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J. Biochem, 1982, Vol. 92, No. 3 765-773
© 1982 Japanese Biochemical Society

research-article

Effect of Tunicamycin on the Biosynthesis of a Glycoprotein Antigen, Contact Site A, in Aggregation-Competent Cells of Dictyostelium discoideum1

Toshihiko HIRANO*, Haruki YAMADA*,2, Toshio MIYAZAKI*, Akira TAKATSUKI** and Gakuzo TAMURA**

*Department of Microbial Chemistry, Tokyo College of Pharmacy Hachioji, Tokyo 192-03
**Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo Yayoi, Bunkyo-ku, Tokyo 113

The effect of tunicamycin (TM) on the biosynthesis of contact site A (cs-A), which is thought to be involved in the cell adhesion of aggregation-competent cells of Dictyostelium discoideum, was investigated. The butanol extract from particles of untreated cells showed a major protein band with a molecular weight of 80,000 daltons on polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). However, this protein band was not detected in the extract from TM-treated cell particles by SDS-PAGE. The monovalent antibodies prepared from the antiserum which was produced against the membrane particles of the aggregation-competent cells (anti P) showed a significant inhibitory activity against EDTA-stable cell adhesion of the aggregation-competent cells. This result indicated that the antibody contained anti cs-A. Upon immunoaffinity chromatography on anti P-immunoglobulin G (IgG)-conjugated protein A/Sepharose, the protein band at 80,000 daltons corresponding to cs-A was detected in the absorbed fraction of the control extract, but it was not detected and a single protein band at 70,000 daltons newly appeared in the same fraction from TM-treated cells. When the radioactive antigens in the[3H]leucine-labeled butanol extract from the control or TM-treated cells were further purified by a second affinity chromatography on concanavalin A (con A) Sepharose, the major radioactive peak at 80,000 daltons was detected in the control, while this peak was not detected in the TM-treated cell extract. However, the minor radioactive peak at 70,000 daltons which was not detected in the control antigen was detected in the con A-reactive antigens from TM-treated cell extract. This protein at 70,000 daltons seemed to be incomplete cs-A, lacking asparagine-Iinked oligosaccharide chains. These results confirmed that the N-glycosylation of an adhesion-mediating glycoprotein, cs-A, was inhibited by the TM treatment of the cells.

2Present address: Laboratory of Biochemistry, Oriental Medicine Research Center, The Kitasato Institute, Minato-ku, Tokyo 108.

1 This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.


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