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.

Abstract

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.