Structure of the glucan-binding sugar chain of Tip1p, a cell wall protein of Saccharomyces cerevisiae

• Published in: Biochimica et biophysica acta Vol. 1427; no. 2; pp. 133 - 144
• Main Authors: Fujii, Tsutomu, Shimoi, Hitoshi, Iimura, Yuzuru
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 19.04.1999
• Subjects: Amino Acid Sequence; Amino Acids; Amino Acids - analysis; analysis; Carbohydrate Sequence; carbohydrate structure; Carrier Proteins; Carrier Proteins - chemistry; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; cell wall components; Cell wall glucan; Cell wall protein; chemical structure; chemistry; Endopeptidases; Ethanolamine; Ethanolamine - analysis; glucans; Glucans - metabolism; glycoproteins; Glycoproteins - chemistry; Glycoproteins - isolation & purification; Glycoproteins - metabolism; GPI anchor; Hexoses; Hexoses - analysis; isolation & purification; Membrane Glycoproteins; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - isolation & purification; Membrane Glycoproteins - metabolism; metabolism; Molecular Sequence Data; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Cell wall glucan; ABOE, 4-aminobenzoic octyl ester; PA, 2-aminopyridine; GPI, glycosylphosphatidylinositol; SDS, sodium dodecyl sulfate; HF, hydrofluoric acid; AQC, N-hydroxysuccinimidyl-6-aminoquinolinyl carbamate; GPI anchor; CBB, Coomassie Brilliant Blue R-250; PAGE, polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; Cell wall protein; TFA, trifluoroacetic acid
• ISSN: 0304-4165; 0006-3002; 1872-8006; 1878-2434
• DOI: 10.1016/S0304-4165(99)00012-4

Tip1p is one of the major cell wall mannoproteins of Saccharomyces cerevisiae and is presumed to be synthesized as a glycosylphosphatidylinositol (GPI)-anchored form. We purified Tip1p from a glucanase extract of yeast cell walls and analyzed the sugar chain involved in the cell wall linkage. One mol of glucanase-extracted Tip1p contained 7.5 mol of glucose derived from glucan and 1 mol of ethanolamine, a component of the GPI anchor. One mol of the C-terminal peptide of Tip1p digested with Achromobacter protease I also contained 7.9 mol of glucose and 1 mol of ethanolamine. On the other hand, Tip1p contained no glucosamine, which is a component of the GPI anchor. The glucan-binding sugar chain of Tip1p was released by hydrazinolysis and isolated. This sugar chain contained ethanolamine with a free amino group and a glucose reducing end, but no mannose reducing end. Phosphodiesterase treatment eliminated the free amino group from this sugar chain, suggesting that a phosphodiester bond exists between the ethanolamine and the glucan remnant. These results indicate (1) the glucan-binding sugar chain of Tip1p is a GPI derivative, and (2) the GPI anchor is cleaved at the glycosyl moiety, and the resultant mannose reducing end is probably used to link Tip1p to cell wall glucan.