Cloning and expression of a cDNA for the T-cell-activating protein TAP.

Abstract

The T-cell-activating protein TAP is a murine phosphatidylinositol-anchored glycoprotein whose expression is controlled by the Ly-6 locus. Previous studies have suggested an important role for this protein in physiological T-cell activation. Using oligonucleotide probes, we have now isolated a cDNA clone whose predicted sequence would encode a protein with an NH2-terminal sequence identical to that of the TAP molecule. Further analysis of the predicted protein sequence revealed a cysteine-rich protein with a hydrophobic domain at the COOH terminus and without N-linked glycosylation sites-all features consistent with our previous analysis of the TAP protein. In Southern blot analysis, the Ly-6.2 cDNA clone detects a multigene family and a restriction fragment length polymorphism that maps precisely to the Ly-6 locus. Expression of the cDNA clone in COS cells demonstrates that it codes for TAP and clarifies the relationship between the epitopes recognized by various aLy-6 monoclonal antibodies.Finally, we have studied the expression of Ly-6 mRNA in a variety of cell lineages. Ly-6 transcripts were detected in all organs examined, including spleen, kidney, lung, brain, and heart. This demonstrates that the Ly-6 locus is transcriptionally active in a wide range of organs and suggests that the role of TAP or TAP-like proteins might extend to other tissues.Activation of T inducer cells is antigen-dependent and major histocompatibility complex (MHC)-restricted (1). The specificity of activation is imparted by a clonotypic receptor, composed of two variable protein chains, Ti a and ,B (2, 3). Cell-surface expression of the T-cell receptor (TCR) has been extensively characterized. It requires coexpression of a set of nonpolymorphic proteins, termed T3, which are physically associated with the antigen receptor (4, 5). In contrast, the molecular mechanisms of signal transduction after TCR triggering is not understood.Recent findings by our laboratory have raised the possibility that the T-cell-activating protein (TAP) (6), a phosphatidylinositol-anchored, murine glycoprotein (7), is involved in this process: First, antibodies against TAP can induce T-cell proliferation and can augment stimulation of the TCR (6). Second, TAP is expressed on virtually all L3T4+ peripheral T cells, and in the thymus, TAP expression defines the immunocompetent compartment (8-10). Finally, T-cell-T-cell (T-T) hybridoma mutants that are deficient in the expression of TAP have significant defects in their responsiveness to TCR stimulation (33).The gene encoding the TAP molecule maps to the Ly-6 locus (8). A polymorphism in the expression of TAP in inbred mouse strains, matches precisely that of the "b" haplotype of the Ly-6 locus. This locus, which consists of two haplotypes, Ly-6.J (Ly-6a) and Ly-6.2 (Ly-6b), encodes multiple lymphoid antigens (8, 11).We have previously reported the nearly complete NH2-terminal sequence of the TAP molecule (12). Based on the obtained protein sequence data, oligonucleotides were synthesized and used to sc...

Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.

“…It should be noted that the basic biosynthetic defect in M43/8 is deficient Dol-P-Man synthesis, which, predictably, affects N-linked glycosylation (Thomas, L., and R. DeGasperi, data not shown). Since Thy-I is N-glycosylated, but Ly-6A is not (37)(38)(39), one would expect Thy-I expression in M43/8 to be more adversely affected than Ly-6A. The observation to the contrary, i.e., that the surface expression ofLy-6A was more diminished than that of Thy-i, suggested that deficient GPI core formation is the primary cause of the differential expression.…”

“…However, the surface expression of Thy-I remained unchanged. This was most likely due to the deleterious effect of tunicamycin on N-linked glycosylation, because Thy-I contained three Nlinked glycosylation sites whereas Ly-6A had none (37)(38)(39). (Fig.…”

Characterization of promoter elements of an interferon-inducible Ly-6E/A differentiation antigen, which is expressed on activated T cells and hematopoietic stem cells.

“…Restriction mapping of these phage DNAs indicated that most of the phages contained the same sequences as previously characterized in the Ly-6C.J gene (5). However, one phage, clone 50, had DNA sequences located in three exons that corresponded exactly to those found in the first 274 bp of the Ly-6E.J cDNA (16) and Ly-6A.2 (19,21). A different library was screened with the entire 750-bp EcoRI cDNA probe, and clone 51, containing the exon 4 of the Ly-6A.2 gene plus an additional 15 kilobase pairs (kb) 3' to that exon, was isolated.…”

“…There is a similar pattern of expression for Ly-6E.1. The cDNAs for the genes encoding Ly-6A.2 and Ly-6E.1 are highly homologous and differ at only three nucleotides in the coding region, resulting in two amino acid differences (16,19,21). One of the amino acid differences is located in the carboxy-terminal end of the protein and is thought to be lost during the addition of phosphatidylinositol anchor.…”

Biosynthesis of a phosphatidylinositol-glycan-linked membrane protein: signals for posttranslational processing of the Ly-6E antigen.

“…The genes encoding these antigens are members of a multigene family of about 20 members located within a region of about 500 kilobases (kb) on chromosome 15 (15; W. Philbrick and A. Bothwell, unpublished data). The Ly-6E molecule studied here is most commonly associated with activated T cells and hence an allele of the encoding gene, Ly-6A.2, has been identified as encoding T-cell activation protein (24)(25)(26). Recently, Ly-6A.2 has also been shown to be equivalent to the Sca-1 antigen found on the hematopoietic stem cell (28.…”