O-linked glycosylation of rat renal γ-glutamyltranspeptidase adjacent to its membrane anchor domain

• Published in: The Journal of biological chemistry 1989-12, Vol.264 (34), p.20718-20722
• Main Authors: Blochberger, T.C., Sabatine, J.M., Lee, Y.C., Hughey, R.P.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Carbohydrates - analysis; gamma-Glutamyltransferase - genetics; Glycosylation; Hydrolases - genetics; kidney; Kidney - enzymology; Microvilli - enzymology; Molecular Sequence Data; Papain; Peptide Fragments - isolation & purification; Rats; Wheat Germ Agglutinins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)47122-6

Large domains rich in serine and threonine, that are likely to exhibit clusters of O-linked oligosaccharides, have been reported adjacent to the anchor of several cell surface proteins. No such domain is evident in the primary sequence of rat renal γ-glutamyltranspeptidase. However, papain treatment of the amphipathic enzyme (Triton-purified γ-glutamyltranspeptidase, TγGT), pretreated with galactose oxidase and NaB3H4 (Frielle, T., and Curthoys, N. P. (1983) Biochemistry 22, 5709-5714), yields the hydrophilic enzyme (papain-treated Triton-purified γ-glutamyltranspeptidase, PTγGT) and a labeled peptide which contains both the amino-terminal membrane anchor and the sequence Pro27-Thr28-Thr29-Ser30. Since [3H]galactose was identified in this peptide, the presence of O-linked oligosaccharides was investigated. Carbohydrate analysis is consistent with the presence of two simple O-linked oligosaccharides on TγGT and one on PTγGT. Lectin blot analysis of TγGT and PTγGT was carried out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The small subunits of both TγGT and PTγGT and the large amphipathic subunit of TγGT all react with the peanut agglutinin lectin, but the large subunit of PTγGT exhibits no such reactivity. The reactivity with PNA is consistent with the presence of one oligosaccharide with the structure galactose β1-3N-acetylgalactosamine α1-Ser/Thr attached to each subunit of TγGT. The papain-sensitivity of the oligosaccharide from the larger subunit is consistent with O-glycosylation at the Thr28-Thr29-Ser30 sequence. The results of lectin blot analysis with wheat germ agglutinin imply that the content of N-linked oligosaccharides is unaffected by papain treatment of the transpeptidase. These data represent the first direct evidence for O-glycosylation of a microvillar hydrolase at a site immediately adjacent to the membrane anchor and indicates that even small clusters of Thr and Ser can be O-glycosylated. Isolated O-linked oligosaccharides may have functional significance since single Ser and Thr residues are consistently found near the membrane anchor of many cell surface proteins.