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The Journal of Immunology, 1999, 162: 4115-4121.
Copyright © 1999 by The American Association of Immunologists

Cloning and Modeling of the First Nonmammalian CD41

Riitta Koskinen2,{dagger}, Urpo Lamminmäki{ddagger}, Clive A. Tregaskes§, Jan Salomonsen, John R. Young§ and Olli Vainio{dagger}

* Turku Immunology Center and Departments of {dagger} Medical Microbiology and {ddagger} Biotechnology, Turku University, Turku, Finland; § Institute for Animal Health, Compton, United Kingdom; and Department of Virology and Immunology, Royal Veterinary and Agricultural University, Frederiksberg, Denmark


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We have cloned and sequenced the first nonmammalian CD4 cDNA from the chicken using the COS cell expression method. Chicken CD4 contains four extracellular Ig domains that, in analogy to mammalian CD4, are in the order V, C2, V, and C2. The molecule is 24% identical with both human and mouse sequences. The extracellular domains were modeled using human and rat CD4 crystal structures as templates. In the first domain there are two extra Cys residues that are at suitable distance to form an intra-ß-sheet disulfide bridge in addition to the canonical one in the V domain. The region responsible for the interaction with MHC class II is relatively nonconserved in chicken. However, there are positively charged amino acids in the C'' region of the N-terminal domain that may mediate the association to the negatively charged residues of the MHC class II ß-chain. Molecular modeling also implies that the membrane-proximal domain mediates dimerization of chicken CD4 in a similar way as it does for human CD4. Furthermore, the cytoplasmic tail is highly conserved, containing the protein tyrosine kinase p56lck recognition site that is preceded by an adjacent di-leucine motif for the internalization of the molecule. Interestingly, there are no Ser residues in the cytoplasmic part, which may explain the slow down-regulation of chicken CD4 after phorbol ester stimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
CD4 is a single chain transmembrane glycoprotein on most thymocytes and on a subset of T cells (1, 2). It has an essential role in thymocyte development as well as in cell-mediated immune defense in mature T cells. In the context of Ag recognition by the TCR, CD4 dimerizes and binds to the {alpha}2 and ß2 domains of MHC class II (3, 4, 5, 6, 7, 8). The interaction triggers a signal to the T cell that is transduced by the protein tyrosine kinase p56lck, which binds to a conserved site in the cytoplasmic domain of CD4 (9, 10). Following T cell activation, CD4 is internalized (11, 12). This requires phosphorylation of cytoplasmic Ser residues that are associated with the di-leucine motif (13, 14). Furthermore, human CD4 is a part of the receptor complex for HIV gp120, and the deficiency of CD4-positive cells plays a role in the pathogenesis of AIDS (15). The three-dimensional structures of the extracellular domains of human and rat CD4 have been determined, revealing the structurally and functionally important features (7, 16, 17, 18, 19).

Here we describe the cloning of the first nonmammalian CD4 cDNA from the chicken. We have also modeled the four extracellular domains of chicken CD4 based on the known three-dimensional structures of human and rat CD4. The model demonstrates that despite the low sequence identity in chicken and mammalian CD4, several similar structural features have remained conserved. Furthermore, our analyses imply that chicken CD4 dimerizes in the same way as human CD4.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Animals

Strain H.B2 chickens (B2 MHC haplotype) were from the colonies of the Department of Medical Microbiology, Turku University (Turku, Finland).

Abs and cell lines

mAb 2-6 (IgG1 isotype), 2-35 (IgG2b), 7-125 (IgG1), 9-11 (IgG1), and 11-33 (IgM) against chicken CD4 were previously described (20, 21). mAb CT4 was purchased from Southern Biotechnology Associates (Birmingham, AL). mAb L22 (IgG1) and 11G2 (IgG1) detect the Bu-1a and Bu-1b molecules, respectively (22). mAb 2-191 recognizes chicken CD5 (IgG1) (23).

Marek’s disease virus-transformed CD4+ T cell lines MDCC-CU32 and MDCC-CU36 were gifts from Drs. B. W. Calnek and K. A. Schat, Cornell University (Ithaca, NY). The reticuloendotheliosis virus-transformed B cell line RECC-RP13 was a gift from Dr. K. Nazerian, U.S. Department of Agriculture (East Lansing, MI).

cDNA cloning and sequencing

A cDNA library was constructed from thymus (8-wk-old H.B2 chicken) mRNA. Transfections of COS-7 cells with the cDNA library and isolation of the positive clone, p2.6, were performed as described previously (23).

Nucleotide sequencing of the cDNA was conducted on both strands using the Sequenase version 2.0 kit (United States Biochemical, Cleveland, OH). Sequence data were analyzed by Genetics Computer Group software (Madison, WI), Lasergene Molecular Biology software (DNASTAR, Madison, WI), and Prosite pattern search (EMBL, Heidelberg, Germany) programs.

Labeling and immunoprecipitation

Metabolic labeling and immunoprecipitation of transfected COS-7 cells were conducted as previously described (23). COS-7 cells were transfected with p2.6 plasmid containing chicken CD4 cDNA. Cell lysate was precleared with a mixture of mAb L22 and 11G2 and immunoprecipitated with mAb 2-6 or anti-CD5-specific mAb 2-191.

Labeling of thymocytes from a 6-wk-old H.B2 chicken was performed using the lactoperoxidase/glucose oxidase method followed by lysis, immunoprecipitation, and analysis on 12% SDS-PAGE as described previously (24, 25).

Northern hybridization

Northern blot hybridization was conducted with mRNA from H.B2 chicken thymus, bursa, spleen, Marek’s disease virus-transformed CD4+ T cell lines MDCC-CU32 and MDCC-CU36, and a reticuloendotheliosis virus-transformed B cell line RECC-RP13. mRNA was isolated using RNeasy and Oligotex mRNA Spin column kits (Qiagen, Chatsworth, CA). After formaldehyde agarose (1.2%)-gel electrophoresis the samples were transferred overnight to a nylon membrane (Hybond-N+, Amersham, Aylesbury, U.K.). Prehybridization and hybridization were performed in a solution containing 0.5% SDS, 5x Denhardt’s solution, 3x SSPE, 10% dextran sulfate, and 100 µg/ml salmon sperm DNA at 60°C for 6 h. Hybridization was conducted at 60°C overnight with a total CD4 cDNA as a probe. The 1029-bp control probe of chicken ß-actin was prepared as previously described (26). The probes were labeled with [{alpha}-32P]dCTP (Amersham) using the Rediprime labeling kit (Amersham). Hybridized membrane was washed in 2x SSC/1% SDS at 60°C for 30 min and in 0.5x SSC/1% SDS at 60°C for 15 min followed by autoradiography for 5 and 18 h.

Molecular modeling

A homology model of the two N-terminal domains of chicken CD4 was built with a HOMOLOGY program included in the Insight II program package (Micron Separations, Westboro, MA). The sequence analysis was made using the GCG program package followed by structural alignment of several conserved molecules. For the V set domains 1 and 3, these structures were human CD4 domains 1 and 3 (7, 16, 17), rat CD4 domain 3 (18), human CD8 (27), human CD2 (28), and Ig light chain variable domain (REI) (29). For the C set domains, structures of human CD4 domain 2 and rat CD4 domain 4 were aligned (17, 18).

The two N-terminal domains were built using human CD4 and CD8 as templates, whereas domains 3 and 4 were built on the rat CD4 template. To combine the models, they were oriented by superimposing them on the structure consisting of all four extracellular domains of human CD4 (7).

The model was energy minimized by program DISCOVER (Molecular Simulation, Waltham, MA). First, the hydrogen atoms were allowed to relax in a 20-step minimization using steepest decent algorithm. Then 100 steps of the steepest decent were run while keeping the C{alpha} atoms of the ß-sheets constrained with a force of 50 kJ/mol/Å2 followed by another 100 steps of steepest decent and 100 steps of conjugate gradient minimization without constraints. Finally, 200 steps of conjugate gradient minimization was performed with formal charges on ionic groups. The CCFC force field and default parameters of DISCOVER were used throughout the minimizations.

Dimerization

To study the potential dimerization interactions between the chicken CD4 molecules, a dimer of the models was constructed. This was performed by superimposing two chicken CD4 models on the structure of human CD4 dimer using C{alpha} atoms of the ß-sheets on the interaction area (residues 341–345, 354–358, 373–378, and 386–382 in chicken and residues 314–318, 324–328, 342–347, and 355–361 in human CD4).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Cloning and identification of an mAb 2-6-specific cDNA

We wanted to clone the cDNA encoding chicken CD4, because of the essential importance of CD4 in both mammalian and avian immune defense. Chicken CD4 has been demonstrated to share functional similarity to the mammalian CD4 homologues (21, 30). Identification of the structure of chicken CD4 will help to reveal evolutionarily important features of the molecule.

To isolate the putative chicken CD4 cDNA, a chicken thymus cDNA library in the pCDM8 expression vector was transfected into COS-7 cells. Transfected cells were stained with mAb 2-6 supposed to detect chicken CD4, and plasmids from a positive cell were isolated. After three rounds of rescreening, a clone, p2.6, was isolated. It contained an insert of 1990 bp containing an open reading frame of 1461 bp between nucleotides 143 and 1603 (Fig. 1Go). The 3' untranslated region contains a putative polyadenylation signal followed by a poly(A) tail. The protein encoded by the open reading frame consists of a 402-amino acid extracellular region including four Ig-like domains, a 24-residue hydrophobic transmembrane region, and a 33-residue cytoplasmic tail. Protein database searches with the predicted amino acid sequences resulted in highest homology, with mammalian CD4 sequences showing 24% identity to both mouse and human sequences. Furthermore, COS cells transfected with p2.6 stained with several proposed anti-chicken CD4 mAb, 2-35, 7-125, 9-11, 11-33, and CT4, but COS cells transfected with a control cDNA did not react with the Abs (data not shown). In addition, a 64-kDa band was immunoprecipitated from metabolically labeled transfected COS cells with mAb 2-6, but not with a control mAb 2-191 recognizing chicken CD5 (Fig. 2GoA). This demonstrates that p2.6-transfected COS cells produce a protein similar to that precipitated from chicken T cells (Fig. 2GoB).



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  		FIGURE 1. Nucleotide sequence of chicken CD4 cDNA and predicted amino acid sequence. Potential N-glycosylation sites, the transmembrane region, and a putative polyadenylation site are underlined. Numbers on the left indicate the locations of nucleotides, and those on the right indicate amino acids. The putative signal peptide is between residues -28 and -1. The mature polypeptide starts at +1. These sequence data are available from EMBL under accession number Y12012.

 


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  		FIGURE 2. Immunoprecipitation of chicken CD4. A, 35S-labeled p2.6-transfected COS cell lysate was immunoprecipitated with mAb 2-6 or control mAb 2-191 against chicken CD5. The precipitated material was analyzed on 12% SDS-PAGE under reducing conditions. The migrations of m.w. standards are indicated to the left. B, Chicken thymocytes were surface 125I-labeled, lysed, and immunoprecipitated with mAb 2-6 or control supernatant from Sp2/0 cells. The samples were run on 12% SDS-PAGE under reducing conditions. The migrations of m.w. standards are indicated to the right.

 
Northern blot analysis with a CD4 cDNA probe revealed a single 2.2-kb transcript in thymus, spleen, and CD4-expressing T cell lines MDCC-CU32 and MDCC-CU36 (Fig. 3Go). No CD4 transcripts were detected in the bursa or in a B cell line, RECC-RP13. In spleen the transcript was expressed at a much lower level than in thymus.



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  		FIGURE 3. Northern blot hybridization. The expression of the CD4 mRNA in indicated chicken tissues and cell lines was detected using total CD4 cDNA as a probe. The exposition time for thymus and cell lines was 5 h; that for bursa and spleen was 18 h. The chicken ß-actin probe was used to assess the amount of mRNA.

 
Analysis of the extracellular domains of chicken CD4

The putative signal peptide of chicken CD4 is 28 amino acids long. Its cleavage site was predicted by the rules of von Heijne (31) and by comparison with mammalian CD4 sequences. The predicted m.w. of the mature polypeptide is 52,039, and the isoelectric point is 10.1.

The chicken CD4 amino acid sequence was compared with the mammalian sequences, and the extracellular part was modeled based on the known three-dimensional structures of human and rat CD4 (7, 16, 17, 18, 19). The sequence alignment suggests that the extracellular region of chicken CD4 consists of four Ig domains in the order V, C2, V, and C2, like mammalian CD4 (Fig. 4Go). In general, the modeled structure of chicken CD4 conformed readily to the mammalian CD4 structure (Fig. 5Go). The deletions and insertions could be accounted for within the loops of the modeled structure, enabling preservation of the basic Ig domain structure. The two N-terminal domains have been of major interest, since they mediate both the binding of MHC class II molecule and the HIV gp120 (8, 32). Recently, the membrane-proximal domain was shown to participate in CD4 dimerization during Ag recognition by the TCR (7).



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  		FIGURE 4. Structurally based alignment of chicken CD4 amino acid sequence with human, mouse, and rat CD4 sequences. The ß strands of V set domains 1 and 3 are marked with ABCC'C''DEFG, and those of C set domains 2 and 4 are marked with ABCC'EFG. The numbered lines above the sequence indicate the ß strands of the particular domain, a bar shows the transmembrane region, and a line indicates the p56lck recognition site. Extra cysteines in the first domain are marked with an asterisk. An identical amino acid to the chicken sequence is shown as a dot, and a gap is shown as a dash. Numbers on the right indicate the location of the amino acid residue of the corresponding CD4 sequence.

 


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  		FIGURE 5. A stereo picture of the four extracellular domains of chicken and human CD4. The chicken sequence is in black, and the human sequence is in red. A circle indicates a putative N-glycosylation site. The model is built using human and rat CD4 crystal structures as templates (7, 17, 19).

 
The typical Ig V domain features of the first domain are well conserved. The canonical disulfide bridge between Cys residues in the B and F strands is conserved as well as the Trp in the C strand (Fig. 2Go). The characteristic pattern for Ig and TCR V domain, Asp-X-Gly/Ala-X-Tyr-X-Cys, precedes the ß strand F (33). The Gly/Ala is not found in human and mouse sequences. The Asp is at a suitable distance to form a salt bridge with the Arg of the D strand (at position 54) in human CD4, as well as with Arg58 in chicken CD4. There are two extra Cys residues in strands C and F at positions 27 and 85, respectively, that, according to the modeling, can form an extra intra-ß-sheet disulfide bridge (Fig. 6Go). Thus, the structural features make the first domain a typical V-like domain with the exception of an extra disulfide bridge.



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  		FIGURE 6. The N-terminal domain of chicken CD4 (left) and human CD4 (right). The chicken domain is built using the corresponding human structure as template (17). The C'/C'' and C''/D loops are marked in both chicken and human structures. The disulfide bridges are colored yellow. The important amino acids involved in MHC class II binding are shown.

 
In the second domain of chicken CD4 the F strand Cys is conserved, but the Cys located noncanonically in the C strand of many mammalian CD4s (at position 130 in the human) is replaced by an Ile residue (Fig. 2Go). This Cys is substituted by a Trp in cat, dog, and rabbit CD4 sequences also (34, 35, 36). Similarly to the human and mouse CD4 there is no disulfide bridge in the third domain of chicken CD4 (Fig. 4Go). However, the F strand of chicken CD4 contains a typical Ig V domain Tyr and a Cys that are not found in mammalian sequences. Instead, the C strand Trp is conserved. In the fourth domain the conserved Cys residues in strands B and F form a typical disulfide bridge (Fig. 4Go). The Ig domain-specific Trp is found in chicken CD4 (at position 344), but is replaced by a Leu residue in human, mouse, and rat. A long loop with 9-residue insertion between A and B strands obviously is located near the membrane (Figs. 4Go and 5Go).

The domain joining sequences are well conserved, suggesting their importance in the maintenance and changes of CD4 conformation (Fig. 4Go). As in mammals, the G strand of the first domain is directly followed by the A strand of the second domain. The hinge region between the second and third domains containing a conserved Val-Leu-Gly-Phe has an essential importance for the conformation, enabling the bending of the molecule (8). The G/A strands connecting domains 3 and 4 have a fully conserved tetrapeptide, Leu-Val-Val-Met, forming the core of the G/A junction. Thus, the structural relationship of these domains is exceptionally conserved in the chicken, implying a strong selective pressure. In summary, the extracellular region of chicken CD4, while having a low level of conservation at the amino acid sequence level, has nevertheless conserved several structural features of the mammalian CD4s. It has also retained some typical Ig superfamily (IgSF) domain features that are absent in mammals.

The high carbohydrate content of chicken CD4 indicates that several of the seven potential N-glycosylation sites are used (21) (Figs. 1Go and 5Go). They form a specific nonconserved glycosylation pattern of chicken CD4 not found in other species. There are three possible N-glycosylation sites in the membrane-proximal domain and two in both the second and third domains (Fig. 5Go). Human CD4 has only two potential N-glycosylation sites, one each in the third and fourth domains (2). Mouse CD4 has four glycosylation sites and rat CD4 has three that are identical with those in the mouse (37, 38).

Putative molecular interactions of chicken CD4

According to several studies the binding of human CD4 to MHC class II is mediated by residues on a broad area in the first IgSF domain and some residues in the second domain (39, 40, 41, 42, 43, 44, 45). The interacting amino acids are situated on either side of the molecule, including the C''/D region amino acids Phe43, Lys46, and Arg59 of the first domain on one side (Fig. 6Go). In the chicken only Lys50 is conserved, but there is also a solvent-exposed Phe at position 49 close to the corresponding location of Phe43 in human CD4 (Figs. 4Go and 6Go). In addition, the insertion and deletion in the C/C' loop and the D/E loop, respectively, may affect the conformation of this region in chicken CD4. However, on the same face around the C'' region positively charged residues, Lys45, Lys50, and Lys52, could associate with negatively charged Glu residues on MHC class II ß-chain that have been shown to be involved in CD4 binding (Fig. 6Go) (46). In human CD4 there are also several positively charged, surface-exposed residues in this region, like Lys46 and Arg59, that may interact with the negatively charged amino acids of the MHC class II. The role of the charge interactions in the binding affinity of CD4 and MHC class II remains to be solved.

On the opposite side of the C''/D region of the first domain of human CD4 there are solvent-exposed residues Ser19 and Gln89 and in the second domain there is Gln165 (Fig. 4Go) that are found to mediate CD4/MHC class II association (47, 48). These residues are not conserved in the chicken. Molecular interactions mediating CD4/MHC class II association on this side of chicken CD4 remain to be clarified.

The amino acids of human CD4 involved in HIV gp120 binding are located mainly near the C'-D region of the first domain (47, 48, 49, 50, 51, 52, 53). Here the chicken CD4 structure is grossly similar (Fig. 4Go). However, its conformation can be affected by the three-residue insertion in the adjacent C/C' loop and the five-residue deletion in the neighboring D/E loop, which still has potential residues for gp120 interaction in human sequence. In addition, the natures of several surface-exposed residues are different, presumably hindering the recognition of chicken CD4 by the virus.

The dimerization of human CD4 through the membrane-proximal domains involves the C/C' loop together with the F and G strands (7). The superimposition of the two chicken CD4 molecules on the human dimeric structure results in a similar clasped hands-like interaction of the concave surfaces (Fig. 7Go). The replacement of Gln344, which is absolutely conserved in mammals, by a shorter Asn375 in the chicken results in loss of the pairwise contact between these residues in the middle of the interface. However, analysis of the contacts mediating possible dimerization shows that several residues from the interacting molecules contact each other (Fig. 7GoA). Leu residues at position 373 seem to have a pairwise hydrophobic interaction similar to that of the corresponding residue Met342 in the human CD4 dimer. The side chains of Lys384 and Glu345 are at a suitable distance for charge-charge interaction. Correspondingly, Lys318 and Glu356 possibly form two pairwise salt bridges in the human dimer (7). Met352 from the C/C' loop seems to pack against residues Ile386 and Ser387 in the G strand of the other molecule. Moreover, Asn348 may interact with Asn389 in the other molecule through hydrogen bonding. Interestingly, both these Asn residues are potential glycosylation sites and are located on the edge of the interaction region. Since these residues from the two molecules are located close to each other in the dimer model, the attachment of a bulky carbohydrate side chain in both of them would result in serious steric hindrance if the dimerization took place in a similar way as that for human CD4. It is most likely that even the glycosylation of Asn389 alone would hinder the dimerization because it is mostly buried in dimer structure (Fig. 5Go). However, Asn348 in the CC' loop is exposed and thus is probably able to accommodate a carbohydrate group. Several plausible interactions, although not very conserved with the human structure, can be observed when supposing a similar dimerization pattern for chicken CD4 as that used for human CD4.



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  		FIGURE 7. A stereo view of the potential dimer interface at the membrane-proximal domain of chicken (A) and human (B) CD4. Side chains are shown for residues able to mediate dimerization.

 
The cytoplasmic region

The cytoplasmic tail has a relatively high homology to mammalian CD4 sequences, with 55 and 52% identities to human and mouse sequences, respectively. The region starting from Arg427 is five amino acids shorter than those in human, mouse, and rat CD4 (Fig. 4Go). It contains the highly conserved protein tyrosine kinase p56lck recognition site KKTCQC (Fig. 4Go) (10, 54). Chicken CD4 has been shown to interact with a cellular tyrosine kinase homologous to mammalian p56lck (55). In chicken the p56lck binding site is also preceded by a di-leucine motif involved in Ag-induced CD4 internalization in the human (13, 14, 56). The internalization requires phosphorylation of the cytoplasmic Ser residues (11, 12, 13). Interestingly, the chicken CD4 sequence does not have the Ser residues that are conserved in mammals (at positions 408 and 415 in human). Their absence may explain the slow down-regulation of chicken CD4 after phorbol ester stimulation (21). Instead, there are two Tyr residues (one of them adjacent to the di-leucine motif) whose phosphorylation may replace the lacking phosphoserines (Fig. 4Go).

Conclusion

We have cloned the first nonmammalian CD4 from the chicken. The composition of the extracellular Ig domains of chicken CD4 as well as the highly conserved cytoplasmic region imply functional mechanisms that have remained conserved across evolutionarily distant species.


   Acknowledgments
 
We thank Ann Sofie Hakulinen, Aija Kaitaranta, and Reija Venho for their excellent technical assistance; Drs. Bruce W. Calnek, Karel A. Schat, and Kevyan Nazerian for the cell lines; Drs. Paavo Toivanen, Mikael Skurnik, Jari Jalava, and Jussi Liippo for discussions; Eija Nordlund and Tuula Närä for secretarial help; and Teuvo Virtanen for help in computing.


   Footnotes
 
1 This work was supported by the Academy of Finland, the Turku University Foundation, and the Human Frontier Science Program (Grant RG 366/96 to O.V.) and by the Danish National Science Research Council (Grants 9401634/9502172 to J.S.). Back

2 Address correspondence and reprint requests to Dr. Riitta Koskinen, Turku Immunology Center and Department of Medical Microbiology, Kiinamyllynkatu 13, 20520 Turku, Finland. Back

Received for publication September 22, 1998. Accepted for publication December 22, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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