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Evidence for a Stepwise Evolution of the CD3 Family¹

Thomas W. F. Göbel^2,* and Jean-Pierre Dangy

^* Institute for Animal Physiology, Munich, Germany; and Basel Institute for Immunology, Basel, Switzerland

	Abstract

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The three CD3 components of the TCR complex are encoded as clustered genes in mammals. The evolution of such a multimeric complex is likely to occur stepwise. The chicken CD3 cluster was entirely sequenced, and, in contrast to mammals, only two chicken CD3 genes were found to be physically linked to the unrelated genes HZW10 and epithelial V-like Ag flanking both sides of the CD3 cluster. Biochemical analyses of CD3 immunoprecipitates confirmed the presence of only two CD3 proteins and revealed an essential role for CD3 glycosylation during assembly. Functional analyses indicated that the chicken TCR/CD3 complex was efficiently down-regulated by phorbol ester treatment, demonstrating the integrity of a CD3-like cytoplasmic internalization motif. These data argue for a stepwise CD3 evolution, with major differences in the TCR/CD3 structure between mammalian and nonmammalian vertebrates setting a basis for the understanding of the CD3 phylogeny and proving the ancestral nature of the CD3 protein.

	Introduction

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Mammalian TCR/CD3 proteins have been the focus of intense research covering aspects of biosynthesis, assembly, structure, and signaling in the past years (1, 2). The clonotypic TCR chains have been characterized in a number of different vertebrate phyla (3). In contrast, the difficulties in cloning of nonmammalian CD3 homologues has limited their detailed analysis. The chicken TCR/CD3 represents the only nonmammalian Ag receptor where two CD3 genes and the -chain have been identified (4, 5, 6). Since data regarding the CD3 biochemistry does not exclude the presence of a third CD3 (7), the present experiments were performed to reveal the actual complexity of the avian TCR.

The CD3 genes are clustered on a 50-kb part of chromosome 11 and 9 in humans and mouse, respectively. CD3 is located in the central position of the cluster and flanked on either side by the oppositely transcribed CD3 and CD3 gene loci. CD3 is less than 2 kb apart from CD3, and CD3 is located about 22 kb downstream from CD3 (8). CD3 is encoded by five exons, whereas seven exons encode CD3 and seven or eight exons encode CD3 depending on the presence of two and three miniexons in humans and mouse, respectively. All CD3 genes have TATA-less, non-tissue-specific promoters, and enhancer elements to ensure T cell-specific expression (9, 10, 11, 12).

To date, only three nonmammalian CD3 homologues and the chicken -chain have been identified (4, 5, 6, 13). The chicken CD3 chain has low extracellular (EX)³ but high cytoplasmic (CY) homology to its mammalian counterparts (5). An additional CD3 chain has been cloned in the chicken and in the amphibian Xenopus laevis. It shares equal homology to both mammalian CD3 and CD3, and it was therefore designated CD3 (4, 13). Calculations based on sequence divergence further demonstrated that human and mouse CD3 and CD3 originate from a gene duplication which occurred about 230 million years ago (14). Since birds and mammals are separated by about 250 million years, this gene duplication could have occurred after the lineage separation.

A recent gene duplication could imply a redundancy of the duplicated genes. This holds true for certain CD3 domains. It has been shown that the CD3 transmembrane (TM) domain can be replaced by that of CD3. In contrast, domain swapping of the CD3 and CD3 EX domains causes loss of TCR surface expression (15). The CD3 and CD3 CY domains are not essential for surface expression (16, 17). CD3 harbors a unique di-leucine based internalization motif mediating protein kinase C-dependent TCR (18). Therefore, phorbol ester-mediated TCR down-regulation is abrogated in T cells with CY-truncated CD3 and chimeric CD3 with a CD3 CY domain, whereas anti-CD3-mediated TCR down-regulation is still intact (15, 17).

In contrast, gene targeting of individual CD3 genes has clearly established unique nonredundant functions during T cell development for both CD3 and CD3. The CD3 chain is essential for a functional pre-TCR since thymocyte development is blocked before selection in the double-negative CD44^-CD25⁺ stage, whereas thymocyte development in the CD3 knockout is arrested at the double-positive stage. Moreover, T cells do develop in the absence of CD3, but are virtually absent in the CD3 knockout (19, 20).

For the first time, we now report the complete sequence and physical linkage of the entire CD3 cluster. These analyses have failed to identify a third CD3 gene in the chicken. In addition, biochemical and functional analyses establish the ancestral hybrid nature of the chicken CD3 protein. These results raise the challenging question why a third CD3 protein evolved as an additional essential component of the mammalian TCR complex.

	Materials and Methods

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Isolation and sequencing of cosmid clones

A commercial chicken cosmid library (Clontech, Palo Alto, CA) was hybridized with a full-length CD3 cDNA probe, and two reactive cosmids were isolated. Cosmid DNA was prepared (Qiagen, Hilden, Germany) and directly used as template for sequencing using CD3-specific and internal oligonucleotides with the ABI Prism dye terminator cycle sequencing ready reaction kit (Perkin-Elmer, Foster City, CA) on an Applied Biosystems 373A Stretch sequencer. The Lasergene (DNAstar, Madison, WI) software package was used for sequence analysis.

TCR down-regulation

The chicken T cell line UG9 was maintained in DMEM supplemented with 5% FCS and 1 mM glutamine. The cells were incubated with the indicated amounts of phorbol 12,13-dibutyrate (PDB; Sigma, St. Louis, MO) for 1 h at 37°C and then collected and labeled using either the chicken CD3-specific CT3 mAb or the chicken V1-specific TCR2 mAb (Southern Biotechnology Associates, Birmingham, AL) (7, 21), followed by goat anti-mouse IgG1-FITC conjugate (Southern Biotechnology Associates). The flow cytometric analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA), and the percentage of TCR down-regulation was calculated [mean fluorescence intensity of PDB-treated cells/mean fluorescence intensity of untreated cells x 100].

Immunoprecipitation, SDS-PAGE, and nonequilibrium pH gradient electrophoresis (NEPHGE)

For metabolic labeling, 50 million UG9 cells were incubated for 3 h in the presence of 500 µCi [³⁵S]methionine/cysteine (Promix; Amersham, Piscataway, NJ) and when indicated in the presence of 1 µg/ml tunicamycin (Boehringer Mannheim, Indianapolis, IN), washed, and lysed with 1% digitonin in 300 mM NaCl, 100 mM Tris-Cl (pH 7.4), 10 mM EDTA, 0.2% NaN₃, 40 mM iodoacetamide, and protease inhibitors (Complete; Boehringer Mannheim). Deglycosylation was performed using PNGase F (New England Biolabs, Berverly, MA). Immunoprecipitations were performed with the CT3 mAb. The immunoprecipitates were either directly separated by 4–20% PAGE or first on a NEPHGE tube gel followed by 9–18% PAGE.

	Results

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The chicken CD3 cluster contains only two CD3 genes flanked by unrelated genes

To test whether a third CD3 gene exists in the chicken CD3 locus, we sequenced the entire CD3 cluster (Fig. 1; accession number AJ250458). The chicken CD3 locus is smaller than in humans or mouse, spanning only 9.5 kb as opposed to 50 kb in humans. The oppositely transcribed CD3 and CD3 genes are only 2 kb apart instead of the 22 kb distance in humans (Fig. 1). The two CD3 genes are flanked on both sides by chicken repeat 1 elements (22). The human and mouse CD3 genes are located less than 2 kb upstream of CD3 (Fig. 1A). In the chicken, however, a highly conserved centromere/kinetochore protein (HZW10) (23) is present in the place of CD3, only 832 bp upstream of CD3. Moreover, the chicken homologue of the recently described epithelial V-like Ag (24) is located 4.2 kb downstream of CD3 on the other side of the CD3 cluster (Fig. 1B). To our knowledge, the human CD3 cluster has not yet been physically linked to other genes; however, both epithelial V-like Ag and HZW10 genes have also been mapped to human chromosomes 11q23 and 11q24, respectively.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Genomic organization of the human and chicken CD3 cluster. A, Sketch representing the human CD3 cluster organization (8 9 12 ). Approximate sizes of the CD3 genes and the intervening sequences are given. Note that the sketch does not represent the exact size. B, Genomic organization of the chicken CD3 cluster and its derived mRNA drawn to scale as indicated. The genomic DNA and mRNA are represented in the top and bottom, respectively. Exons are shown as closed boxes and the chicken repeat (CR1) elements as hatched boxes. EVA, epithelial V-like Ag; SP, signal peptide.

Two CD3 proteins are associated with the chicken TCR heterodimer

Although unlikely, our linkage analysis did not exclude the existence of a third CD3 gene encoded outside of the cluster or even on another chromosome. To exclude this possibility, the CD3 chains were metabolically labeled and immunoprecipitated with the CT3 mAb under mild conditions, preserving the TCR/CD3 interaction. Initially, the immunoprecipitates were analyzed in a one-dimensional gel comparing glycosylated and deglycosylated samples (Fig. 2A). The different CD3 proteins were also identified in parallel Western blot experiments using chain-specific antisera (data not shown). The TCR- migrated around 70 kDa and dropped to 50 kDa following deglycosylation. Additional free TCR-chain was detected at 45 kDa and 29 kDa following deglycosylation. Single CD3 proteins were identified as the glycosylated 19-kDa CD3 protein and the nonglycosylated 16-kDa CD3 protein. After deglycosylation, the CD3 and CD3 proteins were not separated well on a 4–20% PAGE and comigrated. In addition, both CD3 proteins formed a covalently linked CD3-CD3 heterodimer of 35 kDa that migrated faster following PNGase F treatment due to the presence of N-linked carbohydrates in CD3.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Metabolic analyses of the chicken TCR/CD3 complex. A, Metabolically labeled UG9 T cells were lysed in 1% digitonin and immunoprecipitated with a CD3-reactive mAb and either directly analyzed by 4–20% gradient PAGE or treated with PNGase F to remove N-linked carbohydrates. The identity is indicated for the glycosylated proteins and changed following deglycosylation as described in Results. B, Immunoprecipitates were deglycosylated with PNGase F and separated by NEPHGE followed by PAGE on a 9–18% gradient gel. The identity of the proteins is indicated.

An important biochemical difference between mammalian CD3 and CD3 is the dependence of glycosylation for TCR assembly. Although glycosylation of CD3 is essential for TCR assembly, CD3 glycosylation is dispensable (16). To test whether the glycosylation of the chicken CD3 protein is important for the assembly of the TCR complex, the T cell line was metabolically labeled in the presence of tunicamycin to prevent N-linked glycosylation. Immunoprecipitation with CD3-specific mAb revealed no association with CD3 or TCR. Apparently, CD3 glycosylation is required for the assembly with CD3 and TCR and the transport to the cell surface (Fig. 3). Interestingly, in the absence of CD3 association, the CD3 chain formed dimers, as has been reported for mammalian CD3 (Fig. 3) (25). Mammalian CD3 and CD3 proteins drastically differ in their susceptibility to CY degradation (1, 2). When the stability of the chicken CD3 proteins was tested in pulse-chase experiments, both CD3 proteins were stable for several hours like mammalian CD3, but in contrast to mammalian CD3 which is rapidly degraded (data not show). Taken together, these experiments biochemically prove the existence of only two nonmammalian CD3 proteins, a CD3 homologue and a second CD3 protein with hybrid character combining features of mammalian CD3 and CD3.

View larger version (44K): [in this window] [in a new window]   FIGURE 3. Glycosylation of CD3 is essential for the TCR/CD3 assembly. UG9 cells were metabolically labeled in the absence or presence of tunicamycin to prevent N-linked glycosylation as indicated. Immunoprecipitations were done with the CT3 mAb (+) or an isotype control mAb (-). The molecular identity of the proteins and the apparent Mr is indicated.

The chicken CD3 protein shares many characteristics with mammalian CD3 (see below). However, a highly conserved CD3-like feature of the CD3 chain is the presence of an internalization motif within the CY domain, which mediates protein kinase C-dependent TCR down-regulation (15). To demonstrate that this motif has been functionally conserved, the UG9 T cell line was incubated with increasing amounts of PDB, and the degree of TCR down-regulation was determined by CD3 or TCR staining and analyzed by flow cytometry. Within 1 h of incubation, the TCR was down-regulated by PDB treatment in a dose-dependent fashion (Fig. 4). These results demonstrate that the CY CD3-like internalization motif in the CD3 protein is fully intact.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. PDB treatment induces TCR down-regulation. The percentage of TCR/CD3 down-regulation of UG9 cells incubated with increasing concentrations of PDB for 1 h at 37°C as analyzed by CD3 () and TCR surface staining (). Means ± SD of triplicates are shown.

	Discussion

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View this table: [in this window] [in a new window]   Table I. Ancestral nature of chicken CD3

The experimental evidence presented here shows that the CD3 gene resembles an ancestral form that combines typical features of mammalian CD3 and CD3, which are critically relevant for proper TCR assembly and function (Table I). To substantiate this hypothesis, all mammalian CD3 and CD3 chains were aligned with their two nonmammalian homologues to pinpoint diagnostic residues that are only present in mammalian CD3 and the nonmammalian CD3 chains, but not CD3 and vice versa. The EX domains contain few conserved features, including the four cysteines present in all CD3 proteins and the DPRG motif known to create a binding site for CD3 (16). The unique CXXCXE motif in the membrane proximal region present in all CD3 chains is followed by a second negatively charged aspartic acid in mammalian CD3 and CD3, but not CD3 (Table I). Moreover, the glycosylation of CD3 is essential for its assembly with CD3 as in mammalian CD3, whereas CD3 glycosylation is dispensable (Fig. 3). The negatively charged TM residues represent an aspartic acid in CD3 as in the mammalian CD3 proteins, whereas CD3 proteins harbor a glutamic acid at this position. All of these criteria and also the five exon genomic structures and the genomic location would suggest that the chicken CD3 closely resembles a CD3 homologue. However, its CY domain harbors a CD3-like internalization motif. It was therefore critical to demonstrate that this internalization motif in CD3 is functionally active in chicken T cells (Fig. 4). The conserved change of the chicken motif from a leucine to an isoleucine does not affect this function as has been demonstrated by previous mutational studies of human CD3 (26). Moreover, unlike mammalian CD3, the chicken CD3 protein is slowly degraded. In conclusion, the chicken CD3 protein resembles mammalian CD3 in most aspects, with the exception of the CY internalization motif and its slow degradation which are CD3-like features.

The five exon CD3 structures most likely resemble the most primordial form of a CD3 gene, since the basic organization is found in every CD3 gene with variations occurring only at the ends of the CD3 genes. Based on the genomic organization presented here, a model for the CD3 evolution would predict two successive gene duplications where a single CD3 gene first duplicated to form CD3 and CD3 and a second duplication of CD3 has finally generated mammalian CD3 and CD3. Several features argue in favor of such a hypothesis: 1) the central location of CD3 flanked by CD3 and CD3 on either side with opposite transcriptional orientations (Fig. 1) (8, 9, 12), 2) the TATA-less, non-tissue-specific promoters of all CD3 genes, 3) the conserved EX CXXCXE motif close to an exon-intron boundary, 4) the CY ITAM motif consistently interrupted by a type 0 intron, and 5) the 5 exon cores present in all CD3 genes with variations occurring only at the 5' and 3'ends.

All TCR chains cloned to date contain a short CY domain lacking intrinsic enzymatic or signaling activity as well as a positively charged TM residues. It is therefore likely that the TCR heterodimer of lower vertebrates also associate with signal-transducing elements. It should be possible to find an even more rudimentary situation before the initial CD3 gene duplication. In striking difference to the proposed decameric mammalian TCR, where two TCR heterodimers associate with the -homodimer and two CD3 heterodimers, such a primordial receptor could have a pentameric structure consisting of a single TCR heterodimer, -homodimer, and single CD3 chain.

The model proposed for the chicken TCR/CD3 complex closely resembles the decameric model for the mammalian TCR, with the exception that the mammalian CD3-CD3 heterodimer would be replaced by a second CD3-CD3 heterodimer. It is interesting to note that at least two forms of mammalian TCR complexes are not affected by a loss of the CD3, namely, the pre-TCR and the TCR complex on T cells, as demonstrated by the gene targeting of the CD3 chain (19). In this aspect, these TCR complexes might represent a rudimentary form of an ancestral TCR and provide a paradigm where the ontogeny reflects the phylogeny of a cell surface receptor.

The most important question provoked by these results is why an already complex cell surface receptor with apparently normal function was further modified by the addition of a novel essential protein. An additional protein in the complex may be important for the fine tuning of the response by generating new binding sites for coreceptors. It may also change the entire three-dimensional structure of the receptor, thus influencing the MHC/peptide binding. An additional CD3 protein may also alter the TCR/CD3-signaling capacity. It is intriguing to note the correlation of a solvent exposed loop in the TCR-chain located between the V and C region only present in mammals and the occurrence of a third CD3 protein. Thus, this loop may create an important binding site for the CD3 EX domain. The future analyses of these primordial TCR/CD3 complexes will contribute to the understanding of the TCR complex evolution and function.

	Acknowledgments

 
We thank Drs. L. Bolliger and H. Jacobs for critically reading this manuscript, H. Stahlberger for artwork, and H. Spalinger and B. Pfeiffer for photography.

	Footnotes

 
¹ The Basel Institute for Immunology was founded and is supported by Hoffmann-LaRoche & Co. Ltd. (Basel, Switzerland).

² Address correspondence and reprint requests to Dr. Thomas Göbel, Institute for Animal Physiology, Veterinärstrasse 13, 80539 Munich, Germany. E-mail address:

³ Abbreviations used in this paper: EX, extracellular; CY, cytoplasmic; NEPHGE, nonequilibrium pH gradient electrophoresis; PDB, phorbol 12,13-dibutyrate; TM, transmembrane.

Received for publication July 30, 1999. Accepted for publication November 5, 1999.

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