Biochemical characterization of antigen-specific glycosylation-inhibiting factor from antigen-specific suppressor T cells. I. Identification of a 55-kilodalton glycosylation-inhibiting factor peptide with TCR alpha-chain determinant.
Stimulation of OVA-specific suppressor T cell (Ts) hybridoma and bee venom phospholipase A2 (PLA2)-specific Ts hybridoma with Ag-pulsed APC or by cross-linking of CD3 resulted in the formation of Ag-specific glycosylation-inhibiting factor (GIF). Affinity-purified Ag-specific GIF preparations, obtained by using Ag-coupled Sepharose or anti-TCR alpha-chain-coupled Affi-Gel, contained a 55-kDa peptide, which bound both polyclonal anti-GIF Abs and anti-TCR-alpha mAb in immunoblotting. The same hybridomas constitutively secrete 13-kDa bioactive GIF peptide that has no affinity for homologous Ag, but neither the Ag-specific GIF activity nor 55-kDa GIF peptide was detectable in culture supernatants of unstimulated cells. Northern blot analysis of mRNA from the anti-CD3-stimulated hybridoma with 32P-labeled GIF cDNA revealed only 0.6 kb mRNA, which encodes the 13-kDa nonspecific GIF. No mRNA of the 55-kDa GIF was detectable. A representative OVA-specific Th hybridoma, DO 11.10 cells contain the 0.6 kb GIF mRNA and constitutively secrete inactive GIF peptide. However, the Th hybridoma failed to secrete the 55-kDa peptide or any peptide with the TCR-alpha determinant upon stimulation with anti-CD3. It appears that the formation of the 55-kDa peptide with the TCR-alpha determinant is unique for a subset of T cells including Ts cells that form bioactive GIF.
Nakano
T
T
Division of Immunobiology, La Jolla Institute for Allergy and Immunology, CA 92037, USA.
Ishii
Y
Y
Ishizaka
K
K
D000818:Animals; D000939:Epitopes / Q000737:chemistry*; D006031:Glycosylation; D006825:Hybridomas / Q000737:chemistry; D008222:Lymphokines / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D010455:Peptides / Q000276:immunology; D029584:Prostatic Secretory Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology*; D013379:Substrate Specificity; D013491:Suppressor Factors, Immunologic / Q000737:chemistry*; D050378:T-Lymphocytes, Regulatory / Q000737:chemistry* / Q000276:immunology
D000939:Epitopes; D008222:Lymphokines; D010455:Peptides; D029584:Prostatic Secretory Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D013491:Suppressor Factors, Immunologic; C043076:beta-microseminoprotein; C013059:immunoglobulin-binding factors