteins indicated the presence of N-acetylglucosamine residues β-1,2-linked to mannose. Less than 9% of the radioactive oligosaccharide chains was released from the solubilized cell-wall glycoproteins when treated with 0.5 M NaOH at 20°, whereas more than 45% of the radioactivity was released in the presence of 1 M NaOH at 50°. The high hydrolytic sensitivity of cell-wall glycoproteins to peptide N-glycosidase F, N-acetyl-β- d-glucosaminidase NaOH at 50° indicated that most N-acetylglucosamine residues were incorporated into N-linked cell-wall glycoproteins. Further evidence of this was obtained by the use of inhibitors of biosynthesis and processing of N-linked glycoproteins.