monensin and Astrocytoma

Human alpha 2-macroglobulin (alpha 2M) is a high molecular weight plasma proteinase inhibitor exhibiting a broad specificity; in fact it is capable of binding endopeptidases from all known classes of proteases (Barret 1981). Two human glioma cell lines, namely an astrocytoma and a glioblastoma, were found to synthesize and secrete in the culture medium a protein which resembles the serum alpha 2M for immunological, biochemical and biological features. Using polyclonal antibodies to serum alpha 2M, an alpha 2M-like factor could be detected in the cytoplasm and in the culture medium of the tumor cells. Furthermore this factor accumulated in cytoplasmic granules if cells were incubated with monensin and its production was dramatically reduced following a treatment with cycloheximide. This protein behaved like the serum alpha 2M in immunoblotting analysis and exhibited the same antiproteolytic activity. Its role in human brain is unknown at present. Since interactions of proteinases and proteinase-inhibitors appear to influence the host-tumor immune response and to play a crucial role during the migration of metastasizing tumor cells, alpha 2M expression observed in these glioma cells could be involved in tumor cell proliferation and invasion.

Topics: alpha-Macroglobulins; Astrocytoma; Cell Line; Cycloheximide; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Glioma; Humans; Kinetics; Molecular Weight; Monensin; Protease Inhibitors; Time Factors

We have previously shown that gp65 (E3) is a virion structural protein which varies widely in quantity among different strains of mouse hepatitis virus (MHV). In this study, the biosynthetic pathway and possible biological activities of this protein were examined. The glycosylation of gp65 in virus-infected cells was inhibited by tunicamycin but not by monensin, suggesting that it contains an N-glycosidic linkage. Glycosylation is cotranslational and appears to be complete before the glycoprotein reaches the Golgi complex. Pulse-chase experiments showed that this protein decreased in size after 30 min of chase, suggesting that the carbohydrate chains of gp65 undergo trimming during its transport across the Golgi. This interpretation is supported by the endoglycosidase treatment of gp65, which showed that the peptide backbone of gp65 did not decrease in size after pulse-chase periods. This maturation pathway is distinct from that of the E1 or E2 glycoproteins. Partial endoglycosidase treatment indicated that gp65 contains 9 to 10 carbohydrate side chains; thus, almost all of the potential glycosylation sites of gp65 were glycosylated. In vitro translation studies coupled with protease digestion suggest that gp65 is an integral membrane protein. The presence of gp65 in the virion is correlated with the presence of an acetylesterase activity. No hemagglutinin activity was detected.

Topics: Animals; Astrocytoma; Esterases; Glycoproteins; Glycosylation; Golgi Apparatus; Hemagglutinins, Viral; Monensin; Murine hepatitis virus; Precipitin Tests; Protein Biosynthesis; Protein Processing, Post-Translational; RNA, Viral; Transcription, Genetic; Tumor Cells, Cultured; Tunicamycin; Viral Envelope Proteins