Biochemical characterization of antigen-specific glycosylation-inhibiting factor from antigen-specific suppressor T cells. II. The 55-kDa glycosylation-inhibiting factor peptide is a derivative of TCR alpha-chain and a subunit of antigen-specific glycosylation-inhibiting factor.
We isolated a unique TCR alpha-chain derived from the OVA-specific Ts hybridoma, 231F1. The TR alpha-chain consists of V alpha 11.3, a unique J alpha and a complete sequence of C alpha region. Transfection of the TCR-alpha cDNA into a TCR-alpha-, TCR-beta+ T cell line, 175.2, resulted in the expression of TCR-alpha beta, and the transfectant contained a 35-kDa peptide having the TCR- alpha-specific antigenic determinant. However, the stable transfectant failed to release a peptide with the TCR-alpha determinant upon stimulation with anti-CD3. In contrast, overexpression of the cDNA in the 231 F1 cells markedly increased the formation of the 55-kDa peptide, which reacted with both anti-glycosylation-inhibiting factor (GIF) and the mAb H28-710. Definitive evidence for the relationship between the 55-kDa peptide and the TCR alpha-chain was obtained by transfection of the cDNA of the TCR alpha-chain with histidine tag into the 231F1 cells. The 55 kDa GIF peptide formed by stable transfectants of the TCR-alpha-tag cDNA bound to Ni+-nitrilotriacetic acid-agarose. Upon stimulation with anti-CD3, a stable transfectant of the TCR-alpha cDNA formed OVA-specific GIF which contained the 55-kDa GIF peptide, and bound not only to anti-TCR-alpha column but also to anti-TCR-beta column. The results indicate that the OVA-specific GIF consists of the TCR-alpha+ 55-kDa GIF and another peptide with TCR-beta determinant. It was found that the association of the TCR-beta+ peptide with the 55-kDa GIF is required for binding of the factor to OVA, but not essential for the formation and release of the latter peptide.
Ishii
Y
Y
Division of Immunology, La Jolla Institute for Allergy and Immunology, CA 92037, USA.
Nakano
T
T
Ishizaka
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D000939:Epitopes / Q000737:chemistry*; D006031:Glycosylation; D008222:Lymphokines / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010047:Ovalbumin / Q000276:immunology; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D029584:Prostatic Secretory Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000276:immunology*; D013379:Substrate Specificity; D013491:Suppressor Factors, Immunologic / Q000737:chemistry*; D050378:T-Lymphocytes, Regulatory / Q000737:chemistry* / Q000276:immunology
D018076:DNA, Complementary; 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; D010047:Ovalbumin