Association of glycosylation-inhibiting factor with plasma membranes of T suppressor cell hybridomas.
The glycosylation inhibiting factor (GIF) was detected in EGTA extracts of the OVA-specific Ts cell hybridoma, 231F1 cells and 71B4 cells, which constitutively secrete GIF. The lymphokine in both culture supernatants and EGTA extracts failed to bind to OVA-Sepharose. Association of GIF with the plasma membrane was confirmed by surface labeling of the 231F1 cells with 125I. The major species of GIF in the extract was 14.4-kDa peptide as determined by SDS-PAGE, and was identical to that detected in culture supernatants. Pretreatment of the cells with monoclonal anti-GIF switched the cells from the formation of unglycosylated IgE-BF to the formation of glycosylated IgE-BF, indicating that the membrane-associated GIF is involved in the determination of the nature of IgE-binding factor during their biosynthesis. When the hybridoma was stimulated with OVA-pulsed APC, EGTA extracts of the cells contained GIF having affinity for OVA. The binding of the OVA-binding GIF in the EGTA extracts to OVA-Sepharose was inhibited by a synthetic peptide, which corresponds to amino acid residues 307-317 in the OVA molecule and represents the epitope recognized by TCR on the cells. The OVA-binding GIF in the extracts bound to the monoclonal anti-TCR-alpha chain, H-28-710 and the mAb 14-12, which is specific for the Ag-binding chain of effector type suppressor factor, and suppressed the in vivo antibody response of BDF1 mice to DNP-OVA in a carrier-specific manner. Evidence was obtained that indicated that the Ag-binding chain was associated with nonspecific GIF chain on the cell surface of the Ag-stimulated cells.
Iwata
M
M
Johns Hopkins University School of Medicine, Baltimore, MD 21239.
Katamura
K
K
Mori
A
A
Yamagushi
K
K
Grey
H
H
Ishizaka
K
K
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation; D006825:Hybridomas / Q000737:chemistry*; D008222:Lymphokines / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D010047:Ovalbumin / Q000276:immunology; D029584:Prostatic Secretory Proteins; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013491:Suppressor Factors, Immunologic / Q000302:isolation & purification*; D050378:T-Lymphocytes, Regulatory / Q000737:chemistry*
D008222:Lymphokines; D029584:Prostatic Secretory Proteins; D011948:Receptors, Antigen, T-Cell; D013491:Suppressor Factors, Immunologic; C043076:beta-microseminoprotein; C013059:immunoglobulin-binding factors; D004533:Egtazic Acid; D010047:Ovalbumin